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https://openalex.org/W2756060471
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https://www.nature.com/articles/s41598-017-11969-5.pdf
|
English
| null |
Bacteriological diagnosis of childhood TB: a prospective observational study
|
Scientific reports
| 2,017
|
cc-by
| 7,125
|
Bacteriological diagnosis of
childhood TB: a prospective
observational study y
Andrew J. Brent 1,2,3,4, Daisy Mugo1, Robert Musyimi1, Agnes Mutiso1, Susan C. Morpeth1
Michael Levin3 & J. Anthony G. Scott 1,2,5 Received: 16 June 2017
Accepted: 1 September 2017
Published: xx xx xxxx Received: 16 June 2017
Accepted: 1 September 2017
Published: xx xx xxxx Childhood TB diagnosis is challenging. Studies in adults suggest Microscopic Observation Drug
Susceptibility (MODS) culture or the Xpert MTB/RIF assay might be used to expand bacteriological
diagnosis. However data from children are more limited. We prospectively compared MODS and Xpert
MTB/RIF with standard microscopy and culture using the BD MGIT 960 system among 1442 Kenyan
children with suspected TB. 97 specimens from 54 children were TB culture-positive: 91 (94%) by MGIT
and 74 (76%) by MODS (p = 0.002). 72 (74%) culture-positive and 7 culture-negative specimens were
Xpert MTB/RIF positive. Xpert MTB/RIF specificity was 100% (99.7–100%) among 1164 specimens
from 892 children in whom TB was excluded, strongly suggesting all Xpert MTB/RIF positives are true
positives. The sensitivity of MGIT, MODS and Xpert MTB/RIF was 88%, 71% and 76%, respectively,
among all 104 true positive (culture and/or Xpert MTB/RIF positive) specimens. MGIT, MODS and Xpert
MTB/RIF on the initial specimen identified 40/51 (78%), 33/51 (65%) and 33/51 (65%) culture-confirmed
pulmonary TB cases, respectively; Xpert MTB/RIF detected 5 additional culture-negative cases. The high
sensitivity and very high specificity of the Xpert MTB/RIF assay supports its inclusion in the reference
standard for bacteriological diagnosis of childhood TB in research and clinical practice. Definitive diagnosis of tuberculosis (TB) rests on detection of M. tuberculosis from clinical specimens, but is
difficult in children. While sputum smear microscopy underpins the WHO DOTS and Stop TB strategies for TB
diagnosis in adults1, obtaining sputum is more difficult in children, and the yield of microscopy alone is very poor
due to small numbers of bacilli in clinical specimens. Bacteriological diagnosis therefore depends on mycobac-
terial culture2. Strengthening laboratory capacity is a key component of the Stop TB Strategy and Global Plan to Stop TB1,3. Commercial liquid culture methods like the BACTEC Mycobacteria Growth Indicator Tube (MGIT) system
(BD Diagnostics, Sparks, MD, USA) offer higher sensitivity and more rapid results than traditional solid media. However, barriers to their uptake include cost, a lack of technically trained personnel, and the need for biosafety
level 3 facilities3. Correction: Author Correction www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports SCIentIfIC ReporTs | 7: 11808 | DOI:10.1038/s41598-017-11969-5 Bacteriological diagnosis of
childhood TB: a prospective
observational study Provider initiated testing and counseling for HIV was
performed according to Kenyan national guidelines, which recommend testing for all inpatients, and for all
patients investigated for TB, on an opt-out basis. g
Children who were able to expectorate provided up to three spontaneous sputum samples. Sputum induction
was performed on the remainder. If sputum induction was contraindicated (e.g. due to severe respiratory dis-
tress), gastric aspiration was performed. Sputum induction and gastric aspiration were performed according to
international recommendations2. Further investigations including extra-pulmonary or repeat sputum sampling
were performed at the discretion of the clinical team caring for the patient. p
g
p
We classified children as Confirmed TB, Highly Probable TB, Possible TB or Not TB according to their clin-
ical, radiological and microbiological findings. Categories were defined a priori and based closely on published
definitions (Fig. 1)13,14. Treatment protocols followed Kenyan national guidelines. Children treated for TB were
followed up until completion of treatment at 6 months. Other children were also followed up for 6 months, or
until TB could be confidently excluded. Final diagnostic assignments were revised in the light of follow up data. Laboratory procedures. Specimens were transported to the laboratory at 2–8 °C and processed the same
day. Sputum specimens were decontaminated using the modified Petroff’s method and 4% sodium hydroxide15;
centrifuged; and the sediment re-suspended in 2 ml culture broth containing Middlebrook 7H9 medium, oxalic
acid, albumin, dextrose, and catalase (OADC, Becton Dickinson), and polymyxin, amphotericin B, nalidixic acid,
trimethoprim, and azlocillin (PANTA, Becton Dickinson)16. After vigorous vortexing to ensure sample homog-
enization, two drops were used to make a smear for Ziehl Neelsen staining, and the remainder divided equally
for MGIT, MODS, and Xpert MTB/RIF. Samples for Xpert MTB/RIF analysis were promptly frozen at −80 °C for
analysis at the end of the study. y
y
Liquid mycobacterial culture was performed using the MGIT 960 system and MODS assay. A laboratory
technician (RM) first received one month MODS training at the Universidad Peruana Cayetano Heredia in Lima,
Peru. Standard protocols were followed15,16, except that MODS plates were examined 3 times a week rather than
daily for logistic reasons. y
g
Positive cultures from both methods were identified as M. tuberculosis complex (MTBC) or non-MTBC by
the BD MGIT TBc Identification test (BD Diagnostics, Sparks, MD, USA). Bacteriological diagnosis of
childhood TB: a prospective
observational study Microcolony culture techniques such as the Microscopic Observation Drug Susceptibility (MODS) assay have
several potential advantages over conventional culture methods in low resource settings, including high sensi-
tivity; more rapid results that include drug susceptibility testing (DST); lower cost; and less stringent biosafety
requirements making it applicable in biosafety level 2 facilities4. However only limited data exist on their use in
children5–7, and no studies have compared MODS with MGIT for diagnosis of TB in young children; nor eval-
uated MODS yield from induced sputum samples, despite good evidence and international recommendations
supporting sputum induction for childhood TB diagnosis2,8.hf g
g
The Xpert MTB/RIF real-time PCR assay (Cepheid, Sunnyvale, CA, USA) offers a rapid and operationally
simpler alternative to culture for detection of M. tuberculosis and rifampicin resistance9,10. A recent meta-analysis
suggests modest sensitivity and high specificity for paediatric TB diagnosis11, and the latest WHO childhood TB
guidelines (2014) recommend Xpert MTB/RIF as an alternative to conventional microscopy and culture - but
acknowledge the “very low quality of evidence”2. 1KEMRI-Wellcome Trust Research Programme, Kilifi, Kenya. 2University of Oxford, Oxford, UK. 3Imperial College
London, London, UK. 4Oxford University Hospitals NHS Foundation Trust, Oxford, UK. 5London School of Hygiene
& Tropical Medicine, London, UK. Correspondence and requests for materials should be addressed to A.J.B. (email:
dr.a.brent@gmail.com) SCIentIfIC ReporTs | 7: 11808 | DOI:10.1038/s41598-017-11969-5 1 www.nature.com/scientificreports/ We compared microscopy, MGIT and MODS culture, and the Xpert MTB/RIF assay for diagnosis of child-
hood TB in Kenya. Participants. We prospectively assessed each diagnostic method among children presenting to Coast
Provincial General Hospital and Kilifi County Hospital in Coast Province, Kenya. Between July 2010 and
December 2011 children aged <15 years were investigated for TB if one or more of the following features of sus-
pected TB were present and they were not already on TB treatment: unexplained persistent cough for >2 weeks;
pneumonia not responding to first line antibiotics; unexplained fever for >2 weeks; unexplained progressive
weight loss or failure to thrive for >4 weeks; a history of close contact with a suspected or confirmed case of pul-
monary TB; or clinical suspicion of TB for any other reason. Clinical procedures. Each child underwent a structured history and examination, chest radiography and
tuberculin skin testing (TST) according to WHO guidelines2. Severe malnutrition was defined as weight for age
z-score of <−3 or the presence of nutritional oedema12. Bacteriological diagnosis of
childhood TB: a prospective
observational study They were then further speciated and
probed for isoniazid and rifampicin resistance mutations by PCR, using the Hain Genotype® line probe assay
platform (Hain Lifescience GmbH, Nehren, Germany). The Xpert MTB/RIF assay (version G4) was performed
at the end of the study on specimens from all children treated for confirmed, highly probable or possible TB, and
from children in whom TB had been excluded. Laboratory procedures were externally monitored through the UK
NEQAS quality assurance scheme and annual Good Clinical Laboratory Practice audits (Qualogy, UK). q
y
y
gy
In order to compare the performance of MGIT and MODS, and to prevent observer bias in the interpretation
of either result, laboratory technologists were blinded to the identity of the MODS portion of each specimen. Specimens were instead identified by an electronically generated random numeric code, the key to which was
held by the Principal Investigator. Statistical analysis. Data were analyzed at both the patient and the specimen level. We first performed
a per patient analysis to compare the sensitivity of smear microscopy, MGIT, MODS and Xpert MTB/RIF for
identification of culture confirmed pulmonary TB cases. We then included all specimens (pulmonary and
extra-pulmonary) in a per specimen analysis to calculate the sensitivity of each method against the existing refer-
ence standard of a positive M. tuberculosis culture (by MGIT and/or MODS).i p
y
To explore the specificity of Xpert MTB/RIF we calculated the proportion of specimens from Not TB cases
that were Xpert MTB/RIF positive. Having established the very high specificity of the Xpert MTB/RIF assay, we
then repeated the per specimen analysis using a composite reference standard incorporating Xpert MTB/RIF, such
that a specimen was considered positive if either culture or Xpert MTB/RIF identified M. tuberculosis. i
We used McNemar’s χ2 test to compare proportions between tests on paired aliquots of the same sample. To
investigate any learning effect as laboratory staff became more experienced with MODS we performed the χ2 test
for trend in the proportion of M. tuberculosis culture positive specimens identified by MODS in each quarter,
and the Wilcoxon rank sum test to compare MODS time to detection (TTD) in the first and second years of the
study. We used the Wilcoxon signed rank test to compare TTD of MTBC by MODS and MGIT. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Bacteriological diagnosis of
childhood TB: a prospective
observational study Associations with
a positive Xpert MTB/RIF assay were explored using univariable and multivariable logistic regression models
adjusted for clustering at the patient level. SCIentIfIC ReporTs | 7: 11808 | DOI:10.1038/s41598-017-11969-5 2 Figure 1. Childhood TB Case Definitions. Ethical approval and informed consent. The study was approved by the Kenya National Ethics
Committee. All study procedures were performed in accordance with relevant guidelines and regulations. A
parent or guardian provided written informed consent. Data availability. The datasets generated during and/or analysed during the current study are available from
the corresponding author on reasonable request. SCIentIfIC ReporTs | 7: 11808 | DOI:10.1038/s41598-017-11969-5 3 www.nature.com/scientificreports/ Results
We identified 1500 children with features of suspected TB, 1442 of whom were investigated for TB (Fig. 2). Active
Figure 2. Patient enrolment and disease assignments. Confirmed TB
Highly Probable TB
Treated for Possible TB
Not TB (TB excluded)
Not classifiable
(n = 54)
(n = 63)
(n = 95)
(n = 892)
(n = 338)
Patient demographics
Age
Median (IQR1), months
53 (21–112)
32 (13–61)
17 (10–68)
16 (9–40)
17 (9–48)
0 to 4 years
27 (50%)
47 (75%)
67 (70%)
750 (84%)
271 (80%)
5 to 9 years
14 (26%)
10 (16%)
15 (16%)
95 (11%)
45 (13%)
10 to 14 years
13 (24%)
6 (9%)
13 (14%)
47 (5%)
22 (7%)
Male sex
33 (61%)
32 (51%)
48 (51%)
469 (53%)
188 (56%)
Clinical features
Cough > 2 weeks
40 (74%)
37 (59%)
71 (75%)
425 (48%)
194 (57%)
Fever > 2 weeks
37 (69%)
24 (38%)
66 (69%)
375 (42%)
167 (49%)
Weight loss/FTT2 > 4 weeks
32 (59%)
29 (46%)
51 (54%)
385 (43%)
168 (50%)
Pneumonia not responding
to first line antibiotics
22 (41%)
21 (33%)
35 (37%)
260 (29%)
144 (43%)
Close TB contact
24 (44%)
25 (40%)
23 (24%)
145 (16%)
62 (18%)
HIV infected
15 (28%)
15 (24%)
35 (37%)
121 (14%)
88 (26%)
Severely malnourished
19 (35%)
31 (49%)
40 (42%)
303 (34%)
119 (35%)
Pulmonary TB3
51 (94%)
57 (90%)
77 (81%)
—
—
Extra-pulmonary TB3
7 (13%)
6 (10%)
20 (21%)
—
—
Table 1. Baseline characteristics of children with and without TB. 1IQR, interquartile range. 2FTT, failure to
thrive. 3Six patients with pulmonary TB also had extrapulmonary TB (4 with culture confirmed TB, 2 that were
treated for possible TB). Figure 2. Patient enrolment and disease assignments. Figure 2. Patient enrolment and disease assignments. Confirmed TB
Highly Probable TB
Treated for Possible TB
Not TB (TB excluded)
Not classifiable
(n = 54)
(n = 63)
(n = 95)
(n = 892)
(n = 338)
Patient demographics
Age
Median (IQR1), months
53 (21–112)
32 (13–61)
17 (10–68)
16 (9–40)
17 (9–48)
0 to 4 years
27 (50%)
47 (75%)
67 (70%)
750 (84%)
271 (80%)
5 to 9 years
14 (26%)
10 (16%)
15 (16%)
95 (11%)
45 (13%)
10 to 14 years
13 (24%)
6 (9%)
13 (14%)
47 (5%)
22 (7%)
Male sex
33 (61%)
32 (51%)
48 (51%)
469 (53%)
188 (56%)
Clinical features
Cough > 2 weeks
40 (74%)
37 (59%)
71 (75%)
425 (48%)
194 (57%)
Fever > 2 weeks
37 (69%)
24 (38%)
66 (69%)
375 (42%)
167 (49%)
Weight loss/FTT2 > 4 weeks
32 (59%)
29 (46%)
51 (54%)
385 (43%)
168 (50%)
Pneumonia not responding
to first line antibiotics
22 (41%)
21 (33%)
35 (37%)
260 (29%)
144 (43%)
Close TB contact
24 (44%)
25 (40%)
23 (24%)
145 (16%)
62 (18%)
HIV infected
15 (28%)
15 (24%)
35 (37%)
121 (14%)
88 (26%)
Severely malnourished
19 (35%)
31 (49%)
40 (42%)
303 (34%)
119 (35%)
Pulmonary TB3
51 (94%)
57 (90%)
77 (81%)
—
—
Extra-pulmonary TB3
7 (13%)
6 (10%)
20 (21%)
—
—
Table 1. Baseline characteristics of children with and without TB. 1IQR, interquartile range. 2FTT, failure to
thrive. 3Six patients with pulmonary TB also had extrapulmonary TB (4 with culture confirmed TB, 2 that were
treated for possible TB). Table 1. Baseline characteristics of children with and without TB. 1IQR, interquartile range. 2FTT, failure to
thrive. 3Six patients with pulmonary TB also had extrapulmonary TB (4 with culture confirmed TB, 2 that were
treated for possible TB). Resultsi Comparison of MGIT and MODS culture sensitivity against culture reference standard incorporating
both MGIT and MODS. subsequent sputum sample. MGIT, MODS and Xpert MTB/RIF each demonstrated a modest incremental yield
from additional specimens (Table 3). None of the 8 initial culture-negative specimens from confirmed PTB cases
were Xpert MTB/RIF positive. However Xpert MTB/RIF did detect M. tuberculosis in sputum from 5 additional
children with culture negative pulmonary TB (3 with clinically Highly Probable TB and 2 who were treated for
Possible TB). subsequent sputum sample. MGIT, MODS and Xpert MTB/RIF each demonstrated a modest incremental yield
from additional specimens (Table 3). None of the 8 initial culture-negative specimens from confirmed PTB cases
were Xpert MTB/RIF positive. However Xpert MTB/RIF did detect M. tuberculosis in sputum from 5 additional
children with culture negative pulmonary TB (3 with clinically Highly Probable TB and 2 who were treated for
Possible TB). Per specimen analysis of pulmonary and extra-pulmonary specimens combined. A total of 2085
specimens were obtained for mycobacterial culture. We were unable to do MODS on 182 samples due to holiday
staffing shortages (169) or insufficient sample (13). A total of 1903 specimens were therefore included in the anal-
ysis, of which 1802 (95%) were sputum specimens, 33 (2%) were smear positive, and 97 (5%) were M. tuberculosis
culture positive (Supplementary Appendix, Table S1). MGIT was more sensitive than MODS overall (93.8% vs
76.3%, p = 0.002), among all sputum specimens, among induced sputum specimens, and among specimens from
both younger and older children (Table 4).h y
g
(
)
There was weak evidence of an increase in MODS sensitivity (χ2 test for trend p = 0.038) and a decrease in
TTD over time, with a median TTD of 16 (IQR 7 to 21) days in 2010 compared with 10 (7 to 14) days in 2011
(p = 0.052; Supplementary Appendix, Fig. S1). Overall, TTD was slightly shorter for MODS than MGIT (11 [IQR
6 to 15] days versus 12 [7 to 17] days, p = 0.001). ]
y
[
]
y , p
)
Discordant culture results were obtained for 29 specimens. Independent bacteriological confirmation of
M. tuberculosis was available for 21/29 of these specimens, either by the Xpert MTB/RIF assay on the original
specimen (15/29) and/or by isolation of M. tuberculosis from an independent specimen from the same patient
(Supplementary Appendix, Table S2). Resultsi yield*
1st specimen
51
13 (25%)
25%
40 (78%)
78%
33 (65%)
65%
43 (84%)
84%
33 (65%)
65%
2nd specimen
32
10 (31%)
33%
22 (69%)
82%
15 (68%)
73%
24 (75%)
90%
18 (56%)
69%
3rd specimen
23
7 (30%)
33%
14 (61%)
84%
13 (57%)
75%
15 (65%)
92%
13 (57%)
71% Specimen
N
Smear microscopy
MGIT culture
MODS culture
MODS + MGIT
culture
Xpert MTB/RIF
Test + (%)
Cum. yield*
Test + (%)
Cum. yield*
Test + (%)
Cum. yield*
Test + (%)
Cum. yield*
Test + (%)
Cum. yield*
1st specimen
51
13 (25%)
25%
40 (78%)
78%
33 (65%)
65%
43 (84%)
84%
33 (65%)
65%
2nd specimen
32
10 (31%)
33%
22 (69%)
82%
15 (68%)
73%
24 (75%)
90%
18 (56%)
69%
3rd specimen
23
7 (30%)
33%
14 (61%)
84%
13 (57%)
75%
15 (65%)
92%
13 (57%)
71%
Table 3. Cumulative sensitivity (incremental yield) of serial sputum specimens from patients with culture
confirmed pulmonary TB. *Cum. yield = cumulative sensitivity of serial sputum specimens. In 4 (8%) cases of
culture-confirmed pulmonary TB, culture confirmation was obtained from a subsequent specimen (n = 1) or
from a specimen excluded from the analysis because MODS culture was not performed (n = 3). Table 3. Cumulative sensitivity (incremental yield) of serial sputum specimens from patients with culture
confirmed pulmonary TB. *Cum. yield = cumulative sensitivity of serial sputum specimens. In 4 (8%) cases of
culture-confirmed pulmonary TB, culture confirmation was obtained from a subsequent specimen (n = 1) or
from a specimen excluded from the analysis because MODS culture was not performed (n = 3). MGIT positive
MGIT negative
MGIT Sensitivity %
(95% CI)
MODS Sensitivity %
(95% CI)
p value
MODS
positive
MODS
negative
MODS
positive
MODS
negative
Specimen type
All specimens
68
23
6
1807
93.8 (87.0–97.7)
76.3 (66.6–84.3)
0.002
All sputum specimens
61
22
6
1713
93.3 (85.9–97.5)
75.3 (65.0–83.8)
0.003
Induced sputum
40
18
6
1603
90.6 (80.7–96.5)
71.9 (59.2–82.4)
0.014
Age of patient
<5 years
28
14
5
1398
89.4 (76.9–96.5)
70.2 (55.1–82.7)
0.039
5–14 years
36
8
1
380
97.8 (88.2–99.9)
82.2 (67.9–92.0)
0.039
Table 4. Comparison of MGIT and MODS culture sensitivity against culture reference standard incorporating
both MGIT and MODS. Table 4. Comparison of MGIT and MODS culture sensitivity against culture reference standard incorporating
both MGIT and MODS. Table 4. Resultsi We identified 1500 children with features of suspected TB, 1442 of whom were investigated for TB (Fig. 2). Active
TB was diagnosed in 212 (14.7%) children, including 54 (25.5%) with Culture-confirmed TB, 63 (29.7%) with
Highly Probable TB, and 95 (44.8%) with Possible TB. Most cases (185, 87%) had pulmonary TB (PTB). Baseline
demographic and clinical characteristics are summarized in Table 1. In keeping with previous studies, the propor-
tion of cases that were bacteriologically confirmed was higher among older children. Per patient analysis of pulmonary TB cases. There were 51 cases of Culture-confirmed pulmonary TB. Smear microscopy, MGIT and MODS on the initial sputum specimen identified M. tuberculosis in 13 (25%), 40
(78%) and 33 (65%) cases, respectively (Table 2). Sensitivity was highest for each method among smear positive
samples; Xpert MTB/RIF identified 100% of smear-positive cases and 53% of smear-negative cases on the first
sputum specimen (Table 2). p
p
Second and third sputum specimens were obtained from 237 (16%) and 102 (7%) children, respectively. Eight
(16%) culture confirmed PTB cases were culture negative from the initial sample but grew M. tuberculosis from a SCIentIfIC ReporTs | 7: 11808 | DOI:10.1038/s41598-017-11969-5 4 www.nature.com/scientificreports/ AFB smear positive (N = 13)
AFB smear negative (N = 38)
Total (N = 51)
N
Sensitivity (95% CI)
N
Sensitivity (95% CI)
N
Sensitivity (95% CI)
AFB smear microscopy
13
N/A
N/A
N/A
13
25.5 (14.3–39.6)
MGIT culture
13
100.0 (75.3–100.0)
27
71.1 (54.1–84.6)
40
78.4 (64.7–88.7)
MODS culture
13
100.0 (75.3–100.0)
20
52.6 (35.8–69.0)
33
64.7 (50.1–77.6)
MGIT + MODS culture
13
100.0 (75.3–100.0)
30
78.9 (62.7–90.4)
43
84.3 (71.4–93.0)
Xpert MTB/RIF
13
100.0 (75.3–100.0)
20
52.6 (35.8–69.0)
33
64.7 (50.1–77.6)
Table 2. Sensitivity of each method for detection of M. tuberculosis in the initial sputum specimen from
patients with culture confirmed pulmonary TB. Table 2. Sensitivity of each method for detection of M. tuberculosis in the initial sputum specimen from
patients with culture confirmed pulmonary TB. Specimen
N
Smear microscopy
MGIT culture
MODS culture
MODS + MGIT
culture
Xpert MTB/RIF
Test + (%)
Cum. yield*
Test + (%)
Cum. yield*
Test + (%)
Cum. yield*
Test + (%)
Cum. yield*
Test + (%)
Cum. Discussion
Th l
k f
l The lack of reliable diagnostic tools is the single greatest obstacle to improved childhood TB case management,
particularly in low resource settings where the disease burden is greatest. Expansion of TB laboratory diagnostic
capacity provides an opportunity to improve both diagnosis and surveillance in many settings3. However good
quality data on the performance of each method are required to optimize their use and interpretation. q
y
p
q
p
p
Few studies have compared laboratory methods for childhood TB diagnosis. Limitations of existing studies
include small numbers of confirmed TB cases5–7,14,17–22, particularly among young children in whom diagnosis is
h ll
i
5 7 14 19 22
d
l
i
f ‘
ld
d d’
h d f
i
ll
i
d/
b
i l y
Few studies have compared laboratory methods for childhood TB diagnosis. Limitations of existing studies
include small numbers of confirmed TB cases5–7,14,17–22, particularly among young children in whom diagnosis is
most challenging5–7,14,19,22, and exclusion of ‘gold standard’ methods for specimen collection and/or mycobacterial
lt
5 7 17 20 23 26 St
th
f thi
t d i
l d th
f b th
t
i d
ti
d li
id
b
t
i l culture5 7,17,20,23 26. Strengths of this study include the use of both sputum induction and liquid mycobacterial
culture in keeping with current international recommendations2, and rigorous diagnostic assignments based on
detailed clinical assessment and follow up. Prospective recruitment of all children who met broad, pre-defined
inclusion criteria also ensures generalizability to a wide range of settings and clinical syndromes, including among
children under 5 years who accounted for 81% of those investigated and half of all confirmed cases.i y
gi
While comparison of culture yield between studies is hampered by widely varying clinical definitions, speci-
men types and culture methods, the proportion of culture-confirmed cases in our study is consistent with other
studies using liquid mycobacterial culture for paediatric diagnosis5,8,14,18,20,26–30.h In our study MGIT was more sensitive than MODS culture. This is in keeping with the only other published
study comparing MGIT with MODS for paediatric TB diagnosis5. Importantly we were also able to demonstrate
the higher yield of MGIT for culture of paediatric induced sputum samples. These results contrast with other
studies in adults in which MODS compared more favourably with MGIT4. Resultsi The clinical picture strongly supported the presence of M. tuberculosis
complex in the remaining 8 culture-discordant samples: 5 MGIT+/MODS− specimens came from children
with clinically Highly Probable TB prior to culture results; 2 MGIT−/MODS+ specimens came from chil-
dren treated empirically for TB prior to the culture result although they did not meet the stringent definition SCIentIfIC ReporTs | 7: 11808 | DOI:10.1038/s41598-017-11969-5 5 www.nature.com/scientificreports/ Culture positive
Culture negative
Culture Sensitivity %
(95% CI)
Xpert MTB/RIF
Sensitivity % (95% CI)
p value
Xpert
positive
Xpert
negative
Xpert
positive
Xpert
negative
Specimen type
All specimens
72
25
7
1452
93.3 (86.6–97.3)
76.0 (66.6–83.8)
0.002
All sputum specimens
68
21
7
1419
92.7 (85.6–97.0)
78.1 (68.5–85.9)
0.008
Induced sputum
47
17
5
1356
92.8 (83.9–97.6)
75.4 (63.5–84.9)
0.011
Age of patient
<5 years
32
15
7
1152
87.0 (75.1–94.6)
72.2 (58.4–83.5)
0.088
5–14 years
40
10
0
300
100 (92.9–100)
80.0 (66.3–90.0)
0.002
Table 5. Comparison of culture and Xpert MTB/RIF sensitivity against composite reference standard of both
culture and Xpert MTB/RIF. Table 5. Comparison of culture and Xpert MTB/RIF sensitivity against composite reference standard of bot
culture and Xpert MTB/RIF. of Highly Probable TB; and one MGIT+/MODS− specimen that grew M. bovis BCG came from a 4 month
old HIV-infected child who had a clinical syndrome consistent with disseminated BCG disease (Supplementary
Appendix, Table S2). All were the only positive cultures in their batch, arguing against cross-contamination caus-
ing the discordance. Even if cross-contamination were the cause and these specimens considered culture negative,
MGIT would remain more sensitive than MODS (95.5% vs 80.9%, p = 0.005).i of Highly Probable TB; and one MGIT+/MODS− specimen that grew M. bovis BCG came from a 4 month
old HIV-infected child who had a clinical syndrome consistent with disseminated BCG disease (Supplementary
Appendix, Table S2). All were the only positive cultures in their batch, arguing against cross-contamination caus-
ing the discordance. Even if cross-contamination were the cause and these specimens considered culture negative,
MGIT would remain more sensitive than MODS (95.5% vs 80.9%, p = 0.005).i Xpert MTB/RIF identified 72/97M. tuberculosis culture positive specimens, giving a sensitivity compared to
culture of 74.2% (95% CI 64.3 to 82.6). Xpert MTB/RIF was also positive on 5/86 (6%) specimens from patients
with culture-negative but clinically highly probable TB, and 2/179 (1%) specimens from children treated for
possible TB. Resultsi No other specimens were Xpert MTB/RIF positive, including none of 1164 specimens obtained from
children in whom TB was subsequently excluded - giving a specificity of 100% (1 sided 97.5% CI 99.7 to 100%)
and strongly suggesting positive Xpert MTB/RIF results should be interpreted as true positives. Against the composite reference standard of a positive TB culture and/or Xpert MTB/RIF result, the sensi-
tivity of TB culture was 93.3% (86.6 to 97.3), compared with 76.0% (66.6 to 83.8) for Xpert MTB/RIF (p = 0.002;
Table 5). The sensitivity of MGIT and MODS culture alone were 87.5% (79.6 to 93.2) and 71.2% (61.4 to 79.6),
respectively. Table 6 shows Xpert MTB/RIF sensitivity against the composite reference standard, broken down by smear
status, specimen type, age and HIV status. Sensitivity was higher for smear positive specimens (100% compared
with 65% for smear negative specimens, p < 0.001), with a trend towards higher sensitivity among sputum sam-
ples. Neither age nor HIV status were associated with Xpert MTB/RIF yield in univariable or multivariable
analyses.i y
Xpert MTB/RIF identified rifampicin resistance in 4 specimens from the same child, all of which were con-
firmed resistant to rifampicin by MODS and the GenoType MTBDRplus assay. MODS did not identify any other
rifampicin resistant isolates. ignificantly, a combination of culture and Xpert MTB/RIF increased bacteriological yield without compromising
pecificity.ii i
Importantly, we were able to confirm the very high specificity of Xpert MTB/RIF in a large sample of speci-
mens from children in whom TB had been excluded by careful clinical, radiological and laboratory assessment
and follow up. Positive Xpert MTB/RIF results have been reported in a small number of child TB suspects who
did well at follow up without TB treatment11,24. However the very high precision with which we were able to
demonstrate 100% specificity of Xpert MTB/RIF in a carefully characterized group of children without TB sug-
gests that these apparent false positive results from TB suspects in very high burden settings may have represented
self-limiting pulmonary tuberculosis. This is well recognized in the pre-chemotherapy literature31. h
Our primary analysis compared Xpert MTB/RIF sensitivity with the current culture reference standard. Having demonstrated the very high specificity of Xpert MTB/RIF we then repeated the analysis using a compos-
ite reference standard of positive culture or Xpert MTB/RIF. Composite reference standards should be used with
care32. However, given the equivalent high specificities of Xpert MTB/RIF and mycobacterial culture, incorpora-
tion bias in favour of Xpert MTB/RIF is unlikely. Furthermore, misclassification bias arising from the imperfect
sensitivity of culture may bias estimates of the accuracy of Xpert MTB/RIF culture alone is used as the refer-
ence33,34. We believe the composite reference standard therefore provides a fairer comparison of the two methods,
and a better estimate of the true sensitivity of the Xpert MTB/RIF assay. One potential limitation of our study is that molecular typing was not performed on MGIT-MODS discordant
culture isolates to exclude cross-contamination. Nevertheless there was strong microbiological and/or clinical
evidence to corroborate the positive culture in every case. In addition, negative controls in every MGIT batch and
MODS plate provided no evidence of cross-contamination throughout the study, consistent with the very low
prevalence and concentrations of M. tuberculosis in these paediatric specimens.i Due to finite resources, most children only had a single sputum sample obtained. Although this and other
studies show the incremental yield of additional specimens where obtained, analysis of a single specimen is likely
to reflect practice in most low resource settings due to the incremental cost of processing additional specimens. l
p
g
p
g
p
In conclusion, our results underline the imperfect sensitivity of all currently available methods for bacteriolog-
ical diagnosis of childhood TB. ignificantly, a combination of culture and Xpert MTB/RIF increased bacteriological yield without compromising
pecificity.ii The choice of method in any particular setting depends on several factors includ-
ing prevalence of TB and drug resistance; resources available to risk stratify patients for testing; test sensitivity,
cost35, time to detection; and the availability of trained staff and laboratory biosafety facilities. Our data confirm
the superior sensitivity of MGIT compared with MODS and Xpert MTB/RIF. Although MODS may have a role
in some settings, the operationally simpler Xpert MTB/RIF assay was as sensitive as MODS, demonstrated equiv-
alent specificity to MGIT, and combined with MGIT it optimized bacteriological yield. Together these results
strengthen the evidence base for inclusion of Xpert MTB/RIF in the reference standard for bacteriologically
confirmed childhood TB in both WHO clinical guidelines35 and research. Role of the funding source. The study was supported by the Wellcome Trust in the form of research fel-
lowships to AJB (081697) and JAGS (098532), and a core grant to the KEMRI-Wellcome Trust research pro-
gramme (077092); and by a project grant from the Pneumonia Etiology Research for Child Health (PERCH)
project funded by the Bill & Melinda Gates Foundation at Johns Hopkins Bloomberg School of Public Health. The
funders had no role in data analysis or in the decision to publish. Discussion
Th l
k f
l The paucibacillary nature of childhood
TB may have a role, and variation between centres also suggests MODS performance is operator dependent4. In
support of this, even after one month residential training with an experienced team, we were able to demonstrate
a learning effect with increasing MODS yield and reducing time to detection during the two years of the study. We
did not compare MODS with culture on solid media, however other studies have shown MODS to be superior to
Lowenstein Jensen media for diagnosis of childhood TB6,7,26. g
Against a reference standard of mycobacterial culture, Xpert MTB/RIF sensitivity was similar to other paedi-
atric studies11, and comparable to MODS - in keeping with the only other study to compare the two methods29. SCIentIfIC ReporTs | 7: 11808 | DOI:10.1038/s41598-017-11969-5 6 www.nature.com/scientificreports/ No. MTBC positive
specimens
No. Xpert MTB/
RIF positive
Xpert MTB/RIF
Sensitivity, % (95% CI)
p value
Specimen type
sputum specimens
96
75
78.1
(68.5 to 85.9)
0.062
non-sputum specimens
8
4
50.0
(15.7 to 84.3)
Specimen smear status
smear positive
33
33
100.0
(89.4 to 100.0)
<0.001
smear negative
71
46
64.8
(52.5 to 75.8)
Patient age
0–4 years
54
39
72.2
(58.4 to 83.5)
0.461
5–14 years
50
40
80.0
(66.3 to 90.0)
Patient HIV status
HIV positive
19
15
78.9
(54.4 to 93.9)
0.725
HIV negative
85
64
75.3
(64.7 to 84.0)
All MTBC positive specimens
104
79
76.0
(66.6–83.8)
—
Table 6. Sensitivity of the Xpert MTB/RIF assay against the composite reference standard. Table 6. Sensitivity of the Xpert MTB/RIF assay against the composite reference standard. Significantly, a combination of culture and Xpert MTB/RIF increased bacteriological yield without compromising
specificity.ii 2. World Health Organisation. Guidance for national tuberculosis programmes on the management of tuberculosis in children. Second
edition edn, (World Health Organisation, 2014).
3 World Health Organization A Roadmap for Ensuring Quality Tuberculosis Diagnostics Services within National Laboratory References
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of tuberculosis. (WHO Press, 2010).
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3. World Health Organization. A Roadmap for Ensuring Quality Tuberculosis Diagnostics Services within National Laboratory
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malnutrition and pneumonia including an evaluation of Xpert MTB/RIF assay. PloS one 9, e93776, https://doi.org/10.1371/journal. pone.0093776 (2014). 18. LaCourse, S. M. et al. Use of Xpert for the diagnosis of pulmonary tuberculosis in severely malnourished hospitalized Malawian
children. The Pediatric infectious disease journal 33, 1200–1202, https://doi.org/10.1097/INF.0000000000000384 (2014).i 19. Ntinginya, E. N. et al. Performance of the Xpert(R) MTB/RIF assay in an active case-finding strategy: a pilot study from Tanzania. Int J Tuberc Lung Dis 16, 1468–1470, https://doi.org/10.5588/ijtld.12.0127 (2012). ung Dis 16, 1468–1470, https://doi.org/10.5588/ijtld.12.0127 (2012) 20. Pang, Y. et al. Evaluation of the Xpert MTB/RIF assay in gastric lavage aspirates for diagnosis of smear-negative childhood
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23. Bates, M. et al. Assessment of the Xpert MTB/RIF assay for diagnosis of tuberculosis with gastric lavage aspirates in children in sub-
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diseases: an official publication of the Infectious Diseases Society of America 55, 1088–1095, https://doi.org/10.1093/cid/cis598 (2012). 25. Raizada, N. et al. Enhancing TB case detection: experience in offering upfront Xpert MTB/RIF testing to pediatric presumptive TB
and DR TB cases for early rapid diagnosis of drug sensitive and drug resistant TB. PloS one 9, e105346, https://doi.org/10.1371/
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Hanoi, Vietnam. PloS one 8, e72100, https://doi.org/10.1371/journal.pone.0072100 (2013). 27. Hatherill, M. et al. Induced sputum or gastric lavage for community-based diagnosis of childhood pulmonary tuberculosis? Arc
Child 94, 195–201, https://doi.org/10.1136/adc.2007.136929 (2009).hfi p
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28. Marais, B. J. et al. The bacteriologic yield in children with intrathoracic tuberculosis. Clinical infectious diseases: an official public
of the Infectious Diseases Society of America 42, e69–71 (2006). f
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9. Nhu, N. T. et al. Evaluation of Xpert MTB/RIF and MODS assay for the diagnosis of pediatric tuberculosis. BMC infectious disease
13, 31, https://doi.org/10.1186/1471-2334-13-31 (2013). 30. Nicol, M. P. et al. Accuracy of the Xpert MTB/RIF test for the diagnosis of pulmonary tuberculosis in children admitted to hospital
in Cape Town, South Africa: a descriptive study. The Lancet. Infectious diseases 11, 819–824, https://doi.org/10.1016/S1473-
3099(11)70167-0 (2011). 31. Marais, B. J. et al. The natural history of childhood intra-thoracic tuberculosis: a critical review of literature from the pre-
chemotherapy era. Int J Tuberc Lung Dis 8, 392–402 (2004). k
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bmj.f5605 (2013). j
3. Alonzo, T. A. & Pepe, M. S. Using a combination of reference tests to assess the accuracy of a new diagnostic test. Statistics in
medicine 18, 2987–3003 (1999). 4. Togun, T. O. et al. Contribution of Xpert(R) MTB/RIF to the diagnosis of pulmonary tuberculosis among TB-exposed children in
The Gambia. Int J Tuberc Lung Dis 19, 1091–1097, i–ii, https://doi.org/10.5588/ijtld.15.0228 (2015). h
35. World Health Organization. Xpert MTB/RIF assay for the diagnosis of TB. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 4. Leung, E., Minion, J., Benedetti, A., Pai, M. & Menzies, D. Microcolony culture techniques for tuberculosis diagnosis: a systematic
review. Int J Tuberc Lung Dis 16, 16–23, i–iii, https://doi.org/10.5588/ijtld.10.0065 (2012). 5. Ha, D. T. et al. Microscopic observation drug susceptibility assay (MODS) for early diagnosis of tuberculosis in children. PloS o
e8341 (2009). (
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susceptibility method. Pediatrics 118, e100–106 (2006).f 7. Oberhelman, R. A. et al. Diagnostic approaches for paediatric tuberculosis by use of different specimen types, culture methods, and
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(2010). (
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0. Boehme, C. C. et al. Feasibility, diagnostic accuracy, and effectiveness of decentralised use of the Xpert MTB/RIF test for diagnosi
of tuberculosis and multidrug resistance: a multicentre implementation study. Lancet 377, 1495–1505 (2011). g
p
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(
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11. Detjen, A. K. et al. Xpert MTB/RIF assay for the diagnosis of pulmonary tuberculosis in children: a systematic review and m
analysis. The Lancet. Respiratory medicine. https://doi.org/10.1016/S2213-2600(15)00095-8 (2015). h
2. World Health Organisation. Pocket book of hospital care for children: guidelines for the management of common illnesses with limted
resources. (World Health Organisation, 2005). 3. Liebeschuetz, S. et al. Diagnosis of tuberculosis in South African children with a T-cell-based assay: a prospective cohort study
Lancet 364, 2196–2203 (2004). 14. Rachow, A. et al. Increased and Expedited Case Detection by Xpert MTB/RIF Assay in Childhood Tuberculosis: A Prospective
Cohort Study. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America, https://doi.org/10.1093/
cid/cis190 (2012). 15. Siddiqi, S. H. & Ruesch-Gerdes, S. MGIT Procedure Manual. (Foundation for Innovative New Diagnostics (FIND), 2006). 16. Coronel, J., Roper, M., Caviedes, L. & Moore, D. A. Acknowledgements g
We would like to thank Dr. David Moore, Luz Caviedes and the team at Universidad Peruana Cayetano Heredia
in Lima, Peru, for their helpful advice and practical support in MODS training and implementation; and the
clinical, laboratory and administrative staff at Kilifi County Hospital and Coast Provincial General Hospital for
their support of the study. We would also like to thank the following members of the Kilifi Improving Diagnosis &
Surveillance of Childhood TB (KIDS TB) Study Group who contributed to patient recruitment, investigation and
management: Victor Bandika, Jay Berkley, Joyce Langat, Kath Maitland, Carole Mulunda, Charles Newton, John
Paul Odhiambo, Hemed Twahir, Joshua Wambua. SCIentIfIC ReporTs | 7: 11808 | DOI:10.1038/s41598-017-11969-5 8 www.nature.com/scientificreports/ Author Contributions A.J.B., J.A.G.S. and M.L. designed the study with input from D.M., R.M., A.M. and S.C.M. R.M. received
residential training in MODS at Universidad Peruana Cayetano Heredia in Lima, Peru, following which he
trained A.J.B., D.M. and A.M. in the technique. Laboratory work was performed by D.M., R.M., A.M., S.C.M. and A.J.B. A.J.B. had full access to all the data in the study and final responsibility for the decision to submit for
publication. SCIentIfIC ReporTs | 7: 11808 | DOI:10.1038/s41598-017-11969-5 Additional Information dd t o a
o
at o
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-017-11969-5. Competing Interests: The authors declare that they have no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre-
ative Commons license, and indicate if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons license and your intended use is not per-
mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the
copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2017 SCIentIfIC ReporTs | 7: 11808 | DOI:10.1038/s41598-017-11969-5 9
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English
| null | null |
Archives of plastic surgery
| 2,021
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cc-by
| 910
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Kun Hwang Ethics Editor, Archives of Plastic Surgery
Department of Plastic Surgery, Inha University College of Medicine, Incheon, Korea “In the morning, my hair was black like silk bundles. In the eve
ning, it became white like snow (
).”–Li Bai
(
, 701–762 AD, a Chinese poet) case now. The only difference relates to filial duty, as a good son
was expected to avoid looking older than his parents. It is notable that the author wrote, “It is not sad to die, but it is
sad to grow old. People hate aging because they are getting clos
er to death,” which expresses a remarkably modern sentiment,
but was written 200 years ago. With the snapping of smartphone cameras, photographs are
everywhere these days. In our visual society, everybody’s insecu
rities are stamped in time in the annals of the internet. There
fore, troublesome physical features prompt both men and wom
en to undergo plastic surgery. In our times, elderly men dye their gray hair rather than pluck
ing it out. Cosmetic surgery for men is becoming increasingly
common throughout the world. If the author lived in Seoul to
day, he might have written the “Chronicle of Mr. #15 Blade” in
stead of the “Chronicle of Mr. Tweezer.” Recently I read a book written in Korea 200 years ago (during
the Joseon period). The writer, Lee Ok (
, 1760–1815),
wrote Kangnoseonsaeng-Jeon (
, Chronicle of Mr. Tweezer) at the age of 50. This book personifies a tweezer as a
narrator, and is structured as a set of questions and answers that
express psychological anxiety about aging. The author was sen
sitively aware of aging and described the aging process of the
body in detail. The descriptions of changes in hair color and lo
cations are especially impressive [1]. The author would like to
delay aging—if not the aging process itself, then at least aging of
one’s external appearance—by plucking out gray hairs (Fig. 1). Fig. 1. An old writer is tweezing gray hairs. Illustration by Hye
Won Hu, MFA, Division of Biomedical Art, Incheon Catholic
University Graduate School (Incheon, Korea). Why do elderly men desire youth instead of
aging? An analysis of Kangnoseonsaeng-Jeon
(却老先生傳) by Lee Ok (李鈺) Why do elderly men desire youth instead of
aging? An analysis of Kangnoseonsaeng-Jeon
(却老先生傳) by Lee Ok (李鈺) Editorial Copyright © 2021 The Korean Society of Plastic and Reconstructive Surgeons py g
y
g
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/
licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
www.e-aps.org This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.
censes/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is prop Article published online: 2022-05-22 Article published online: 2022-05-22 Article published online: 2022-05-22 Copyright © 2021 The Korean Society of Plastic and Reconstructive Surgeons
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/
licenses/by-nc/4 0/) which permits unrestricted non-commercial use distribution and reproduction in any medium provided the original work is properly cited
www.e-aps.org buted under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/
unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
www.e-aps.o y g
y
g
is an Open Access article distributed under the terms of the Creative Com py g
y
g
This is an Open Access article distributed under the terms of the Creative Commons Attribution N
licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproductio Correspondence: Kun Hwang
Department of Plastic Surgery, Inha University College of Medicine, 27 Inhang-ro, Jung-gu, Incheon
22332, Korea
Tel: +82-32-890-3514, Fax: +82-32-890-2918, E-mail: jokerhg@inha.ac.kr
This study was supported by a grant from National Research Foundation of Korea
(NRF-2020R1I1A2054761).
Received: August 5, 2021 • Revised: August 22, 2021 • Accepted: August 23, 2021
pISSN: 2234-6163 • eISSN: 2234-6171
https://doi.org/10.5999/aps.2021.01578 • Arch Plast Surg 2021;48:575-576 Kun Hwang In this book, the following reasons for plucking out gray hairs
are given: (1) in order to serve in a governmental post, men
must avoid looking old; (2) if one’s parents are still alive, their
son should not look older than his parents; (3) if a man marries
a young wife, the husband’s old appearance must be avoided; or
(4) society treats elderly men poorly. From this book, we realize that 200 years ago in Korea, aristo
crats (
) desired to look young for reasons related to their
jobs, social treatment, and marital relationships, just as is the Fig. 1. An old writer is tweezing gray hairs. Illustration by Hye
Won Hu, MFA, Division of Biomedical Art, Incheon Catholic
University Graduate School (Incheon, Korea). Fig. 1. An old writer is tweezing gray hairs. Illustration by Hye
Won Hu, MFA, Division of Biomedical Art, Incheon Catholic
University Graduate School (Incheon, Korea). 575 Hwang K Why do elderly men desire youth Conflict of interest 1. Ahn SH. A study on Kangnoseonsaeng-Jeon of Lee Ok: fo
cusing on the aging and autobiography. J Korean Literature
Chin 2016;45:253-76. Kun Hwang is an editorial board member of the journal but was
not involved in the peer reviewer selection, evaluation, or deci
sion process of this article. No other potential conflicts of inter
est relevant to this article were reported. ORCID https://orcid.org/0000-0002-1994-2538 https://orcid.org/0000-0002-1994-2538 Kun Hwang 576
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Supplementary Fig. S1 from Dual targeting of Raf and VEGF receptor 2 reduces growth and metastasis of pancreatic cancer through direct effects on tumor cells, endothelial cells, and pericytes
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5µM
Suppl. Figure 1: Effect of NVP-AAL881 on endothelial cell proliferation.
Antiproliferative effects were evaluated by MTT analysis, demonstrating that NVP-
AAL881 significantly reduced cell proliferation after 48 hours in full serum medium
AAL881 significantly reduced cell proliferation after 48 hours in full serum medium
(*P<0.01). 250.0
300.0
24h
48h
D. 150.0
200.0
48h
change in O.D
*
*
*
0 0
50.0
100.0
%-c
*
*
0.0
Control
0.25µM
0.5µM
1µM
2µM
5µM
Suppl. Figure 1: Effect of NVP-AAL881 on endothelial cell proliferation. Antiproliferative effects were evaluated by MTT analysis, demonstrating that NVP-
AAL881 significantly reduced cell proliferation after 48 hours in full serum medium
AAL881 significantly reduced cell proliferation after 48 hours in full serum medium
(*P<0.01). 250.0
300.0
24h
48h
D. 150.0
200.0
48h
change in O.D
*
*
*
0 0
50.0
100.0
%-c
*
*
0.0
Control
0.25µM
0.5µM
1µM
2µM
5µM Suppl. Figure 1: Effect of NVP-AAL881 on endothelial cell proliferation. Antiproliferative effects were evaluated by MTT analysis, demonstrating that NVP-
AAL881 significantly reduced cell proliferation after 48 hours in full serum medium
AAL881 significantly reduced cell proliferation after 48 hours in full serum medium
(*P<0.01).
|
https://openalex.org/W2003525528
|
https://europepmc.org/articles/pmc2979596?pdf=render
|
English
| null |
5-Methyl-3,6,7,8a-tetrahydro-2<i>H</i>-diimidazo[1,2-<i>c</i>:1′,2′-<i>e</i>]pyrido[1,2-<i>a</i>][1,3,5]triazin-5-ium iodide
|
Acta crystallographica. Section E
| 2,010
|
cc-by
| 3,022
|
organic compounds Experimental
Crystal data
C12H16N5
+I
Mr = 357.20
Monoclinic, P21=n
a = 7.6299 (2) A˚
b = 15.3939 (4) A˚
c = 11.4503 (3) A˚
= 92.204 (2)
V = 1343.89 (6) A˚ 3
Z = 4
Mo K radiation
= 2.37 mm1
T = 100 K
0.2 0.2 0.1 mm
Data collection
Oxford Diffraction Xcalibur-E CCD
diffractometer
Absorption correction: multi-scan
(CrysAlis PRO; Oxford
Diffraction, 2009)
Tmin = 0.496, Tmax = 0.789
25955 measured reflections
4406 independent reflections
4018 reflections with I > 2(I)
Rint = 0.026
Refinement
R[F 2 > 2(F 2)] = 0.029
wR(F 2) = 0.070
S = 1.20
4406 reflections
165 parameters
H-atom parameters constrained
max = 1.74 e A˚ 3
min = 1.24 e A˚ 3 Acta Crystallographica Section E
Structure Reports
Online
ISSN 1600-5368 Acta Crystallographica Section E
Structure Reports
Online
ISSN 1600-5368 V = 1343.89 (6) A˚ 3
Z = 4
Mo K radiation
= 2.37 mm1
T = 100 K
0.2 0.2 0.1 mm Table 1 Table 1
Hydrogen-bond geometry (A˚ , ). D—H A
D—H
H A
D A
D—H A
C3—H3B I1i
0.99
3.15
4.007 (3)
145
C6—H6A I1ii
0.99
3.05
4.010 (3)
163
C9—H9 I1iii
0.95
3.08
4.025 (3)
172
C12—H12 I1iv
0.95
3.14
3.887 (3)
137
C17—H17C I1i
0.98
3.16
4.025 (3)
148
Symmetry codes: (i) x þ 1
2; y þ 1
2; z 1
2; (ii) x þ 1; y; z; (iii) x þ 1
2; y þ 1
2; z þ 1
2; (iv)
x; y þ 1; z þ 1. The structure of the title compound, C12H16N5
+I, shows that
the methylation reaction with CH3I occurred at the imine N
atom at position 5 of the 3,6,7,8a-tetrahydro-2H-diimidazo-
[1,2-c:10,20-e]pyrido[1,2-a][1,3,5]triazine system. In the cation,
the sp3-hybridized C atom belonging to the fused dihydro-
pyrine and dihydro-1,3,5-triazine rings deviates by 0.514 (3) A˚
from the best plane defined by the remaining cationic non-H
atoms. The fused dihydropyridine and dihydro-1,3,5-triazine
rings are each in a half-chair conformation with the sp3-
hybridized C atom as a flap. The iodide anion is 3.573 (2) A˚
from the methylated N atom and exhibits five short C—H I
contacts with distances less than 3.16 A˚ . The structure has
been determined from a non-merohedral twin with twin law
[1 0 0 0 1 0 0.115 0 1], minor domain = 0.1559 (12). Data collection: CrysAlis PRO (Oxford Diffraction, 2009); cell
refinement:
CrysAlis
PRO;
data
reduction:
CrysAlis
PRO;
program(s) used to solve structure: SHELXS97 (Sheldrick, 2008);
program(s) used to refine structure: SHELXL97 (Sheldrick, 2008)
and WinGX (Farrugia, 1999); molecular graphics: ORTEP-3 for
Windows (Farrugia, 1997); software used to prepare material for
publication: SHELXL97. Related literature For the synthesis and data reported earlier for the title
compound, see: Sa˛czewski & Foks (1981). For the programs
used to derive the twin law, see: Cooper et al.(2002); Farrugia
(1999). Supplementary data and figures for this paper are available from the
IUCr electronic archives (Reference: TK2676). 5-Methyl-3,6,7,8a-tetrahydro-2H-
diimidazo[1,2-c:1000,2000-e]pyrido[1,2-a]-
[1,3,5]triazin-5-ium iodide 5-Methyl-3,6,7,8a-tetrahydro-2H-
diimidazo[1,2-c:1000,2000-e]pyrido[1,2-a]-
[1,3,5]triazin-5-ium iodide Data collection
Oxford Diffraction Xcalibur-E CCD
diffractometer
Absorption correction: multi-scan
(CrysAlis PRO; Oxford
Diffraction, 2009)
Tmin = 0.496, Tmax = 0.789 25955 measured reflections
4406 independent reflections
4018 reflections with I > 2(I)
Rint = 0.026 Aleksandra Wasilewska,a Franciszek Sa˛czewskia and
Maria Gdaniecb* Cooper, R. I., Gould, R. O., Parsons, S. & Watkin, D. J. (2002). J. Appl. Cryst.
35, 168–174.
Farrugia, L. J. (1997). J. Appl. Cryst. 30, 565.
Farrugia, L. J. (1999). J. Appl. Cryst. 32, 837–838.
Oxford Diffraction (2009). CrysAlis PRO. Oxford Diffraction Ltd, Yarnton,
England.
Sa˛czewski, F. & Foks, H. (1981). Synthesis, pp. 151–152.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Aleksandra Wasilewska,a Franciszek Sa˛czewskia and
Maria Gdaniecb* aDepartment of Chemical Technology of Drugs, Medical University of Gdan´sk,
80-416 Gdan´sk, Poland, and bFaculty of Chemistry, Adam Mickiewicz University,
60-780 Poznan´, Poland
Correspondence e-mail: magdan@amu.edu.pl aDepartment of Chemical Technology of Drugs, Medical University of Gdan´sk,
80-416 Gdan´sk, Poland, and bFaculty of Chemistry, Adam Mickiewicz University,
60-780 Poznan´, Poland
Correspondence e-mail: magdan@amu.edu.pl Refinement
R[F 2 > 2(F 2)] = 0.029
wR(F 2) = 0.070
S = 1.20
4406 reflections
165 parameters
H-atom parameters constrained
max = 1.74 e A˚ 3
min = 1.24 e A˚ 3 165 parameters
H-atom parameters constrained
max = 1.74 e A˚ 3
min = 1.24 e A˚ 3 Received 11 May 2010; accepted 24 May 2010 Received 11 May 2010; accepted 24 May 2010 Key indicators: single-crystal X-ray study; T = 100 K; mean (C–C) = 0.004 A˚;
R factor = 0.029; wR factor = 0.070; data-to-parameter ratio = 26.7. Table 1
Hydrogen-bond geometry (A˚ , ). References Cooper, R. I., Gould, R. O., Parsons, S. & Watkin, D. J. (2002). J. Appl. Cryst. 35, 168–174. Farrugia, L. J. (1997). J. Appl. Cryst. 30, 565. Farrugia, L. J. (1999). J. Appl. Cryst. 32, 837–838. Oxford Diffraction (2009). CrysAlis PRO. Oxford Diffraction Ltd, Yarnton,
England. Sa˛czewski, F. & Foks, H. (1981). Synthesis, pp. 151–152. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. o1486
Wasilewska et al. Acta Cryst. (2010). E66, o1486 doi:10.1107/S1600536810019495 Comment Biguanide derivatives are known to possess diverse biological activities, including antidiabetic, antibacterial, germicidic,
antiviral and antimalarial. On the other hand, quaternary ammonium salts constitute a well known class of bacteriostatic
agents. Therefore, we have decided to synthesize some N-alkylated cyclic biguanide derivatives for biological testing, based
on the previously described procedure (Sączewski & Foks, 1981) which consists in the reaction of 2,3,6,7,8a,13-hexahy-
dropyrido[1,2-a]diimidazo[1',2'-c:1'',2''-e]-1,3,5-triazine (1) with an alkyl halide. As shown in Fig. 1, the course of the reac-
tion of 1 with methyl iodide has not been established and two products, 2 or 3, arising from either N1 or N5 alkylation have
been proposed. In this work, based on X-ray structure analysis (Fig. 2) and hetero-correlation NMR experiments (HSQC
and HMBC), the structure of the title compound (2) is determined unambiguously. Regioselectivity of N-alkylation of the
cyclic biguanide derivative 1 could not be predicted on the basis of calculated electrostatic potential and charge distribution. The structure of the N5 alkylated product 2 was also confirmed by 2D NMR spectroscopic data. Thus, assignment of signals
observed in 1H and 13C-NMR spectra was possible using HSQC spectrum (see numbering scheme in Fig. 3). The crucial
signals of quaternary carbon atoms C13a and C4a were found at 145.7 and 152.6 p.p.m., respectively. 3-Bond correlation
from the latter carbon to a singlet of three protons at 3.26 p.p.m. observed in the HMBC spectrum (Fig. 4) indicated the
placement of methyl group at the N5 nitrogen atom. 5-Methyl-3,6,7,8a-tetrahydro-2H-diimidazo[1,2-c:1',2'-e]pyrido[1,2-a][1,3,5]triazin-5-ium iodide A. Wasilewska, F. Saczewski and M. Gdaniec A. Wasilewska, F. Saczewski and M. Gdaniec Experimental 1D and 2D NMR spectra were recorded on a Varian Unity 500 spectrometer. The title compound was prepared according to
the previously described procedure (Sączewski & Foks, 1981); m.p. 492–494 K (decomp.); 1H NMR (500 MHz, DMSO-d6,
see Fig. 3 for numbering scheme) δH 6.94 (1H, d, J = 7.2 Hz, H12), 6.22–6.19 (1H, ddd, J=9.8, 6.8, ~1 Hz, H10), 5.86 (1H,
t, J = 1 Hz, H8a), 5.67 (1H, dd, J = 9.8, ~1 Hz, H9), 5.41–5.38 (1H, dd, J = 7.2, 6.8 Hz, H11), 4.42 (1H, dt, J = 9.3, 6.8 Hz,
H2), 4.20 (1H, dt, J = 9.3, 6.8 Hz, H2), 4.01–3.83 (4H, m, H7, H6, 2xH3),3.78–3.71 (1H, m, H6), 3.49–3.43 (1H, m, H7),
3.26 (3H, s, CH3); 13C NMR (125 MHz, DMSO-d6, see Fig. 3 for numbering scheme) δC 152.6 (C4a), 145.7 (C13a), 125.4
(C12), 124.8 (C10), 113.6 (C9), 103.4 (C11), 67.0 (C8a), 51.7 (C3), 50.7 (C6), 46.6 (C2), 43.3 (C7), 33.7 (C14); IR (KBr,
cm–1): 3090, 3079, 3025, 2936, 2880, 1684, 1661, 1550, 1429, 1321, 1301, 1186, 677. 3.26 (3H, s, CH3); 13C NMR (125 MHz, DMSO-d6, see Fig. 3 for numbering scheme) δC 152.6 (C4a), 145.7 (C13a), 125.4
(C12), 124.8 (C10), 113.6 (C9), 103.4 (C11), 67.0 (C8a), 51.7 (C3), 50.7 (C6), 46.6 (C2), 43.3 (C7), 33.7 (C14); IR (KBr,
cm–1): 3090, 3079, 3025, 2936, 2880, 1684, 1661, 1550, 1429, 1321, 1301, 1186, 677. supplementary materials supplementary materials Acta Cryst. (2010). E66, o1486 [ doi:10.1107/S1600536810019495 ] Acta Cryst. (2010). E66, o1486 [ doi:10.1107/S1600536810019495 ] Acta Cryst. (2010). E66, o1486 [ doi:10.1107/S1600536810019495 ] Refinement The twin matrix, -1 0 0/0 - 1 0/0.115 0 1, corresponding to 180° rotation about [0 0 1] direct lattice direction has been
determined with the program ROTAX (Cooper et al., 2002). For the refinement with the SHELXL97 program (Sheldrick,
2008), the reflection data file was prepared in the HKLF 5 format using the 'Make HKLF5' function of the WinGX program
(Farrugia, 1999). The overlapping reflections and those belonging to only one twin domain are used in the refinement (HKLF
5 format of SHELXL97). Those which were excluded, 132 reflections, are partial overlaps which could not be integrated
properly at the data processing stage. The BASF parameter refined at 0.1559 (12). The H atoms bonded to C atoms were sup-1 supplementary materials HMBC spectrum of the title compound 2 Crystal data
C12H16N5
+·I−
F(000) = 704
Mr = 357.20
Dx = 1.765 Mg m−3
Monoclinic, P21/n
Mo Kα radiation, λ = 0.71073 Å
Hall symbol: -P 2yn
Cell parameters from 19213 reflections
a = 7.6299 (2) Å
θ = 2.7–32.3°
b = 15.3939 (4) Å
µ = 2.37 mm−1
c = 11.4503 (3) Å
T = 100 K
β = 92.204 (2)°
Block, colourless
V = 1343.89 (6) Å3
0.2 × 0.2 × 0.1 mm
Z = 4 sup-2 supplementary materials Data collection
Oxford Diffraction Xcalibur-E CCD
diffractometer
4406 independent reflections
Radiation source: fine-focus sealed tube
4018 reflections with I > 2σ(I)
graphite
Rint = 0.026
ω scan
θmax = 32.4°, θmin = 4.1°
Absorption correction: multi-scan
(CrysAlis PRO; Oxford Diffraction, 2009)
h = −9→11
Tmin = 0.496, Tmax = 0.789
k = −23→23
25955 measured reflections
l = 0→16 Data collection
Oxford Diffraction Xcalibur-E CCD
diffractometer
4406 independent reflections
Radiation source: fine-focus sealed tube
4018 reflections with I > 2σ(I)
graphite
Rint = 0.026
ω scan
θmax = 32.4°, θmin = 4.1°
Absorption correction: multi-scan
(CrysAlis PRO; Oxford Diffraction, 2009)
h = −9→11
Tmin = 0.496, Tmax = 0.789
k = −23→23
25955 measured reflections
l = 0→16 Data collection
Oxford Diffraction Xcalibur-E CCD
diffractometer
Radiation source: fine-focus sealed tube
graphite
ω scan
Absorption correction: multi-scan
(CrysAlis PRO; Oxford Diffraction, 2009)
Tmin = 0.496, Tmax = 0.789
25955 measured reflections 4406 independent reflections
4018 reflections with I > 2σ(I)
Rint = 0.026
θmax = 32.4°, θmin = 4.1°
h = −9→11
k = −23→23
l = 0→16 4406 independent reflections Refinement
Refinement on F2
Least-squares matrix: full
R[F2 > 2σ(F2)] = 0.029
wR(F2) = 0.070
S = 1.20
4406 reflections
165 parameters
0 restraints Primary atom site location: structure-invariant direct
methods Primary atom site location: structure-invariant direct
methods Refinement on F2
Least-squares matrix: full
R[F2 > 2σ(F2)] = 0.029
wR(F2) = 0.070
S = 1.20
4406 reflections
165 parameters
0 restraints Secondary atom site location: difference Fourier map
Hydrogen site location: inferred from neighbouring
sites H-atom parameters constrained w = 1/[σ2(Fo
2) + (0.0196P)2 + 3.1405P]
where P = (Fo
2 + 2Fc
2)/3
(Δ/σ)max = 0.001
Δρmax = 1.74 e Å−3
Δρmin = −1.24 e Å−3 supplementary materials placed at calculated positions, with C—H = 0.95–1.00 Å, and refined as riding on their parent atoms, with Uiso(H) = x
Ueq(C), where x = 1.5 for the H atoms from the methyl group and x = 1.2 for the remaining H atoms. The maximum and
minimum residual electron-density peaks of 1.74 and -1.24 eÅ-3 were located 0.72 Å and 1.84 Å from H6B and I1 atoms,
respectively. Fig. 1. Reaction of 2,3,6,7,8a,13-hexahydropyrido[1,2-a]diimidazo[1',2',-c:1",2"-e]-1,3,5-
triazine (1) with methyl iodide. Fig. 2. Asymmetric unit for the title salt with displacement ellipsoids shown at the 50% prob-
ability level. Fig. 3. HSQC spectrum of the title compound 2 Fig. 4. supplementary materials pp
y
sup-4
H3A
0.1869
0.3569
0.2173
0.025*
H3B
0.3846
0.3898
0.1973
0.025*
N4
0.3083 (3)
0.40681 (15)
0.36610 (19)
0.0155 (4)
N5
0.4690 (3)
0.27675 (15)
0.4159 (2)
0.0162 (4)
C6
0.5326 (4)
0.23510 (18)
0.5250 (2)
0.0189 (5)
H6A
0.6623
0.2316
0.5289
0.023*
H6B
0.4837
0.1759
0.5322
0.023*
C7
0.4659 (4)
0.29506 (18)
0.6205 (2)
0.0201 (5)
H7A
0.3677
0.2679
0.6613
0.024*
H7B
0.5609
0.3097
0.6785
0.024*
N8
0.4068 (3)
0.37245 (14)
0.55467 (19)
0.0147 (4)
C9
0.3144 (4)
0.4560 (2)
0.7238 (2)
0.0202 (5)
H9
0.3752
0.4175
0.7759
0.024*
C10
0.2718 (4)
0.5354 (2)
0.7605 (3)
0.0237 (6)
H10
0.2938
0.5510
0.8400
0.028*
C11
0.1922 (4)
0.59810 (19)
0.6798 (3)
0.0237 (6)
H11
0.1462
0.6510
0.7082
0.028*
C12
0.1842 (4)
0.58081 (18)
0.5651 (3)
0.0197 (5)
H12
0.1363
0.6226
0.5118
0.024*
N13
0.2459 (3)
0.50171 (14)
0.5233 (2)
0.0162 (4)
C14
0.2675 (3)
0.42666 (17)
0.6013 (2)
0.0151 (4)
H14
0.1556
0.3928
0.6010
0.018*
C15
0.3916 (3)
0.35088 (16)
0.4392 (2)
0.0136 (4)
C16
0.2403 (3)
0.48697 (17)
0.4045 (2)
0.0156 (5)
C17
0.4863 (4)
0.2302 (2)
0.3064 (3)
0.0242 (6)
H17A
0.3854
0.1916
0.2934
0.036*
H17B
0.5944
0.1957
0.3102
0.036*
H17C
0.4909
0.2719
0.2420
0.036*
Atomic displacement parameters (Å2)
U11
U22
U33
U12
U13
U23
I1
0.01712 (8)
0.01865 (8)
0.01406 (7)
0.00055 (6)
0.00212 (5)
−0.00050 (6)
N1
0.0239 (12)
0.0181 (11)
0.0207 (11)
0.0010 (9)
−0.0007 (9)
0.0040 (9)
C2
0.0251 (14)
0.0244 (14)
0.0170 (12)
0.0007 (11)
−0.0009 (10)
0.0062 (11)
C3
0.0271 (15)
0.0233 (14)
0.0114 (11)
0.0015 (11)
−0.0001 (9)
0.0007 (10)
N4
0.0198 (11)
0.0150 (10)
0.0117 (9)
0.0004 (8)
0.0010 (7)
0.0016 (8)
N5
0.0174 (11)
0.0158 (9)
0.0155 (9)
0.0029 (8)
0.0033 (8)
0.0009 (8)
C6
0.0189 (13)
0.0187 (12)
0.0191 (12)
0.0056 (10)
0.0012 (9)
0.0035 (9)
C7
0.0245 (13)
0.0194 (12)
0.0161 (11)
0.0044 (10)
−0.0008 (9)
0.0045 (9)
N8
0.0157 (10)
0.0150 (9)
0.0134 (9)
0.0007 (8)
−0.0001 (7)
0.0017 (7)
C9
0.0213 (13)
0.0242 (13)
0.0152 (11)
−0.0016 (10)
0.0003 (9)
−0.0024 (10)
C10
0.0223 (14)
0.0297 (15)
0.0191 (13)
−0.0052 (11)
0.0023 (10)
−0.0092 (11)
C11
0.0241 (15)
0.0162 (12)
0.0312 (15)
−0.0029 (10)
0.0048 (11)
−0.0088 (11)
C12
0.0178 (13)
0.0126 (11)
0.0289 (14)
−0.0021 (9)
0.0045 (10)
−0.0026 (10)
N13
0.0212 (11)
0.0111 (9)
0.0164 (10)
0.0001 (8)
0.0001 (8)
−0.0002 (8)
C14
0.0166 (12)
0.0152 (11)
0.0135 (11)
−0.0006 (9)
0.0001 (8)
−0.0001 (9)
C15
0.0113 (11)
0.0147 (11)
0.0148 (10)
−0.0024 (8)
0.0022 (8)
0.0012 (8) Special details Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The
cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds
in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used
for estimating esds 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, convention-
al 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. actional atomic 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
I1
0.04305 (2)
0.194494 (11)
0.473423 (14)
0.01657 (5)
N1
0.1806 (3)
0.53774 (16)
0.3251 (2)
0.0209 (5)
C2
0.2057 (4)
0.4944 (2)
0.2120 (2)
0.0222 (5)
H2A
0.2910
0.5272
0.1660
0.027*
H2B
0.0931
0.4914
0.1663
0.027*
C3
0.2755 (4)
0.40179 (19)
0.2385 (2)
0.0206 (5) sup-3 supplementary materials Atomic displacement parameters (Å2) sup-4 supplementary materials supplementary mate
C16
0.0157 (12)
0.0130 (11)
0.0181 (11)
−0.0021 (9)
0.0012 (9)
0.0011 (9
C17
0.0316 (16)
0.0213 (13)
0.0201 (12)
0.0047 (11)
0.0075 (11)
−0.0047 (
Geometric parameters (Å, °)
N1—C16
1.270 (3)
C7—H7B
0.9900
N1—C2
1.476 (4)
N8—C15
1.364 (3)
C2—C3
1.547 (4)
N8—C14
1.468 (3)
C2—H2A
0.9900
C9—C10
1.336 (4)
C2—H2B
0.9900
C9—C14
1.504 (4)
C3—N4
1.475 (3)
C9—H9
0.9500
C3—H3A
0.9900
C10—C11
1.453 (5)
C3—H3B
0.9900
C10—H10
0.9500
N4—C15
1.344 (3)
C11—C12
1.340 (4)
N4—C16
1.415 (3)
C11—H11
0.9500
N5—C15
1.317 (3)
C12—N13
1.397 (3)
N5—C17
1.455 (4)
C12—H12
0.9500
N5—C6
1.470 (3)
N13—C16
1.378 (3)
C6—C7
1.533 (4)
N13—C14
1.466 (3)
C6—H6A
0.9900
C14—H14
1.0000
C6—H6B
0.9900
C17—H17A
0.9800
C7—N8
1.471 (3)
C17—H17B
0.9800
C7—H7A
0.9900
C17—H17C
0.9800
C16—N1—C2
107.1 (2)
C10—C9—C14
121.1 (3)
N1—C2—C3
107.4 (2)
C10—C9—H9
119.4
N1—C2—H2A
110.2
C14—C9—H9
119.4
C3—C2—H2A
110.2
C9—C10—C11
120.5 (3)
N1—C2—H2B
110.2
C9—C10—H10
119.7
C3—C2—H2B
110.2
C11—C10—H10
119.7
H2A—C2—H2B
108.5
C12—C11—C10
119.6 (3)
N4—C3—C2
101.0 (2)
C12—C11—H11
120.2
N4—C3—H3A
111.6
C10—C11—H11
120.2
C2—C3—H3A
111.6
C11—C12—N13
120.4 (3)
N4—C3—H3B
111.6
C11—C12—H12
119.8
C2—C3—H3B
111.6
N13—C12—H12
119.8
H3A—C3—H3B
109.4
C16—N13—C12
118.9 (2)
C15—N4—C16
122.4 (2)
C16—N13—C14
118.1 (2)
C15—N4—C3
129.9 (2)
C12—N13—C14
120.6 (2)
C16—N4—C3
107.6 (2)
N13—C14—N8
107.0 (2)
C15—N5—C17
131.1 (2)
N13—C14—C9
110.4 (2)
C15—N5—C6
110.0 (2)
N8—C14—C9
111.1 (2)
C17—N5—C6
118.7 (2)
N13—C14—H14
109.4
N5—C6—C7
103.6 (2)
N8—C14—H14
109.4
N5—C6—H6A
111.0
C9—C14—H14
109.4
C7—C6—H6A
111.0
N5—C15—N4
129.4 (2)
N5—C6—H6B
111.0
N5—C15—N8
112.7 (2)
C7—C6—H6B
111.0
N4—C15—N8
117.9 (2)
H6A—C6—H6B
109.0
N1—C16—N13
127.0 (3)
N8—C7—C6
103.0 (2)
N1—C16—N4
116.1 (2) sup-5 supplementary materials supplementary materials N8—C7—H7A
111.2
N13—C16—N4
116.9 (2)
C6—C7—H7A
111.2
N5—C17—H17A
109.5
N8—C7—H7B
111.2
N5—C17—H17B
109.5
C6—C7—H7B
111.2
H17A—C17—H17B
109.5
H7A—C7—H7B
109.1
N5—C17—H17C
109.5
C15—N8—C14
117.1 (2)
H17A—C17—H17C
109.5
C15—N8—C7
108.2 (2)
H17B—C17—H17C
109.5
C14—N8—C7
119.1 (2)
Hydrogen-bond geometry (Å, °)
D—H···A
D—H
H···A
D···A
D—H···A
C3—H3B···I1i
0.99
3.15
4.007 (3)
145
C6—H6A···I1ii
0.99
3.05
4.010 (3)
163
C9—H9···I1iii
0.95
3.08
4.025 (3)
172
C12—H12···I1iv
0.95
3.14
3.887 (3)
137
C17—H17C···I1i
0.98
3.16
4.025 (3)
148
Symmetry codes: (i) x+1/2, −y+1/2, z−1/2; (ii) x+1, y, z; (iii) x+1/2, −y+1/2, z+1/2; (iv) −x, −y+1, −z+1. sup-6 supplementary materials Fig 1 Fig. 1 Fig. 1 sup-7 supplementary materials Fig. 2 sup-8 supplementary materials Fig. 3 sup-9 supplementary materials Fig. 4 Fig. 4 sup-10
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Professional Negligence, Mismanagement and Malpractice. Polish Companies in the Light of Supreme Audit Office Materials in the Years 1976-1980
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Studia Historiae Oeconomicae
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Krzysztof L es iako w s ki (The University of Łódź, Łódź) Krzysztof L es iako w s ki (The University of Łódź, Łódź) Krzysztof L es iako w s ki (The University of Łódź, Łódź) PROFESSIONAL NEGLIGENCE,
MISMANAGEMENT AND MALPRACTICE.
POLISH COMPANIES IN THE LIGHT
OF SUPREME AUDIT OFFICE MATERIALS
IN THE YEARS 1976-1980 The birth of the “Solidarity” Independent Self-governing Trade Union in 1980 was accompanied
by a strong revisionist movement. The Polish society expected punishment for the previous admin-
istration, led by Edward Gierek, as it was their policy up to that point that resulted with enormous
external debt and ubiquitous shortages of consumer goods. A lot was said about wastefulness, cor-
ruption and professional negligence, both in the state administration and in the managements of
Polish companies. And these opinions were somehow justified, as evidenced by the results of audits
performed by the Supreme Audit Office (Najwyższa Izba Kontroli, NIK) in the years 1976–1980. Information recorded in the audit documentation was in line with the social attitudes. Due to for-
mal and legal conditions back then, NIK auditors could not inspect the most important companies,
however they managed to prove that the scale of illegal actions committed by directors and manag-
ers was large and increased year by year. Keywords: Poland 1976-1980, state control, national companies, management. doi:10.1515/sho-2016-0009 doi:10.1515/sho-2016-0009 Studia historiae oeconomicae
UAM
Vol. 34
Poznań 2016
zhg.amu.edu.pl/sho Studia historiae oeconomicae
UAM
Vol. 34
Poznań 2016
zhg.amu.edu.pl/sho INTRODUCTION The Supreme Audit Office (NIK) was the key supervisory institution
in Poland, therefore, materials prepared by its auditors are very useful
when investigating the problem of financial crime. During their statuto-
ry auditory activities, the inspectors detected various negative incidents,
which were evidence that some aspects of operation of Polish companies,
cooperatives and budgetary institutions were of a criminal or quasi-crim-
inal nature. Many of these problems were the fault of the management –
though dysfunctions in public institutions and the abnormal behavior of Krzysztof Lesiakowski 150 the authorities, although were nothing new, they achieved a whole new
level at that time [Jarosz M. 2004: 42]. This was evidenced by a particularly
strong revisionary movement in the first sixteen months of the existence of
“Solidarity”, in the years 1980-1981, when the society sought revenge for
frauds and private criminal activities that people in broadly understood
“positions of power” were involved in. This circumstance begs the ques-
tion – what had actually happened before that time? Did NIK inspectors,
during audits, detect the fact that managerial staff abused their positions
and were guilty of mismanagement? It must be noted at this point that
from the year 1976, NIK was subordinated to the Prime Minister, which
significantly limited its authority, planning capabilities and the course of
the audits performed [Sierpowska I. 2004: 55-56; Sylwestrzak A. 2006: 90;
Konaszyc A. 1978: 96-100]. Furthermore, the fact that this problem was not
noticed in literature on financial crime in Poland is an additional encour-
agement to investigate it [Jarosz M. 2004: 236]. This subject was either ig-
nored or belittled [Górniok O. 1986: 101]. NEGLIGENCE OF PROFESSIONAL DUTIES Polish companies in the years 1976-1980 functioned in the conditions
of state-controlled economy, whose priority was the implementation of
the economic plan. In the course of this implementation, many illegal
acts were committed, including excessive tolerance towards negligence
of professional responsibilities and false reporting. The gap between the
official policy of fighting with these problems and the actual impunity of
those who committed similar acts meant that the problem kept escalating
[Karklins R. 2009: 134-137]. According to NIK, one of the main reasons
why the audited companies incurred losses was the fact that the system
of professional supervision was poor and the enforcement of responsi-
bility for mistakes in company management – inconsistent. This result-
ed from the fact that in state-controlled economy, the division of respon-
sibilities in particular units of the organization was not clearly defined,
particularly in smaller plants [ANIK (Archive of Supreme Audit Office),
GP (President’s Office), 294/1, f. 2]. Furthermore, the managers did not
use their authority to punish those guilty of negligence due to general
problems with employment. On the other hand, on the level of minis-
tries, associations or similar units, unreliable reports and information on Professional Negligence 151 the alleged economic achievements of companies were very often accept-
ed indiscriminately. Weaker supervision in this respect results from the fact that also the superior units
wanted to present economic results as better than they actually were [ANIK, GP. 1627/7l, ff. 248-249]. Improper wage bill management was one of the aspects of manage-
rial negligence frequently pointed out by NIK auditors. For example, in
1979, the “Organika-Azot” Chemical Plant in Jaworzno overstated the ex-
ecution of the production plan and paid workers undue bonuses in the
amount of 6.4 million PLN. The “Stomil” Rubber Company in Wolbrom –
made similar undue payments totaling 3.2 million PLN. The director of
the Light Industry Construction Company in Dzierżoniów forged doc-
uments concerning the works performed, the overtime worked etc. The
money he made this way was split with other conspirators operating in
the company [ANIK, GP, 148/14, f. 25; 294/1, f. 12]. Although formally
this behavior was heavily punished: e.g. NEGLIGENCE OF PROFESSIONAL DUTIES with immediate termination of
employment, termination of employment with notice, transfer into a dif-
ferent position with different working conditions and salary, a fine, re-
ferring the matter to a penal and administrative court, and, in exception-
al circumstances, reporting the matter to law enforcement agencies, the
problem was recurring. i The audits showed that insufficient professional supervision or even
abnormalities in its functioning in terms of wage bill management (pay-
ing undue base salaries, benefits, bonuses and awards) was also the prob-
lem in state-controlled agricultural farms. Particularly shocking arbitrar-
iness and violations were observed in 1979 in the Agriculture and Food
Cooperative in Szczekociny (Częstochowa region). Manipulations in the
number of daily rates in the period from July 1978 until December 1979
meant that this company paid 18 people 10.7 million PLN. Notably, the
cooperative did not turn a profit in 1979, and thus obtained a 21.5 million
PLN subsidy from the state budget. The director of the company single-
handedly made decisions concerning bonuses and awards, without the
approval of the general meeting. He was also the main recipient of the
money distributed this way. Another problem was the fact that manageri-
al staff were paid undue compensation for using their private cars for pro-
fessional purposes. In the whole of 1979, it was 600,000 PLN for 9 people,
out of which 360,000 PLN went into the director’s pocket. In the follow-
ing year (from January until September), the aforementioned director also Krzysztof Lesiakowski 152 granted himself another 211,000 PLN by way of such compensation de-
spite being on sick leave at that time [AAN (Archive of New Files), PZPR
(Polish United Workers’ Party), 4014, ff. 133-134]. According to NIK, improper professional supervision was the weak-
est link in company management in Poland. This malfunction entailed
further adverse consequences: poor work discipline (drinking alcohol at
work, thefts, bad performance), fictional turnover, substituting allegedly
used machines with operational ones while decommissioning them, lack
of supervision with regards to materials used in production (in food in-
dustry this meant creating surpluses of e.g. coffee or meat which were not
recorded anywhere). The latter problem was related to illegal transactions
in fluid fuels. How could it be any different, though, if e.g. NEGLIGENCE OF PROFESSIONAL DUTIES in the “Wega”
Wool Company in Bielsko-Biała an audit of drivers’ logs performed by
NIK inspectors showed that they reported different distances covered on
the same Bielsko-Biała – Straconka route: 20, 23, 38, 48, 50, and even 61
km, while the actual distance is 12 km [ANIK, GP, 1569/10, f. 417]? This
was not an isolated case of course. NIK assessed that the worst situation
in terms of fuel turnover was observed in agriculture. Tests showed that,
officially, farmers bought only 10–15% of the fuel needed, and “presuma-
bly obtained the rest of the fuel needed from illegal sources” [ANIK, GP,
1569/12, f. 56]. A clear proof of the terrible state of management in Polish companies
was the frequency of sham reporting. In any case it had to be at least toler-
ated , and quite probably, even inspired by directors, managers, and pres-
idents. In the 4th quarter of 1977, and in the 1st and 2nd quarters of 1978, out
of 323 companies audited in this respect, in 234 (72%) unreliable and in-
accurate reports were found. The problem was therefore much more se-
rious that the Central Statistical Office reported – according to them, 10%
of reports contained data inconsistent with the records found in source
documentation [ANIK, GP, 1627/4, f. 16]. It must be added that the prob-
lem of false reporting concerned all companies: small and large, new and
long established, from agricultural cooperatives to coal mines. By manip-
ulating reports, the management usually tried to conceal problems with
implementing the production plan or achieving the planned level of in-
come. Large scale manipulations were reported in national agricultural com-
panies and farmers’ cooperatives. Data regarding the number of livestock
were falsified, its value was manipulated, also fictional renovations and
irrigation works were reported [ANIK, GP, 294/3, f. 27]. Some mines hid Professional Negligence 153 their problems with executing coal extraction plans in the same way. This
was the case for example in the “Generał Zawadzki” mine, which in just 5
months of 1980 issued 223 fictional invoices, claiming to have sold 189,000
tons of coal worth 41.5 million PLN. Other mines hid the fact that their
yield was lower than expected by overestimating own usage of coal and
its alleged stock in heaps [AAN, PZPR, 4014, f. 16]. NEGLIGENCE OF PROFESSIONAL DUTIES The record in this re-
spect was probably held by the Jelcz Car Company, which unjustly in-
cluded 479 cars worth 333 million PLN in their production figures for the
year 1976, while some of the cars were sold as late as April 1977 [ANIK,
GP, 1627/3, f. 367]. Dismissal of three vice presidents of the Jelcz compa-
ny and of several employees responsible for the incorrect reports proved
that NIK findings were correct. NIK also made a request to reconsider the
employment of the chief director of the company because, clearly, he ne-
glected his professional duties. For similar reasons, based on NIK findings
in 1979, the prosecutor’s office arrested the chief director of the “Organika
Azot” Chemical Company in Jaworzno [ANIK, GP, 148/14, f. 27]. ABUSES One of the most prevalent problems in Polish companies was the abuse
of managerial positions for private purposes. In this respect, the behavior
of the management of the Radio and Television Committee was particular-
ly telling. The audit in the Committee began in April 1980, because previ-
ous proceedings initiated in 1977 and 1979 had been suspended upon the
request of the Prime Minister. NIK accused the management of this insti-
tution of diminishing currency-related income to the state budget [AAN,
PZPR, 4014, f. 147] and incurring losses as a result of unreasonable cur-
rency purchases, including those made for personal benefit [Lesiakowski
K. 1998: 418]. In particular this involved the creation of two companies
abroad by the management of the Committee. Foreign cooperators made
payments due for broadcasts (for example from the Pope’s visit in Poland)
and for commissioned films and television programs to the accounts of
these companies. From May 1979 until September 1980, these payments
amounted to USD 1.6 million, which the Committee’s management had
at their free disposal. NIK also questioned the purchase of equipment and
personal items from the so-called 2nd payment area, without an in-depth
analysis of needs and justification of the purchase: 154 Krzysztof Lesiakowski As a result, a large part of purchased items made was either not used or used very lit-
tle. Some were handed over to the management or other employees of the Committee
for their personal use [AAN. PZPR, 4014, f. 149]. As a result, a large part of purchased items made was either not used or used very lit-
tle. Some were handed over to the management or other employees of the Committee
for their personal use [AAN. PZPR, 4014, f. 149]. This included sports equipment, radio and television equipment, cassette
recorders or calculators. Based on the results of the audit, NIK made a re-
quest to the prosecutor’s office to initiate penal proceedings against the
management of the Committee [Komisja 2013: 309]. NIK also reported massive abuses by and privileging of the people in
power in the housing construction industry. The situation in this sector
was difficult – in the late 1976 in Poland there were 700,000 members of
housing cooperatives waiting for flats [Jarosz D. 2010: 258] – which gave
rise to abuses in institutions making decisions on flat allocation. ABUSES The people responsible for allocating flats were found guilty of non-compliance with
the transparency of allocation procedures and of excluding some flats from social su-
pervision [ANIK. GP, 1569/6, f. 228]. For example, the construction of 13 single-family detached houses by
the Housing Cooperative in Rzeszów for selected employees of the Agri
cultural Construction Association in Rzeszów and associated companies
had criminal features. Furthermore, interested workers of the Association,
including the director and his two deputies, led to a situation where the
executive companies set the house prices clearly below the actual costs of
construction [ANIK, GP, 1627/3 f. 75]. l Similar problems, including allocating flats to ineligible people or priv-
ileging people in positions of power, occurred during allocation of flats
from the so-called company pool. In 1978, NIK inspectors established that
the “Stomil” Automotive Rubber Company in Dębica allocated a dozen or
so flats to workers who had already received their own cooperative flats. Even more shocking evidence for privileging the management was found in
the Industrial Construction Company No. 1 in Wrocław. The construction
of 14 single-family homes for the managerial staff of the company and oth-
er associated entities (including the Construction Association) consumed
77% of the total number of working hours in all sites, despite the fact that
this investment comprised only 9% of the total housing construction plan
of the company until the end of 1976. As a result, in the 1st quarter of 1977,
the construction of the 14 houses was finished, but none of the 214 flats in
multi-family buildings for the employees of the company were built until
the middle of the year, even though, according to the plan, 90 flats should Professional Negligence 155 had been finished a year earlier, that is by July 30, 1976. Notably, the hous-
es for directors were built in a much better standard than stipulated in the
design and pricing documentation [ANIK, GP, 1627/3, f. 331-332]. The problems indicated in NIK reports also included irregularities in
allocating construction and recreational plots. After an audit conducted in
1977 in the Land Management and Environment Protection Department
at the Regional Administration Office in Szczecin, the director of this in-
stitution was dismissed. Also in Ełk the scale of abnormalities in sales of
plots for building single-family homes was so large that the head of the
city had to be replaced [ANIK, GP, 294/3, f. ABUSES 31; 294/4, k. 26]. NIK inspec-
tors also noticed quite a commotion regarding allocating plots in Piastów,
near Warsaw. These were allocated to people whose housing needs had
already been satisfied elsewhere. Also the order of examining the requests
was not observed, and the value of the property or plants located in plots
was underestimated [ANIK, GO, 294/5, f. 47]. Later, it turned out that the
problem escalated, and that people from the highest party and state ad-
ministration positions took part in this despicable criminal activity. p
p
p
y
According to NIK materials, another consequence of the directors’ or
managers’ acquisition of plots was subsequent use of the production capa-
bilities of their mother companies and their materials for building private
residential and recreational houses. This was done, for example, by the di-
rector and the main engineer of the “Orzeł Biały” Mining and Foundry
Company in Piekary and the director of the “Lenko” Linen Company in
Bielsko-Biała [ANIK, GP, 294/1, f. 13, 16; 294/4, f. 17]. In this situation, it
is hardly surprising that companies which fundamentally sponsored the
construction investments of the “elites” generated massive losses. For ex-
ample, the “Insbud” Construction Cooperative in Suwałki lost 18 million
PLN in similar circumstances in the year 1978 and the 6 months of the year
1979. Interestingly, when the company’s main accountant, probably un-
willing to permanently endorse giving away construction materials to in-
dividuals for free or at wholesale prices and underestimating payments
for the single-family homes built, informed her superiors of this situation,
she lost her job and was dismissed from the PZPR. Only after NIK had
been alarmed and had performed a new audit, the scale of abnormalities
in “Insbud” was confirmed, leading to the dismissal of the cooperative’s
director and several other people [ANIK, GP, 294/5, ff. 48-49]. How could
it have been any different, though, if the Minister of Construction and
Construction Materials Industry himself, Adam Glazur, was such a bad
role model? When building his own recreational house, he used materi- Krzysztof Lesiakowski 156 als and workers from various companies worth 1.7 million PLN without
any authorization. In October 1972, he was sentenced to 7 years in pris-
on and the house was confiscated by the Treasury [Seidler B.1988: 380,
389]. ABUSES In the case of the management
of the Residential Construction Company in Kielce, which, for two years, Professional Negligence 157 organized trips to Bulgaria for privileged employees, the “only” problem
was the fact that these trips were settled from the social fund as domes-
tic trips. In 1977, the management of the Poultry Company in Prochów fi-
nanced foreign trips – and to be more precise, a voyage on the “Stefan
Batory” cruise ship for 13 white-collar workers, including 9 people in
managerial positions – from the company’s current assets. NIK also noticed the problem of bribery. In its classic form, it oc-
curred when state institutions met private business. This was evidenced
by convicting, in 1979, two deputy presidents of the “Lech” Leather Goods
Company in Poznań, to 1.5 and 1 year imprisonment and fines of 20,000
and 80,000 PLN, for having accepted a 180,000 PLN bribe for selling acryl-
ic imported from France to private manufacturers [ANIK, GP, 148/14,
f. 28].i A specific form of bribery involved “gifts” from directors and manag-
ers to their superiors and more important cooperators. The fact that those
who were receiving those gifts due to their positions treated them as their
private property and not the property of the company they represented
was even more telling: The court you had was the court you could afford. The higher you are, the larger your
court, and the more substantial the privilege at your disposal [Seidler B. 1988: 412]. The court you had was the court you could afford. The higher you are, the larger your
court, and the more substantial the privilege at your disposal [Seidler B. 1988: 412]. The court you had was the court you could afford. The higher you are, the larger your
court, and the more substantial the privilege at your disposal [Seidler B. 1988: 412]. This criminal business thrived in particular – as it was revealed by an au-
dit in 1980 – in the Radio and Television Committee. The management of
the Committee presented selected people with gold and silver watches
and pens and valuable works of art, while – by means of comparison – the
“Sintur” Invalid Cooperative in Turek in 1977 gave away only tapestried
bed covers, rugs and decorative throws. ABUSES Edward Gierek, until September 1980 the First Secretary of the Polish
United Worker’s Party Central Committee, managed to avoid convic-
tion, though. Based on NIK materials, the prosecutor’s office accused him
of spending money from the funds of the Regional Entertainment Park
Construction Committee in Chorzów on his residential house in Katowice
[Gajdziński P. 2014: 343]. [
j
]
Investigation of the large-scale use of state company resources for
building private residential and recreational houses of prominent people
was started by NIK in accordance with the resolution of the 6th Plenary
Session of PUWP Central Committee of October 6, 1980, which obliged
NIK and the state financial supervision institutions to verify the financing
sources of all dubious private construction investments executed in the
1970s – and in particular all single-family homes (apart from those in agri-
cultural farms) and recreational houses. For this purpose, the president of
NIK, Mieczysław Moczar, appointed a special team headed by Władysław
Pilatowski, a member of the Chamber’s council. The team operated un-
til June 30, 1981. 7149 investments were investigated, out of which 2819
(39.4%) belonged to people in managerial or administrative positions, in-
cluding the First Secretary, 2 Central Committee secretaries, 23 first secre-
taries of Regional Committees, 34 secretaries of Regional Committees, 7
deputy prime ministers, 18 ministers, 31 deputy ministers, 21 governors
and 31 deputy governors [Smith H., Smolar A., Wosleński M. 1983: 19]. 3422 people were found guilty (47.9%), including 2245 (79.6%) in promi-
nent positions [Korupcja 1983: 148–206; Mac J. S. 1981: 5; ANIK, GP, 148/1,
ff. 14-14v; 148/2, f. 8]. This meant that the scale of abuses by prominent
people in the area of private construction was particularly large. Abuse of authority and positions for personal purposes went beyond
matters related to the construction of residential and recreational houses. It
also involved the use of company cars, tools, typewriters, recorders, loud-
speakers or portable television sets for private purposes. Another exam-
ple of abuse was the use of managerial positions to arrange foreign trips. In 1979, the aforementioned agriculture and food company in Szczekociny
financed a trip to Canary Islands for three people who were not employ-
ees of the company, including the daughter and the son-in-law of the com-
pany president [AAN, PZPR, 4014, f. 134]. 1 Thus, Edward Gierek was wrong when he said many years later that during his ad-
ministration, only licenses that had been implemented for production were bought. ABUSES This indicates that the mechanism
of giving and accepting “gifts” was common, and the value of those items
depended on the company’s capabilities, but surely this phenomenon can-
not be justified by the Polish society’s fairly tolerant attitude towards sim-
ilar behaviors at the time [Kutyłowski A., Rzepliński A. 1981: 152-154]. On
a similar basis, the “Silesian Insurgents” Coking Plant in Zdzieszowice
(as revealed during an audit in 1978) gave away crystals and paintings
that had been bought earlier, and the management of the Copper Mines
and Foundries Company in Lubin, which had more resources, gifted sil-
ver items worth almost 500,000 PLN that had been specially manufactured
in the company’s prototype workshop [AAN, PZPR, 4008, f. 9; ANIK, GP,
294/4, ff. 4-5; 294/3, ff. 16-17, 23]. 158 Krzysztof Lesiakowski MISMANAGEMENT MISMANAGEMENT The scale of the problem is ev-
idenced by the fact that during the NIK council sitting on March 14, 1979
it was concluded that the cases of professional negligence “can be qual-
ified as economic sabotage”. Every case of such negligence led to tangi-
ble financial losses. For example the “Minex” export and import company
paid its Brazilian supplier for goods (magnesite) based on the weight de-
clared by the exporter and not the actual weight confirmed in the Polish
port once the products had been unloaded. As a result, they paid USD
273,000 more than needed for the delivery of 1.6 t of magnesite in the years
1977-1978; the money could not be recovered. Furthermore, it turned out
that the bosses of trade companies sent different goods than stipulated
in contracts, which also meant losses – they had to provide discounts or
cover return costs. For example, the “Textilimpex-Tricot” company lost
USD 150,000 after having sent to Libya 60,000 men’s coats made of a dif-
ferent fabric than stipulated in the contract (discount). Polish companies
also incurred losses by carelessly delivering products to unreliable com-
panies. Although in 1976 the “Skórimpex” leather company had not re-
ceived payment of SEK 92,400 for shoes delivered to a Swedish compa-
ny, the following year they sent another batch of shoes to the same buyer
[ANIK, GP, 1569/8, ff. 465-568]. Negotiation of prices for exported goods
was also often neglected. In the years 1977–1978, the “Unitra” Foreign
Trade Company exported silver powder at a price that did not cover the
raw material and production costs. The conduct of the “Ciech-Siarkopol”
sulfur company was also despicable – not only was the research of foreign
market limited to information provided in the bi-monthly trade magazine
“Sulphur”, but also, despite actual increase in sulfur prices in 1978, the
company sold it at lower prices than in the previous year [Rurarz Z.1990;
f. 59; ANIK, GP, 1569/8, ff. 467-468]. What is more, NIK also pointed out serious irregularities with regard
to organizing foreign business trips. These trips were planned in a partic-
ularly extravagant way. MISMANAGEMENT The information materials drafted by NIK in January 1977 reported
that the actual losses resulting from mismanagement and wastefulness
were much higher than what the Prosecutor’s General Office had found,
amounting to around 750 million PLN a year. NIK admitted, though, that
a large part of the losses was hard to establish, because the system of reg-
istration, settlements and supervision in production and sales made it
possible to include them into operational costs of the companies. As a re-
sult, the manufacturing or trading costs in companies grew, but the in-
crease did not affect the assessment of their activity in the form of gener-
al financial results, because many of them achieved or even exceeded the
results planned. However, this was not due to rational management or
decreasing prime costs, but, for example, to price manipulation and falsi-
fying the quality and types of resources and materials [ANIK, GP, 1627/3,
ff. 60-61]. The most frequently encountered examples of mismanagement, very
often punishable by law, included: making irrational and unnecessary pur-
chases of machines and tools (including imports); insufficient protection
of property; not issuing the required documents for materials and resourc-
es sold and dispatched from warehouses; unjustified settling of materials
at underestimated prices; using improper technologies in production (e.g. during roadworks); improper storage or processing of agricultural prod-
ucts; neglect of livestock in cooperative and state agricultural farms. When it comes to buying equipment for foreign currency it must be
noted that the “Pioma” Mining Equipment Company in Piotrków Try
bunalski was particularly careless. An audit in 1976 showed that the com-
pany bought in West Germany three complete welding stations for manu-
facturing automated machines whose production had been canceled even
before the stations were imported [ANIK, GP, 2941, f. 12]. In 1979, the
“Bumar” and “Energopol” Foreign Trade Companies paid in foreign cur-
rency for the import from Belgium of two completely useless prototypes
of extracting machines [Rolicki J. 1990: 96]1, which were supposed to be
used in Gdańsk during the construction of a refinery [ANIK, GP, 148/14,
f. 27]. In this case, the NIK audit led to charging the people responsible. 159 Professional Negligence NIK made many mismanagement accusations towards the bosses of
foreign trade centers who were responsible for negotiating prices and en-
forcing payments for the exported goods. MISMANAGEMENT Some were completely unnecessary, as evidenced
by the actions of the “Kolmex” foreign trade company, which in July,
August and September of 1976 sent three consecutive teams to Turkey in
order to prepare a tender proposal for delivering Diesel locomotives for
this country – even though it had been known from the very beginning
that the Polish party could not enter the tender because they were not able
to offer engines as powerful as the Turks required. Another problem was Krzysztof Lesiakowski 160 the fact that, as found during audits, teams were too large and included
unnecessary employees, for example, too many members of the manage-
ment. In 1977, a NIK report stated that in some cases, directors spent over
100 days abroad! [AAN, PZPR, 3852, ff. 44-45; ANIK, GP, 1569/12, ff. 564,
568]. If we add that some of these trips were organized at the expense of
foreign companies, but prior to signing the contract, then the situation
must have risen suspicion of corruption [Madej K. 2003: 262]. PERSONAL RESPONSIBILITY
IN THE LIGHT OF NIK AUDITS Identifying those personally responsible for the detected cases of neg-
ligence, abuses and mismanagement was very difficult for auditors. It was
much easier to say that the situation is abnormal and define the conse-
quences than to specify who should be held responsible (professionally,
legally and financially). Difficulties in this respect resulted mainly from
poor organization and functioning of companies, a complicated (collec-
tive) decision-making processes and the weakness of professional super-
vision. In many cases obstacles were created by informal, local groups of a crim-
inal nature, the so-called cliques [Górniok O. 1986: 65-66]. This is evidenced
by the case of punishing those guilty of delivering bad meat products in the
Nisk Meat Company (including return of exported canned ham worth 2.7
million PLN). The district unit of NIK in Rzeszów made requests to pun-
ish the company’s management twice, in 1978 and 1979. The first time, the
prosecutor’s office discontinued proceedings, and the second time, no ver-
dict was given due to the amnesty. Only the third attempt proved effec-
tive. The vice director of the company for production was sentenced to two
years in prison (verdict suspended for 3 years) and a 30,000 PLN fine, and
the export production manager – to 1 year and 6 months in prison (verdict
suspended for 3 years) and a 20,000 PLN fine. However, it was impossible
for NIK to lead to the dismissal of the manager in the “Wasiułki” brick com-
pany (the worst unit of the Construction Ceramics Company in Olsztyn),
which, as a result of numerous acts of negligence, for a long time intro-
duced bricks of very bad quality to the market. The manager was defended
by the Polish United Workers’ Party committee in Nidzica and the Workers
Council [ANIK, GP, 148/14, f. 27]. NIK also failed to enforce professional Professional Negligence 161 punishment (not to mention any other consequences) for people from the
so-called research and extraction coal mine in the Lublin region who al-
lowed for shaft deepening works to be conducted contrary to the applicable
technological and geotechnical procedures, which resulted in a temporary
flooding of the mine in October 1979 [AAN, PZPR, 4014, f. 18]. Despite similar obstacles, NIK managed to enforce personal responsi-
bility in many cases. PERSONAL RESPONSIBILITY
IN THE LIGHT OF NIK AUDITS If the shortage caused by mismanagement or waste-
fulness was qualified as “significant”, then the case was transferred to
law enforcement agencies [ANIK, GP, 1586/6, f. 5].2 In 1979, NIK audited
7769 units. 5396 people were sanctioned for abuses and mismanagement
proved during audits. Professional sanctions were applied against 4624
people, including 803 directors, deputy directors and main accountants. 58 people were dismissed immediately (including 24 directors and deputy
directors), 350 were dismissed with notice, 312 people (including 78 direc-
tors and deputy directors) were transferred to different positions with dif-
ferent salaries. Furthermore, 85 cases were transferred to prosecutor’s of-
fice as qualifying for a criminal investigation. NIK also imposed 772 fines
in the total amount of 3.2 million PLN. In 1980, NIK also imposed professional sanctions against 4872 people,
out of whom 777 were in managerial positions. In total, 460 people were
dismissed, out of whom 100 without notice. Fines in the total amount of
3.4 million PLN were administered towards 807 people found guilty, in-
cluding 397 in managerial positions. Furthermore, 180 cases were trans-
ferred to the prosecutor’s office [AAN, PZPR, 4014, f. 9]. 2 Shortage was referred to as “significant” on the basis of comparison between its size
and the overall stock of the goods that the perpetrator supervised or managed. CONCLUSIONS The aforementioned data show that NIK had proven criminal incli-
nation among part of the Polish managerial staff in the second half of the
1970s, before the problem was publicized by “Solidarity”. The number of
people in managerial positions who were punished professionally, finan-
cially, by dismissal or by request for launch of a criminal investigation by
the prosecutor’s office gradually increased. Auditors concentrated mainly
on lower ranking managers, though – at company, cooperative or commu- Krzysztof Lesiakowski 162 nal levels. Managers in higher positions – in associations and ministries –
guilty of professional negligence, abuses and mismanagement on a much
larger scale than those lower in the hierarchy, were beyond the reach of
NIK auditors due to formal considerations (as NIK reported to the Prime
Minister). Even more important is the fact that the government and in-
dividual ministries did not draw any conclusions from this information. Therefore, NIK audits were not an effective preventive measure and did
not lead to any significant decrease in criminal practices among the direc-
tors and managers of Polish companies. ARCHIVE MATERIALS 1569/6, Information for NIK council on some important housing prob-
lems in the light of analyses and audits performed by NIK, May 1977, f. 228. g
y
y
y
ANIK – GP, sign. 1627/3, A note to the President of the Council of Ministers regarding
some conclusions from the audit, October 8, 1977, f. 75. ANIK – GP, sign. 1627/3, Information on the condition of collective property protection
against abuses, wastefulness and mismanagement, January 1977, ff. 331-332. ANIK – GP, sign. 294/3, Information on the mode of examining and handling complaints
and requests sent to NIK in 1977, February 1978, f. 31; ibidem, sign. 294/4, Report on
complaints handled in 1978 r., February 1979, f. 26. p
y
ANIK – GP, sign. 294/5, Report on complaints and requests handled in the year 1979,
[1980], f. 47. ANIK – GP, sign. 294/1, Information on the mode of examining and handling complaints
and requests sent to NIK in 1976, February 1977, f. 13, 16; ibidem, sign. 294/4, sign. 294/4, Report on complaints handled in 1978, February 1979, f. 17. y
ANIK – GP, sign. 294/5, Report on complaints and requests handled in the year 1979,
[1980], f. 48-49. ANIK – GP, sign. 148/1, Decision no. 1/90 of the president of NIK, October 21, 1980, f. 14-
14v; ibidem, sign. 148/2, Decision no. 11 of the president of NIK, June 30, 1981, f. 8. g
p
AAN – PZPR, sign. 4014, Report on NIK operation in the year 1980 [1981], f. 134. g
y
ANIK – GP, sign. 294/3, Information on the mode of examining and handling complaints
and requests sent to NIK in 1977, February 1978, f. 3, 16. ANIK – GP, sign. 148/14, Information on problems resulting from the report of Prosecutor
General and cooperation between NIK and law enforcement agencies in 1979, March
1980, f. 28. AAN – PZPR, sign. 4008, A note to the president of NIK on the audit in the Radio and
Television Committee [1980], f. 9; ANIK – GP, sign. 294/4, Report on complaints han-
dled in 1978, February 1979, ff. 4-5; ibidem, sign. 294/3, Information on the mode of
examining and handling complaints and requests sent to NIK in 1977, February 1978,
ff. 16-17, 23. ANIK – GP, sign. 1627/3, A note to the President of the Council of Ministers regarding
some conclusions from the audit, October 8, 1977, ff. 60-61. ANIK – GP, sign. ARCHIVE MATERIALS Archiwum Akt Nowych (Archive of New Files, AAN) – Polska Zjednoczona Partia
Robotnicza (Polish United Workers’ Party, PZPR), sign. 3852, 4008, 4014 i Archiwum Najwyższej Izby Kontroli (Archive of Supreme Audit Office, ANIK) – Gabinet
Prezesa (Chairman’s Office, GP), sign. 148/1, 148/2, 148/14, 294/1, 294/3, 294/4,
294/5, 1569/6, 1569/8, 1569/10, 1569/12, 1586/6, 1627/3, 1627/4, 1627/7, 2941 ANIK – GP, sign. 294/1, Information on the mode of examining and handling complaints
and requests sent to NIK in 1976, February 1977, f. 22. q
y
ANIK – GP, sign. 1627/7, Responsibility in the light of NIK audits, September 7, 1978,
ff. 248-249. ANIK – GP, sign. 148/14, Information on problems resulting from the report of Prosecutor
General and NIK cooperation with law enforcement agencies in 1979, March 1980,
f. 25; ibidem, sign. 294/1, Information of the mode of examining and handling com-
plaints and requests sent to NIK in 1976, February 1977, f. 12. AAN – PZPR, sign. 4014, Report on NIK operation in the year 1980 [1981], ff. 133-13 ANIK – GP, sign. 1569/10, Information on the results of personal management audit, June
1978, f. 417. ANIK – GP, sign. 1569/12, Information of NIK council on the results of the audit on ex-
panding and efficiency of operation of liquid fuels and oil distribution network in the
country, October 4, 1978, f. 56. ANIK – GP, sign. 1627/4, Information on the results of the audit of accuracy of statistical
reports, June 1978, f. 16. ANIK – GP, sign. 294/3, Information on the mode of examining and handling complaints
and requests sent to NIK in 1977, February 1978, f. 27. q
y
AAN – PZPR, sign. 4014, Report on NIK operation in the year 1980 [1981], f. 16. ANIK – GP, sign. 1627/3, A note to the President of the Council of Ministers regarding
some conclusions from the audit, October 8, 1977, f. 367. ANIK – GP, sign. 148/14, Information on problems resulting from the report of Prosecutor
General and cooperation between NIK and law enforcement agencies in 1979, March
1980, f. 27. AAN – PZPR, sign. 4014, Report on NIK operation in the year 1980 [1981], f. 147. NIK operation in the year 1980 [1981], f. 14 AAN – PZPR, sign. 4014, Report on NIK operation in the year 1980 [1981], f. 149. Professional Negligence 163 ANIK – GP, sign. ARCHIVE MATERIALS 2941, Information on the mode of examining and handling complaints
and requests sent to NIK in 1976, February 1977, f. 12. ANIK – GP, sign. 148/14, Information on problems resulting from the report of Prosecutor
General and cooperation between NIK and law enforcement agencies in 1979, March
1980, f. 27. ANIK – GP, sign. 1569/8, Minutes of the sitting of NIK council on March 14, 1979, f. 436; ibi-
dem, Information on the results of the audit of the efficiency of foreign trade, February
1979, ff. 465-468.i ANIK – GP, sign. 1569/8, Information on the results of the audit of the efficiency of foreign
trade, February 1979, ff. 467-468. AAN – PZPR, sign. 3852, A report from NIK operation in the year 1977 [1978], ff. 44-45;
ANIK – GP, sign. 1569/12, Information the conclusions from the audit on foreign busi-
ness trips [1977], ff. 564, 568. ANIK – GP, sign. 148/14, Information on problems resulting from the report of Prosecutor
General and cooperation between NIK and law enforcement agencies in 1979, March
1980, f. 27. AAN – PZPR, sign. 4014, Report on NIK operation in the year 1980 [1981], f. 18. ANIK – GP, sign., 1586/6, A bulletin from NIK president, January 6, 1977, f. 5. g
p
y
AAN – PZPR, sign. 4014, Report on NIK operation in the year 1980 [1981], f. 9. Krzysztof Lesiakowski 164 LITERATURE
Gajdziński P. (2014), Gierek. Człowiek z węgla [Gierek. A man of coal], Wydawnictwo
Poznańskie, Poznań. dziński P. (2014), Gierek. Człowiek z węgla [Gierek. A man of coal], Wydawnictwo
Poznańskie, Poznań. rniok O. (1986), Przestępczość gospodarcza: wybrane przejawy i uwarunkowania [Financia
crime: selected symptoms and conditions], Uniwersytet Śląski, Katowice. Jarosz D. (2010), Mieszkanie się należy... Studium z peerelowskich praktyk społecznych [A house
is your right... A study of social practices in the Polish People’s Republic], Oficyna
Wydawnicza Aspra-JR, Warsaw. y
p
Jarosz M. (2004), Władza, przywileje, korupcja [Power, privilege, corruption], Wydawnictwo
Naukowe PWN, Warszawa. Karklins R. (2009), Wszystkiemu winien system. Korupcja w krajach postkomunistycznych [The
system is to blame. Corruption in post-communist countries], Wydawnictwo Sic!,
Warsaw. Komisja Tadeusza Grabskiego 1981 [Tadeusz Grabski Commission 1981] (2013), Jabłonowski
M., Janowski W. (ed.), Faculty of Journalism and Political Science, University of
Warsaw, Warsaw. Konaszyc A. (1978), Kontrola parlamentarna w PRL, Zakład Narodowy im. Ossolińskich
[Parliamentary control in the Polish People’s Republic], Wrocław-Warszaw-Kraków. Korupcja polskiej „nomenklatury”. ARCHIVE MATERIALS Z tajnych akt Najwyższej Izby Kontroli PRL [Corruption
of the Polish “nomenklatura”. From the secret files of Supreme Audit Office] (1983),
“Zeszyty Historyczne” (Paris), no. 64, p. 148-206. Kutyłowski A., Rzepliński A. (1981), Opinie na temat łapownictwa jako przestępstwa i zjawis-
ka społecznego [Opinions on bribes as a crime and a social problem], [in:] “Opinia pub-
liczna i środki masowego przekazu a ujemne zjawiska społeczne” [Public opinion and
mass media and negative social phenomena], Wydawnictwo Prawnicze, Warszawa,
p. 142-156. p
Lesiakowski K. (1998), Mieczysław Moczar „Mietek”. Biografia polityczna [Mieczysław Moczar,
“Mietek”. A political biography], Oficyna Wydawnicza „Rytm”, Warszawa. p
g
p yi
y
y
y
Mac J. S. (1981), Duże pranie [Big laundry], “Prawo i Życie”, no. 28, p. 5. Madej K. (2003), Siermiężna i dolarowa – korupcja w PRL w latach 1956-1980 [Poverty and dol-
lars – corruption in socialist Poland in the years 1956-1980], [in:] Stola D., Zaremba M. (ed.), “PRL. Trwanie i zmiana” [Socialist Poland. Stagnation and change] , Wydawnictwo
Wyższej Szkoły Przedsiębiorczości i Zarzadzania im. Leona Koźmińskiego, Warsaw,
p. 249-280. p
Rolicki J. (1990), Edward Gierek. Replika (wywiad rzeka) [Edward Gierek. A replica (an inter-
view)], Polska Oficyna Wydawnicza ”BGW”, Warsaw. i )i
y
y
Rurarz Z. (1990), Byłem doradcą Gierka [I used to be Gierek’s advisor], Andy Grafik,
Warszawa. Seidler B. (1988), Ludzie i paragrafy [People and law], Wydawnictwo Literackie, Kraków. Sierpowska I. (2003), Funkcje kontroli państwowej. Studium prawno-porównawcze [Function of
state controls. A legal and comparative study], Kolonia Limited , Wrocław. Smith H., Smolar A., Wosleński M. (1983), Władza i przywileje [Power and privileges],
Oficyna WE, Warsaw Sylwestrzak A. (2006), Najwyższa Izba Kontroli. Studium prawnoustrojowe [Supreme Audit
Office. Systemic and legal study], Wydawnictwo Sejmowe, Warszawa. Professional Negligence 165 Krzysztof Lesiakowski – PhD, Profesor, who works at the Polish and World History
Faculty at the University of Łódź. His main research interests include the relationship
between authorities and society in Poland after the Second World War, Polish People’s
Republic, policy towards the young generation, Polish biographies in the 20th century. His
publications include: Mieczysław Moczar „Mietek”. Biografia polityczna [Mieczysław Moczar,
“Mietek”. A political biography] (Warszawa 1998); Jarocin w obiektywie bezpieki [Jarocin
Festival through the lenses of the Security Services] (Warszawa 2004) (co-written with two
authors); Powszechna Organizacja “Służba Polsce” 1948–1955 – powstanie, działalność, likwidac-
ja”, [The Common Organization “Service for Poland” (1948–1955): origin, activity, liqui-
dation] vol. ARCHIVE MATERIALS 1–2 (Łódź 2008); Strajki robotnicze w Łodzi 1945–1976 [Workers’ strikes in Łódź
1945–1976] (Łódź 2008). He is also the author of numerous articles and the editor of sever-
al collected works, including Łódzki strajk studencki styczeń-luty 1981. Spojrzenie po latach”
[Students’ strikes in Łódź January–February 1981. A perspective after the years] (Łódź-
Warsaw 2014).
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Accomplishments in wheat rust research in South Africa
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South African journal of science
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cc-by
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FUNDING: FUNDING:
Biotechnology and Biological
Sciences Research Council /
Department for International
Development (BBSRC-DfID; BB/
F004125/1); Sustainable Crop
Production Research for International
Development, Biotechnology and
Biological Sciences Research Council
(BBSRC-SCPRID; BB/J011525/1) The objective of this review is to provide a summary of recent accomplishments in wheat rust research in
South Africa. EMAIL:
pretorza@ufs.ac.za •
Collaboration among plant pathologists, geneticists and breeders has provided momentum in rust
research and control in South Africa in recent years. •
Collaboration among plant pathologists, geneticists and breeders has provided momentum in rust
research and control in South Africa in recent years. DATES:
Received: 29 Nov. 2019
Revised: 16 Mar. 2020
Accepted: 18 Mar. 2020
Published: 26 Nov. 2020 Accomplishments in wheat rust research in
South Africa AUTHORS:
Zacharias A. Pretorius1
Renée Prins2,3
Elsabet Wessels2
Cornel M. Bender1
Botma Visser1
Willem H.P. Boshoff1
AFFILIATIONS:
1Department of Plant Sciences,
University of the Free State,
Bloemfontein, South Africa
2CenGen (Pty) Ltd., Worcester,
South Africa
3Department of Genetics,
Stellenbosch University, Stellenbosch,
South Africa Rust diseases, although seasonal, have been severe constraints in wheat production in South Africa
for almost 300 years. Rust research gained momentum with the institution of annual surveys in the
1980s, followed by race identification, an understanding of rust epidemiology, and eventually a focused
collaboration amongst pathologists, breeders and geneticists. Diversity in South African populations of
Puccinia triticina, P. graminis f. sp. tritici and P. striiformis f. sp. tritici has been described and isolates are
available to accurately phenotype wheat germplasm and study pathogen populations at national, regional
and global levels. Sources of resistance have been, and still are, methodically analysed and molecular
marker systems were developed to incorporate, stack and verify complex resistance gene combinations
in breeding lines and cultivars. Vigilance, capacity, new technologies, collaboration and sustained funding
are critical for maintaining and improving the current research impetus for future management of these
important diseases. © 2020. The Author(s). Published
under a Creative Commons
Attribution Licence. ARTICLE INCLUDES:
☒ Peer review
☐ Supplementary material No mention is made of rust during the foundational years of cereal production in South Africa but, according to
Theal3, a critical shortage of wheat in 1727 was ascribed in the previous year to rust – a disease known in South
Africa only on rye at the time. The regular occurrence of rust led Neethling2 to conclude: ‘There is no doubt that rust,
owing to the severe damage caused, is the most important factor which caused the extinction and origin of varieties
in South Africa’. Nhemachena and Kirsten4 gave a detailed account of wheat cultivar development in South Africa,
Smit et al.5 summarised wheat research between 1983 and 2008, and overviews of wheat rust research in South
Africa were provided by De Jager6, Lombard7 and Pretorius et al.8 Early milestones were interspecies crosses to
transfer stem rust resistance genes to bread wheat (Triticum aestivum L.) in 1912 followed by pathotyping isolates
of Puccinia graminis f. sp. tritici Erikss. & E. Henn. (Pgt) and P. triticina Erikss. (Pt) in the 1920s and 1930s.8 The
establishment of a centre for dedicated small grains research at Bethlehem in 1976, currently named Agricultural
Research Council – Small Grain (ARC-SG), resulted in appropriate training in rust methodologies, surveillance, race
analysis and germplasm evaluation. These initiatives were expanded with the formation of a rust laboratory at the
University of the Free State in 1989. DATA AVAILABILITY:
☐ Open data set
☐ All data included
☒ On request from author(s)
☐ Not available
☐ Not applicable
EDITORS:
Teresa Coutinho
Salmina Mokgehle
KEYWORDS:
Puccinia, resistance, rust,
surveillance, wheat In recent decades, notable events and initiatives in South African wheat rust research include annual surveys8,
Sr24 virulence9, the appearance of stripe rust (caused by P. striiformis Westend. f. sp. tritici, Pst)10, Ug99 stem
rust studies11-15, the mapping of quantitative resistance loci16,17, genetic characterisation of Puccinia isolates18-20,
comprehensive phenotyping of wheat germplasm, and establishment of a marker service laboratory with a
particular focus on rust resistance genes (https://www.cengen.co.za). Background The sowing of small grain cereals in South Africa occurred within 2 months after the United (Dutch) East India
Company set foot on land in present-day Cape Town in 1652.1,2 Crop failures, in particular due to damage caused
by heavy rains, wind storms and unadapted cultivars, were common occurrences. Nonetheless, efforts to
successfully grow wheat continued and systematically included new production areas, different sowing times,
new cultivars – not only from Europe but also from India, and exports when grain supplies allowed. Varietal
assessments during the early years provided evidence for the first selection of higher-yielding types in South
Africa.2 The pioneering wheat cultivars are not well documented, but reference is made of ‘white’ wheat in 1659,
‘Sarut’ from India in 1673, ‘Roode’ and ‘Grijse Winter’ in 1677, until names based on phenotype (e.g. ‘Bloukoring’,
‘Kleinkoring’, ‘Baardkoring’, ‘Zwartbaard’, ‘Vroeëbaard’), origin (e.g. ‘Ciciliaans’, ‘Bengaalsch’), or growers (e.g. ‘Du Toits’, ‘Niewoudts’, ‘Tautes’) became customary.2 HOW TO CITE:
Pretorius ZA, Prins R, Wessels E,
Bender CM, Visser B, Boshoff WHP. Accomplishments in wheat rust
research in South Africa. S Afr J
Sci. 2020;116(11/12), Art. #7688,
8 pages. https://doi.org/10.17159/
sajs.2020/7688 CORRESPONDENCE TO:
Zacharias Pretorius •
Rust diseases threaten wheat crops worldwide, including in South Africa. •
Management of rusts includes regular surveillance, pathogen diversity studies, rigorous screening of
wheat germplasm, and efficient breeding and selection for resistance. Rust surveillance and phenotypic analysis Surveillance and race typing are routinely conducted by the ARC-SG to determine rust distribution, impact and
pathogenicity in the major wheat-producing areas of South Africa. Recent reports of similarities in races between
southern African countries have also emphasised the importance of regional sampling.21-23 © 2020. The Author(s). Published
under a Creative Commons
Attribution Licence. Handling obligate rust fungi in controlled experiments such as race typing or host plant screening requires
specific infrastructure. In addition to facilities for plant growth, inoculation and incubation, equipment for collection Volume 116| Number 11/12
November/December 2020 Review Article
https://doi.org/10.17159/sajs.2020/7688 Volume 116| Number 11/12
November/December 2020 Volume 116| Number 11/12
November/December 2020 Review Article
https://doi.org/10.17159/sajs.2020/7688 1 Wheat rusts in South Africa
Page 2 of 8 Thew (Lr20) and Agent (Lr24), all Lr genes occur in a Thatcher wheat
background. New races are further characterised on an additional set
containing 23 Lr genes.27 Infection types on the lines RL6011 (Lr12),
CT263 (Lr13), RL6044 (Lr22a), RL6058 (Lr34), RL6082 (Lr35) and
Thatcher control (Lr22b) are determined on flag leaves of adult plants. Thew (Lr20) and Agent (Lr24), all Lr genes occur in a Thatcher wheat
background. New races are further characterised on an additional set
containing 23 Lr genes.27 Infection types on the lines RL6011 (Lr12),
CT263 (Lr13), RL6044 (Lr22a), RL6058 (Lr34), RL6082 (Lr35) and
Thatcher control (Lr22b) are determined on flag leaves of adult plants. and application of small amounts of urediniospores is essential. Because
these specialised items are not commercially available, Pretorius et al.24
developed an additive manufacturing process to assemble spore
collectors and atomisers through 3D printing. Using these devices,
traditional race analysis is done by infecting seedlings of a predetermined
(differential) set of wheat host lines with a rust isolate. An appropriate
experimental set-up and experience in achieving accurate seedling
infection types are essential for reliable phenotyping. Examples of
infection types are shown in Figure 1. (differential) set of wheat host lines with a rust isolate. An appropriate
experimental set-up and experience in achieving accurate seedling
infection types are essential for reliable phenotyping. Examples of
infection types are shown in Figure 1. Figure 1:
Seedling (top, left to right: wheat leaf rust, stem rust and stripe
rust) and adult plant phenotypes commonly encountered for
leaf rust (second from top), stem rust (third from top) and
stripe rust (bottom). Rust surveillance and phenotypic analysis Its broad virulence and subsequent specialisation
in 13 pathotypes have raised serious concerns about sustained wheat
production in many regions of the world.15 Stem rust race 2SA106
(TTKSP North American race code) detected in 2007, 2SA107 (PTKST,
2009), 2SA88+ (TTKSF+, 2010) and 2SA42 (PTKSK, 2017) all show
phenotypic similarities to race 2SA88 (TTKSF, 2000), which was the first
stem rust race in the Ug99 lineage detected in South Africa.12,13,18,21,32,35
These races are phenotypically characterised by differences in virulence
for Sr9h, Sr21, Sr24 and Sr31.13,32 Sr24 and Sr31 have been reported
to occur in South African wheat germplasm36 and virulence was not
unexpected. Likewise, the virulence adaptation of TTKSF+ was recently
confirmed by the endorsement of Sr9h in the wheat cultivar Matlabas.37
Despite being less virulent compared to the more recently detected
Ug99 races, TTKSF remains the dominant variant.8,31,32,38 Stem rust
races TTKSF (2009), TTKSF+ (2010) and PTKST (2010) were also
identified in samples collected in Zimbabwe and PTKST was confirmed
in Mozambique.15,21 The most significant change in the Pgt population since 2005 was
the regular appearance of new races in the Ug99 lineage. African race
Ug99, named after the country of first detection (Uganda) and year of
description (1999)11, was the first race with virulence for the widely used
Sr31 resistance gene. Its broad virulence and subsequent specialisation
in 13 pathotypes have raised serious concerns about sustained wheat
production in many regions of the world.15 Stem rust race 2SA106
(TTKSP North American race code) detected in 2007, 2SA107 (PTKST,
2009), 2SA88+ (TTKSF+, 2010) and 2SA42 (PTKSK, 2017) all show
phenotypic similarities to race 2SA88 (TTKSF, 2000), which was the first
stem rust race in the Ug99 lineage detected in South Africa.12,13,18,21,32,35 These races are phenotypically characterised by differences in virulence
for Sr9h, Sr21, Sr24 and Sr31.13,32 Sr24 and Sr31 have been reported
to occur in South African wheat germplasm36 and virulence was not
unexpected. Review Article
https://doi.org/10.17159/sajs.2020/7688 Rust surveillance and phenotypic analysis No new Pt races were detected between 1988 and 2008 in South
Africa.8,28 This situation changed with the report of race 3SA145
(CCPS North American race code) in 2009, followed by races 3SA146
(MCDS, 2010), 3SA147 (FBPT, 2010), 3SA115 (CBPS, 2012), 3SA10
(CFPS, 2016), 3SA38 (CDPS, 2016) and 3SA248 (CFPS, 2016).27-
30 The frequency of Pt races with virulence to Lr3, Lr12, Lr13, Lr15,
Lr26 and Lr37 is high and varied between 79% and 98% during recent
surveys.27 The Pt population was dominated for many years by race
3SA133 (PDRS) which initially was common on winter wheat in the Free
State. This changed significantly with the appearance of races 3SA145,
3SA146 and 3SA115 which accounted for >80% of isolates typed
during the 2012–2016 surveys.27 The more recently described races
3SA38 and 3SA10 are increasing in prevalence and comprised more
than 50% of the isolates typed from the 2018 growing season.31 Pt race
MCDS was common in Zimbabwe and Zambia with FBPT and SCDS
detected in Zimbabwe and Malawi.23 Twenty differential wheat lines are used for stem rust pathotyping. Although the resistance genes are similar to the proposal of Jin et al.26,
Acme (Sr9g), Renown (Sr17) and Trident (Sr38) have replaced CnSr9g,
Combination VII and VPM1, respectively. Additional differentials include
Barleta Benvenuto (Sr8b), the triticales Coorong (Sr27), Kiewiet (SrKw)
and Satu (SrSatu), and either LcSrWst-2Wst (Sr9h) or Matlabas (Sr9h).32
New races are further characterised on an extended set of tester lines.33
Although differential lines grown in the field can provide an indication
of prevailing Pgt races, Boshoff et al.34 showed that certain resistance
genes are not well expressed in adult plants whereas other lines contain
resistance in addition to that observed in seedling assays. The most significant change in the Pgt population since 2005 was
the regular appearance of new races in the Ug99 lineage. African race
Ug99, named after the country of first detection (Uganda) and year of
description (1999)11, was the first race with virulence for the widely used
Sr31 resistance gene. Genetic analysis of Puccinia isolates In a recent study,
Li et al.48 provided genomic evidence of somatic hybridisation in Pgt,
shedding light on the origin of Ug99 through the exchange of nuclei
between standard race 21 and an unknown race. This is an important
discovery to understand the formation of new diversity in the absence of
sexual recombination. The Ug99 lineage on the other hand, first detected in South Africa in 2000
with the description of TTKSF12, has expanded into five variants13,14,21,35. I
t
t t th
U 99 li
ll fi
S
th Af i
i
t
d Stem rust assessments for local germplasm are summarised in Figure 2. Only cultivars with seedling infection types <2 (0 to 4 scale)53, and a
coefficient of infection54 <20, were considered to carry true ASR. Some
cultivars regarded as resistant as seedlings showed an intermediate
stem rust reaction in the field and were thus not classified as displaying
true ASR. Inoculum loads in the Greytown field nursery are extremely
high and not all ASR genes provide complete rust protection under
such conditions. In most cases it is assumed that these cultivars will
be acceptable in commercial fields where inoculum pressure is lower. The opposite was also observed where some cultivars were classified
as intermediate in the seedling stage but stem rust resistant in the field. The effect of using race PTKST in the field from 2011 onwards is clear
from the initial decrease in resistance before a gradual improvement
in resistant entries as breeders adapted their selection and breeding
strategies. Collectively such information, also for leaf and stripe rust,
adds to an understanding of disease risk and management at production
level. To support field data, protocols for accelerated and reliable
greenhouse assays have been developed for stripe rust16,55,56, leaf rust57
and stem rust58. The current South African Pt population consists of two primary genetic
lineages20, but at least five were evident according to Pt isolates collected
during the previous century49. Rust surveillance and phenotypic analysis However, it is not yet known if Pst will successfully
establish in Zimbabwe and, as anticipated, migrate to South Africa. throughout the lifespan of the plant whereas adult plant resistance
(APR), often polygenic and partial in manifestation, becomes effective
at more mature growth stages.51 Phenotypes commonly encountered on
adult plants are shown in Figure 1. As some APR genes are considered
durable, this resistance type is frequently preferred in breeding and
selection. Wheat cultivars carrying the pleiotropic race non-specific APR
genes Sr2/Yr30, Lr34/Yr18/Sr57, Lr46/Yr29/Sr58 and Lr67/Yr46/Sr55
have maintained moderate levels of rust resistance under epidemic field
trial conditions in South Africa and might not provide adequate protection
when deployed singly under high disease pressure. Soko et al.52 recorded
grain yield losses due to stem rust of between 10.1% and 19.5% for APR
cultivars as opposed to a 6.4% loss in an ASR line. Previously, Pretorius
et al.8 mentioned losses as high as 65% for susceptible wheat cultivars
infected with stripe rust and a 56% yield gain was obtained when leaf
rust was controlled by fungicide application on a susceptible cultivar. Breeders are therefore encouraged to either combine APR sources or
stack them with ASR genes, the latter especially in areas prone to early-
season infection. It has been suggested that wheat cultivated at a higher elevation in
Lesotho during summer serves as a source of Pst inoculum for winter-
grown crops in South Africa.8 Although not customary, some hectares
may also be sown to wheat in the Free State during summer. The impact
of these formal off-season productions on the epidemiology of the rusts
has, however, not been studied in detail. Volunteer wheat has generally
been assumed to provide a green bridge for the survival of these
biotrophic pathogens between seasons. Genetic analysis of Puccinia isolates Three of these appear to be extinct while
only one lineage is expanding.27,28,30 Similar to Pgt, these new races
probably represent exotic introductions as races with similar phenotypes
and genotypes were found in countries to the north of South Africa.23
Globally, the South African Pt races grouped significantly with isolates
from the Middle East, Pakistan and New Zealand.50 Based on microsatellite analysis, the four Pst races described in South
Africa represent a single, clonal lineage.19 As opposed to these races,
the recently identified Pst race in Zimbabwe was genetically very similar
to two Kenyan isolates45, indicating a southerly expansion of stripe rust
diversity in Africa. Genetic studies of host resistance provide information on the monogenic
or polygenic nature of genes involved, their identity and chromosome
location, association with known genes or quantitative trait loci (QTL), and
molecular markers for tracking the resistance. Together this knowledge
contributes to assumptions of durability and targeted attempts to achieve
long-lasting resistance. Ramburan et al.16 were the first to map rust
resistance in a South African wheat cultivar. They identified three major
stripe rust resistance loci in the spring wheat cultivar Kariega and paved
the way for fine mapping and marker development for QYr.sgi-2B.1 and
QYr.sgi-4A.1, and confirmation of the pleiotropic resistance gene Lr34/
Yr18/Sr57.57,59 In a similar approach, the durable stripe rust resistance
of the European wheat cultivar Cappelle Desprez was mapped17 with
subsequent identification of the major effect QTL QYr.ufs-2A along with
three QTL of smaller effect, QYr.ufs-2D, QYr.ufs-5B and QYr.ufs-6D. Using
histological techniques, Maree et al.60,61 investigated fungal behaviour
in lines containing different combinations of the stripe rust resistance Due to the unique ability of markers to distinguish genotypes independently
of their associated phenotypes, genetic screening of field isolates can
detect variants before a new phenotype becomes evident. While Pt races
3SA38, 3SA10 and 3SA248 were first detected as phenotypic variants
in 201627, their unique genotypes were already abundant in field isolates
collected in 201549. These markers also indicated that within each
phenotype, significant genetic variation was present, making genetic
markers an effective supplementary tool to race phenotyping. Genetic analysis of Puccinia isolates Analysis of Pt, Pgt and Pst with microsatellite markers has contributed
to explaining genetic diversity within the three populations. In the
absence of functional alternate hosts for wheat rust pathogens in South
Africa, wind dispersal, human activities, mutation and possibly somatic
recombination are considered as drivers of variation. The South African
Pgt population consists of two highly diverse genetic lineages.18 In the
absence of viable historical samples in South Africa, the close genetic
similarity of members of the non-Ug99 genetic lineage with Australian
standard races 21-0 collected in 1954, and 326 and 194 collected in
1969, respectively, suggested that this lineage represents the original
South African population.46 Included in this lineage are races that are
specific for both wheat and triticale. The acquisition of virulence within
this group appears to be the result of step-wise mutations.32,33 On a global
scale, this lineage grouped closest with Pgt samples from Pakistan,
Czech Republic47,48 and Australia due to the proposed movement of
urediniospores on high-altitude westerly winds46. The damage potential of wheat rusts is a reality, and it remains
important to verify the resistance status of local germplasm and embark
on appropriate breeding and selection programmes. As part of risk
assessment and compilation of production guidelines, all commercially
recommended wheat cultivars in South Africa, as well as leading breeding
lines, are tested annually against a panel of rust races. These tests
comprise seedling assays for ASR and field tests under high inoculum
pressure in carefully managed rust nurseries. The University of the Free
State has implemented rust nurseries with great success at the research
facilities of Corteva AgriscienceTM at Greytown in KwaZulu-Natal since
the early 1990s. The Greytown environment is highly conducive to the
vigorous development of both spring and winter wheat types as well as
rust development. In a typical year, stripe rust would be first to establish
during the cooler months of August and September, followed by leaf rust
in October and finally stem rust, which peaks at the end of the season. The Ug99 lineage on the other hand, first detected in South Africa in 2000
with the description of TTKSF12, has expanded into five variants13,14,21,35. In contrast to the non-Ug99 lineage, all five South African variants and
the original TTKSK11 shared more than 85% genetic similarity and fall
within the bigger Ug99 race group from east Africa15. Rust surveillance and phenotypic analysis Likewise, the virulence adaptation of TTKSF+ was recently
confirmed by the endorsement of Sr9h in the wheat cultivar Matlabas.37
Despite being less virulent compared to the more recently detected
Ug99 races, TTKSF remains the dominant variant.8,31,32,38 Stem rust
races TTKSF (2009), TTKSF+ (2010) and PTKST (2010) were also
identified in samples collected in Zimbabwe and PTKST was confirmed
in Mozambique.15,21 Seedling infection types produced on the World and European differential
sets39,40, followed by an A+ or A- suffix to describe virulence or avirulence
for the YrA gene in Avocet R41, are used for Pst race designations in
South Africa. Near-isogenic lines with Avocet S as the recurrent parent
are used as additional tester lines for race characterisation and in field
plots.42 Following the detection of Pst race 6E16A- in 199610, proposed
to be a foreign introduction from Central or Western Asia either by wind
or human intervention43,44, there is strong evidence that adaptation to
the host genes Yr25 (race 6E22A-, cultivar Hugenoot, 1998) and YrA
(6E22A+, PAN 3195, 2005) resulted from selection pressure10,19. The Pst population has remained relatively stable since the detection
of race 6E22A+ on winter wheat in the eastern Free State in 200519
with 6E22A+ persisting as the most dominant race, comprising 58% of
isolates in 201831. Figure 1:
Seedling (top, left to right: wheat leaf rust, stem rust and stripe
rust) and adult plant phenotypes commonly encountered for
leaf rust (second from top), stem rust (third from top) and
stripe rust (bottom). Figure 1: Based on the pathogenicity of an isolate on entries in the differential set, a
race (pathotype) name is allocated. Apart from an alpha-numerical code
to name leaf and stem rust races in South Africa8, the North American
system of nomenclature25,26 is used to place races in an international
context. The standard South African differential set for determining
seedling infection types to Pt isolates contains 20 entries.27 Except for Volume 116| Number 11/12
November/December 2020 Review Article
https://doi.org/10.17159/sajs.2020/7688 Volume 116| Number 11/12
November/December 2020 2 Wheat rusts in South Africa
Page 3 of 8 Wheat rusts in South Africa
Page 3 of 8 The outbreak of stripe rust on irrigated spring wheat in 2018 represented
the first report of the disease in Zimbabwe.45 Showing virulence to Yr3a,
Yr4a, Yr9 and Yr27, race 30E142A+ was distinctly more virulent on
South African wheat cultivars than 6E22A+ and poses a potential threat
to the local industry. Breeding and selection Table 1:
Yield performance of selected marker-assisted selection (MAS)
lines of Corteva AgriscienceTM compared to commercial cultivars Table 1:
Yield performance of selected marker-assisted selection (MAS)
lines of Corteva AgriscienceTM compared to commercial cultivars
Entry
Relative yield (%)a
Genes incorporated through MAS
Cultivar 01
102
Confidentialb
Cultivar 02
97
Confidential
Cultivar 03
94
Confidential
Cultivar 04
100
Confidential
Cultivar 05
106
Confidential
Cultivar 06
106
Confidential
MAS Line 01
99
Fhb1 Qfhs.ndsu-3BS; FHB Qfs.ifa-5A
MAS Line 02
96
Fhb1 Qfhs.ndsu-3BS; FHB Qfs.ifa-5A
MAS Line 03
98
Fhb1 Qfhs.ndsu-3BS; FHB Qfs.ifa-5A
MAS Line 04
99
Fhb1 Qfhs.ndsu-3BS; FHB Qfs.ifa-5A
MAS Line 05
96
Fhb1 Qfhs.ndsu-3BS; FHB Qfs.ifa-5A
MAS Line 06
102
Fhb1 Qfhs.ndsu-3BS; FHB Qfs.ifa-5A
MAS Line 07
95
Sr2/Yr30; Lr34/Yr18/Sr57/Pm38
aYields measured in tons/ha are expressed relative to Cultivar 04 which was taken as
the benchmark (100%). bDeveloped through traditional breeding without MAS. Marker-assisted selection (MAS) is widely accepted as a key strategy
to pyramid resistance genes into wheat genotypes, in particular, genes
that do not exhibit easily distinguishable phenotypes.72 In South Africa,
large-scale MAS was not implemented by breeding companies in the
early 2000s8, although it was routinely used to select for several traits in
countries such as Australia, Mexico, USA, the UK and India72. In 2011,
a proposal by CenGen (Pty) Ltd. titled ‘Establishment of a molecular
marker service laboratory for routine application of marker-assisted
selection in South African wheat breeding programs’ (WCT/W/2009/02),
was approved for funding by the Winter Cereal Trust. The capital expense
of establishing a MAS laboratory and routine maintenance justified a
central facility at CenGen for all wheat breeding programmes. The project
is based on (1) purity testing of donor lines and confirmation of the target
trait, (2) planning of breeding schemes and crosses to transfer the new
trait, and (3) tracking the trait in subsequent filial generations. Host resistance Resistance phenotypes in wheat are typically growth stage mediated. All-
stage resistance (ASR), conferred by major genes, is clearly expressed Volume 116| Number 11/12
November/December 2020 Review Article
https://doi.org/10.17159/sajs.2020/7688 3 Wheat rusts in South Africa
Page 4 of 8 Wheat rusts in South Africa
Page 4 of 8 QTL characterised in Kariega and Cappelle Desprez, respectively. These
studies confirmed the value of gene stacking and careful selection of
lines with the best ability to mitigate fungal invasion. or commercial cultivars and from the F1-generation doubled haploid
lines are developed to integrate the genes/QTL into better adapted
backgrounds. This approach has proven to be successful in pyramiding
rust resistance genes/QTL (Figure 3). They have managed to develop a
line containing multiple genes for resistance to all three rust pathogens,
which is now used as a key donor line to incorporate complex resistance
into existing cultivars. Figure 2:
The frequency of South African wheat varieties expressing a
low seedling response, adult plant resistance (APR) and true
all-stage resistance (ASR) to stem rust over 8 years. In 2009
and 2010, entries were tested with Puccinia graminis f. sp. tritici pathotype UVPgt59 (TTKSP), and since 2011 with the
more virulent UVPgt60 (PTKST) pathotype. Number of lines containing at least
2 target genes/QTL
Figure 3:
Complement of rust resistances incorporated in the Sensako
doubled haploid marker-assisted selection programme. Numbers in brackets indicate the maximum number of
genes/quantitative trait loci (QTL) screened for in the specific
combination. For each combination, lines containing at least
two target genes/QTL were selected for field evaluation in
2019. For all combinations, at least one line was recovered
containing all targets. Number of lines containing at least
2 target genes/QTL Figure 2:
The frequency of South African wheat varieties expressing a
low seedling response, adult plant resistance (APR) and true
all-stage resistance (ASR) to stem rust over 8 years. In 2009
and 2010, entries were tested with Puccinia graminis f. sp. tritici pathotype UVPgt59 (TTKSP), and since 2011 with the
more virulent UVPgt60 (PTKST) pathotype. Complement of rust resistances incorporated in the Sensako
doubled haploid marker-assisted selection programme. Numbers in brackets indicate the maximum number of
genes/quantitative trait loci (QTL) screened for in the specific
combination. For each combination, lines containing at least
two target genes/QTL were selected for field evaluation in
2019. For all combinations, at least one line was recovered
containing all targets. Host resistance Figure 3: Prins et al.62 assessed stem rust response in an African wheat collection
and identified several marker-trait associations in a genome-wide study. Two lines with exceptional APR were identified and biparental mapping
populations developed. Marker-trait associations on chromosomes 6AS
and 3BS and the Lr34/Yr18/Sr57 resistance locus were confirmed, along
with stem rust resistance QTL not detected in the association study, one
of which was the significant QTL QSr.ufs-4D. This emphasises the value
of applying multiple approaches to unravel host resistance, particularly
in cases where marker coverage in certain chromosomal areas is too
low to detect QTL. The South African wheat breeding programme of Corteva AgriscienceTM
follows a more traditional approach of gradually incorporating multiple
genes/QTL into their breeding lines. Gene enrichment is done at the F2-
generation, and the presence of the target genes is confirmed in the
F5-generation after three cycles of selection for agronomic traits. Pure
lines containing the target genes are then either used to generate new
resistance gene combinations or, if within the tolerance levels set
for the different milling and baking quality criteria, are considered for
commercial release. This programme has been successful in combining
APR genes for stem, stripe and leaf rust resistance into elite breeding
material that performs similarly to current commercial cultivars in yield
trials (Table 1). The availability of Pgt races with virulence attributes appropriate for
targeting certain sources of resistance has contributed to several studies. These projects addressed phenotyping and genetics of resistance to Ug99
races63-66, resistance characterisation of triticale67 and lines with genes
transferred from Aegilops sharonensis68 and Thinopyrum ponticum69,70. Furthermore, Pretorius et al.71 demonstrated the application of remote
sensing and the normalised difference vegetation index in reliably
phenotyping wheat stripe rust response in the field. Review Article
https://doi.org/10.17159/sajs.2020/7688 We declare that there are no competing interests. Figure 5:
Number of data points generated since inception of the marker-
assisted selection programme. Acknowledgements Substantial funding by BBSRC-DfID for the project ‘Assessment of
genetic biodiversity of durable disease resistance in African wheat
genotypes, leading to the development of novel marker systems for wheat
breeding’ (BB/F004125/1) and by BBSRC-SCPRID for ‘Implementing
effective marker technologies into disease resistance wheat breeding
programmes within Africa’ (BB/J011525/1) is gratefully acknowledged. Additional support by the Winter Cereal Trust, University of the Free
State, National Research Foundation, Cornell University (DRRW project)
and many local and international colleagues is also acknowledged. Conclusions The relatively frequent introduction of new rust races into South Africa
strongly suggests the possibility of further incursions. Stem rust and
stripe rust, in particular, are extremely damaging diseases and the
description of highly virulent and aggressive Pst and Pgt races in other
wheat regions77,78 emphasises continued vigilance. The introduction of
such races could impact severely on cultivar response with a consequent
increase in production risk and cost. The survival of rust on off-season
wheat crops and ancillary hosts such as wild rye (Secale strictum
subsp. africanum) in the Roggeveld Mountains of the southwestern
Karoo79, requires further attention. Although samples collected from wild
rye revealed Pst, the stem and leaf rust forms were those of cultivated
rye and not bread wheat.79 Wild rye is, however, moderately susceptible
to Pgt and could serve as an inoculum source. The occurrence of both
Pt and Pgt on a summer wheat crop in the eastern Free State in January
2020 (WHP Boshoff, unpublished) is of concern and supports the
expansion of surveys to this period. Scientists should thus continue with
surveillance, studies of pathogen variability, characterisation of cultivars,
genetic analyses, resistance discovery, focused breeding and selection,
and communication of research outcomes to producers. Overarching
activities include international, regional and national collaboration;
capacity building and training; embracing of new technologies;
resistance gene stewardship; and sourcing sustained funding. Figure 4:
Targets of rust resistance genes screened for in the marker-
assisted selection programme at CenGen. Molecular markers for target genes/QTL are obtained from the public
domain and research articles, or from in-house mapping projects by
CenGen and collaborators. These include simple sequence repeat (SSR),
sequence-tagged site (STS), cleaved amplified polymorphic site (CAPS)
and single nucleotide polymorphism (SNP) markers. Since 2013, the
implementation and upgrade of KASPTM SNPLlineTM instruments (LGC,
UK) at CenGen greatly enhanced high-throughput capacity. The number
of data points (calculated as the number of samples x number of markers
tested per sample) that are generated annually continues to increase
(Figure 5) despite a decrease in industry funding. Figure 5:
Number of data points generated since inception of the marker-
assisted selection programme. Breeding and selection Entry
Relative yield (%)a
Genes incorporated through MAS
Cultivar 01
102
Confidentialb
Cultivar 02
97
Confidential
Cultivar 03
94
Confidential
Cultivar 04
100
Confidential
Cultivar 05
106
Confidential
Cultivar 06
106
Confidential
MAS Line 01
99
Fhb1 Qfhs.ndsu-3BS; FHB Qfs.ifa-5A
MAS Line 02
96
Fhb1 Qfhs.ndsu-3BS; FHB Qfs.ifa-5A
MAS Line 03
98
Fhb1 Qfhs.ndsu-3BS; FHB Qfs.ifa-5A
MAS Line 04
99
Fhb1 Qfhs.ndsu-3BS; FHB Qfs.ifa-5A
MAS Line 05
96
Fhb1 Qfhs.ndsu-3BS; FHB Qfs.ifa-5A
MAS Line 06
102
Fhb1 Qfhs.ndsu-3BS; FHB Qfs.ifa-5A
MAS Line 07
95
Sr2/Yr30; Lr34/Yr18/Sr57/Pm38 South African seed companies use different strategies to breed for
rust resistance, dependent on their approach, resources and location. Yet there is a collective focus on pyramiding rust resistance genes, in
particular those that confer durable APR, to uphold the international
drive of gene stewardship. Sensako (Pty) Ltd., a private breeding
company with headquarters in Bethlehem (Free State, South Africa),
follows a strategy in which they combine target genes/QTL in doubled
haploid donor lines. This is followed by a top cross with their elite lines Volume 116| Number 11/12
November/December 2020 Volume 116| Number 11/12
November/December 2020 4 Wheat rusts in South Africa
Page 5 of 8 Wheat rusts in South Africa
Page 5 of 8 The MAS programme commenced in 2011 with the capacity to screen
for 19 genes/QTL, of which 13 were related to rust resistance. These
targets included the popular APR genes Lr34/Yr18/Sr57/Pm38 (Pm is
the notation for powdery mildew resistance genes) and Sr2/Yr30, the
leaf rust resistance gene Lr1973 as well as QTL previously identified for
stripe rust resistance in the cultivars Kariega16 and Cappelle-Desprez17. Since its inception, the programme has grown to include 63 genes/
QTL of which 29 are associated with rust resistance (Figure 4). These
are obtained by breeders through international collaboration with
organisations such as CIMMYT, or are newly identified sources from
ongoing local research projects.62 by-sequencing in wheat were published, creating yet another avenue
for genomics-assisted breeding (Figure 6).76 The challenge remains for
South African breeders and geneticists to follow international trends in
genomics-assisted breeding and sensibly implement selection strategies
for multi-locus traits. MAS, marker-assisted selection; GS, genomic selection; GBS, genotyping by
sequencing Figure 4:
Targets of rust resistance genes screened for in the marker-
assisted selection programme at CenGen. MAS, marker-assisted selection; GS, genomic selection; GBS, genotyping by
sequencing Figure 6:
Timeline of MAS in South Africa (bottom) compared to
international programmes (top). Competing interests We declare that there are no competing interests. Review Article
https://doi.org/10.17159/sajs.2020/7688 References ‘n Oorsig oor die koringsiektesituasie in Suid-Afrika,
met spesiale verwysing na stamroes, en oorwegings vir ‘n nasionale
koringsiekteprogram [An overview of the wheat disease situation in South
Africa, with special reference to stem rust and considerations for a national
wheat disease programme] [PhD thesis]. Stellenbosch: Stellenbosch
University; 1980. Afrikaans. 24. Pretorius ZA, Booysen GJ, Boshoff WHP, Joubert J, Maree GJ, Els J. Additive
manufacturing of devices used for collection and application of cereal rust
urediniospores. Front Plant Sci. 2019;10, Art. #639. https://doi.org/10.3389/
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in South Africa from 1983 to 2008. S Afr J Plant Soil. 2010;27(1):81–96. https://doi.org/10.1080/02571862.2010.10639973 6. De Jager JNW. Authors’ contributions Z.A.P. developed the outline, wrote the Abstract, Background, ‘Host
resistance’ section and Conclusions; R.P. and E.W. wrote the ‘Breeding
and selection’ section including table and figures; C.M.B. provided long-
term cultivar data; B.V. wrote ‘Genetic analysis of Puccinia isolates’;
W.H.P.B. wrote ‘Rust surveillance and phenotypic analysis’. All authors
contributed to editing of the final manuscript. Z.A.P. and W.H.P.B. provided
photographs of rust phenotypes. Notwithstanding the success of the implementation and application of
the MAS programme for single locus traits such as rust resistance,
the status of MAS in South Africa trails behind that of international
programmes, which are exploring an integrated genomics-assisted
breeding approach.74 In 2010, crop geneticists started to investigate
genomic selection in wheat to select for complex, multi-locus traits.75
By 2012, reports of the value of genomic selection using genotyping- Volume 116| Number 11/12
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Page 6 of 8 19. Visser B, Herselman L, Pretorius ZA. Microsatellite characterization of South
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selected South African races of Puccinia graminis f. sp. tritici. Mol Plant Pathol. 2009;10(2):213–222. https://doi.org/10.1111/j.1364-3703.2008.00525.x Review Article
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Page 7 of 8 37. Wessels E, Prins R, Boshoff WHP, Zurn JD, Acevedo M, Pretorius ZA. Mapping
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06-19-0186-R 76. Poland J, Endelman J, Dawson J, Rutkoski J, Wu S, Manes Y, et al. Genomic
selection in wheat breeding using genotyping-by-sequencing. The Plant Genome. 2012;5(3):103–113. https://doi.org/10.3835/plantgenome2012.06.0006 79. Pretorius ZA, Bender CM, Visser B. The rusts of wild rye in South Africa. S Afr J Bot. 2015;96(1):94–98. https://doi.org/10.1016/j.sajb.2014.10.005 79. Pretorius ZA, Bender CM, Visser B. The rusts of wild rye in South Africa. S Afr J Bot. 2015;96(1):94–98. https://doi.org/10.1016/j.sajb.2014.10.005 79. Pretorius ZA, Bender CM, Visser B. The rusts of wild rye in South Africa. S Afr J Bot. 2015;96(1):94–98. https://doi.org/10.1016/j.sajb.2014.10.005 Volume 116| Number 11/12
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Auch diese Annahme ist insofern nieht ganz
ricbtig, weft sie in m~nchen Fallen nicht-zut~ifft, Es.zeigt sich dies namenflieh in dem Verhalten yon Farbstoffen gegen Wolle undSeideT),
wobei die Regel gilt, dat~ bei niedriger Temperatur die Affinitiit f/Jr Seide grSfier a|s f/Jr
Wolle ist, wahrend bei hSherer Temperatur der
Farbstoff haupts~tchlich yon der Wollfaser aufgenommen wird. Abet-au, ch bei Kohle zeigt
sich in ihrer mannigfaltigen ArtWendung zum
Entfiirben yon L6sungen eine deutliche Abh~ingigkeit der Sorption yon der Temperatur.
Endlich sei noch konstatiert, dab Sorption und
LSslichkeit sicher nicht immer ~im umgekehrten
Verh~itnis zueinander stehen" ( L u n d e l i u s
S. 147). Denn- meine Fiirbeve~suche mit den
Azofarbstoffen aus ~-Naphthol und den a Naphthylaminsulfosiiurena) hatten ergeben, dag die
aus 1,4 und 1,5 Naphthylaminsulfos[iuren erzeugten Farbstoffe, welche sowohl im Wasser
wie auch in verdtinnter Essigsiiure leichter 16s.
Itch als ihre lsomeren sind, yon Wolle (aus essigsaurem Bad) in viel grSt~eren Mengen als
diese aufgenommen werden.
5) Siehe diesbezfiglich auch Koll:Zeitsehr. 14,
7) Ioc. r 191i, 479.
8) G. O e o r g i e v i c s u. L. S p r i n g e r , Monatsh.
f. Chem. 1900, 626.
72]73 (1914).
~) loc. cit. 1911, 478'79.
Prag (BOhmen).
Deutsche tedmische Hochschule.
Kolloidchemische Studien iiber die Reimwirkung.
Yorl[iufige
Von
Mitteilung.
F. S e k e r a
Die AuslSsung iibers~ttigter LSsungen, unterkfihlter Schmetzen und dergleichen durch Keimwirkung wurde vielfach in den Rahmen katalytischer Betrachtungen einbezogen. Erst in
letzter Zeit, da man einen, wenn auch vorderhand nur oberflfichlichen Einblick in den Mechanismus der heterogenen Katalyse gewinnen
konnte, zeig~en sich ~in vieler Hinsicht Verschiedenheiten zwischen den beiden Klassen
von Erscheinungen, so dafl sch!iet~lich das
Problem der Keimwirkung als ,,terra incognita"
yore Gesichtsfeld der katalytischen Forschung
abgetrennt wurde. Die Frage, ob es sich hier
um katalytische Erscheinungen handelt oder
nieht, muB wohl so lange often bleiben so
lange der Mechanismus. der Keimwirkung unaufgekl/irt ist.
(W~en).
(~ing~ng~n a,n t. Vet,r,a~r l ~ t . )
Es schien nun ein aussichtsreiches Beginnen
dem Problem der Keimwirkung vom Standpunkt
der Kolloidchemie hither zu treten, um so mehr,
als sich damit gleichzeitig die Gelegenheit erg a b , in die Natur des Orenzgebietes kolloider
und molekularer Dimensionen tiefer einzudringen ~).
I. U e b e r s i i t t i g t e
LSsungon.
Zuwiederholten Malen 2) wurde der Zustand der Ueber............
1) Die folgenden Dar!egungen sind das TeilergebhiS ether noch im Oange befindlichen Arbeit fiber
,,Die M o r p h o l o g i e der N i e d e r s c h l a g s f o r m e n". Die experimentellen Daten werden im l~ahmen
dieser Arbeit verOffentlicht werden; hier beschr~nke
ich reich darauf, meine Anschauungen fiber die Keimwirkun~ zur Diskussiou zu stellen.
t) ~3iehe insbesondere die zahlreichen Arbeiten
P. P. v. Wet m arn's in dieser Zeitschrift.
255
s~ttigung einer L0sung Ms der Uebergang vom
molekular- zum koltoiddlspersen System gekennzeichnet. Schon in konzentrierten L6sungen beginnen die L6sungsnlolekiile zu'Komp|exen zusammenzutreten, die an Or6ge um so mehr
zunehmen, je .hS-her die Lfsungskonzentrationen
sind. Nach den Anschauungen, zu denen uns
die Laue'schen Photogramme geliihrt haben,
diirften auch schon diese em.bryonalen Formen
einer stoffllchen Aggregation an gewisse Oesetzm.~fligkeiten gebunden sr
Das Raumgitter, das
im festen K6rper zu voUkommener Starrheit
entwickelt ist, wird .man sich auch bet den
Molek/ilkomplexen einer konzentrierten LOsung
vorstellen kOnnen, nur mit dem Unterschied,
da~ die Orientierung noch nicht voUk0mmen
is[, und auch die Starrheit noch nicht [enen
Grad erreicht hat, wie sic sich im Raumgitter
des festen Teilehens erketinen liiBt. Der Austausch der L/$sungsmolekfile an der enorm entwickelten spezifischeri Oberfliiche dieser Molekfilkomplexe~ sowie das auf dieselbe Oberfl~iche
einwirkende Bombardement der LSsungsmolekfile lassen diese Vorstellungen berechtigt erscheinen. Je gr~Ber die Mo!ekfilkomplexe werden
(d. h. ie konzentrierter die LOsung.ist), urn so
widerstandsfiihiger wird ihre Oberfliiche den
Einwirkungen yon au6en, um so ungestSrter
und starter wird das"Raumgitter, his end[ich,
sobald kolloide Dimensionen erreicht sird, die.
Raumgitterstrukturexperimentel.I eJ-kennbar wird.
An' der Hand dieser Vorstellungen dfirfte die
Kontinuit~t zwischen Molekii! und Kotloid am
klarsten sein.
Die eben betrachtete TeilchenvergrOgerung
erleidet nun, wie es nach den bisher fiblichen
Anschauungen-~tiber die LOslichkeit der Stoffe
den Anschein hatte, irgendeine unvermittelte
St~rung. Sobald der ,LOslichkeitsgrad ~ des vorliegenden Stoffes erreicht ist, fiihrt iede weitere
KonzentrationserhtShungzur Bildun g ether zweiten
Phase, die wohl auch den Weg der Dispersit~,tsverminderung ei.nschliigt, aber in ganz anderer
Weise, als er bis zumL6stichkeitspunkt geffihrt
hat. Inwieweit und w.arum dieser Knick in der
Dispersitiitsverminderung eintritt, soil sptiter erOrtert werden. Hier interessieren wir uns besonders daffir, da6 es unter Wahrung besonderer
VorsichtsmaBregeln gelingt fiber diesen kritischen
Punkt hinwegzukommen und die Verringerung
des Dispersitiitsgrades weiterzutreiben, ohne die
Bildung ether zweitenPhase zu beobachten. Man
erreicht dann den Zustand der ,Uebers.;ittigung"
und lernt diesen Zustand als einen unstabilen
Zustand kenrien Tats/ichlich geniigen auch die
bescheidensten Unregelm[it3igkeiten an den GefiiBwiinden, Staubteilchen usw, um das Zustandekommen einer Uebersiittigung zu verhindern.
Besonderes Interesse aber land das Phiinomen
der Ausl6sung einer Uebersiittlgung dutch die
Berfihrung mit einem festen Keim des gel6steu
Stoffes. Unwiigbare a) Mengen desselben genfigen,
um eine Uebers~tiigung aufzuheben und eine dem
L6slichkeitsgrad entsprechende Trennung in gesattigte L~sung und Bodenk0rper herbeizuffihren.
Fiir das Verst~indnis dieser Erscheinung fehlte
bisher iede Orund|age, um so mehr als sich die
Vorg~nge einer quantitativen Untersuchung kaum
zugiinglich erwiesen. Der diskrete Zusammenhang zwischen Keim und dem durch ihn a~s'gel6sten Vorgang brachte es mit sich, eine
katalytische Erscheinung zu vermuten. Erst
glaubte man, die Uebers~ttigung, da" sie sich
als eine verz0gerte Bildung der heterogenen
Phase zeigt, auf einen Widerstand zurfickffihren
zu k6nnen, der sich denkristallbauenden Kriiften
entgegenstellt. Dieser an und ffir sieh recht
subiektive Erkl~irungsversuch versagt abet vo|lkommen bet den fibers~ttigten L0sungen yon
Gasen, bet denen an einen derartigen Widerstand nicht gedacht werden kann.
Welchen Weg hat nun ein kolloidchemischer
Erkl~irungsversuch zu gehen? ~ Zwei Falle sind
zu berficksichtigen: Die Aufhebung ether Uebersiittigung dutch einen kfinstliChen Keim und die
langsame, yon selbst verlaufende Entmischung,
die den fibersattigten L6sungen die Eigenschaft
der Unbest.~indigkeit aufpragt. S c h o n eingangs
wurde eine fibersattigte L~sung als ein.sehr
hochdisperses System charakterisiert, desseu disperse Phase aus den blolekfilkomplexen des ge16sten Stoffes besteht. In diese L0sung wird
nun ein KristaU des festen Stoffes eingetragen;
es besteht dann neben einei aut~erst hochdispersen Phase eine grobdisperse desselben
Stoffes. Ein solcher Unterschied des Dispersitatsgrades bedingt naturgernaB entsprechende
Unterschiede in den physikalischen Eigenschaften
der beiden Phasen. Beim Studium der iibersatJigten LOsungen war es wohl das naheliegendste, die Dispersit|itsfunkti0n der L 6 s I i c h k ei t zu berficksichtigen. Bekanntlich ist
der LSslichkeitsdruck fiber e!ner wenig gekriimmten Oberflache geringer als fiber ether
stark gekrfimmten ; m. a. W. kieine Teilchen~sind
18slicher als grot~e. Diese Abhangigkeit der LOslichkeit yore Dispersitatsgrad ist im Gebiete grober
Dispersitlit kaum merkbar, wird bet fortschreitend
a) Siehe Nachtrag.
256
abnehme/ader kolloider TeilchengrSfle immer deutlieher, urn im Grenzgebiet kolloider und molekularer Dimensionen eine Hauptrolle bei allen
Vorgiingen, die sich in diesem DimensiOns
bereich abspielen, zu ttbernehmen, tn der beigefiigten Skizze "ist diese Funktion schematisch
dargestellt. Die L0slichkeitskurve steigt im
Bereiche grober Dispersitat nut allm~,hl.ich an
u.nd wird:im Gebiete der kolloiden..Dimensionen
zunehmend steiler, urn schliefilich asymptotisch
zu enden. Uebertragen wir nun dieses Bild
auf unsere iibersiittigte L6sung. Das Verhii/tnis
der Molekiilkomplexe zurn etngetragenen Keim
sei durch die Punkte A und B d~r Kurve .fes'cgel~egt, wobei die Ordinaten dieser Punkte die
Menge yon L S s u n g s m o l e k i i l e n
vorstelien,
mit" denen der Keim einerseits und die Molekiitkomplexe andrerseits im Gleichgewicht stehen.
B
s
Fig. 1
Es ergibt sich nun als selbstverstandlieh, dab
zwischen den beiden Phasen mit verschiedener
LSsltchkeit, da sic sich im selben LSsungsmittel
befinden, ein Ausgleich erfolgen wird. So langr
Teflchen vorhanden sind, die mit mehr L 6 s u n g sm o l e k f i l e n im G|eichgewicht stehen als tier
Keim, wird letzterer dieses Plus an MOleklilen
in sein Raumgitter aufnehmen und damit so
lange wachsen, bis die kleinen Teilehen (die
Molekiilkomplexe) aufget6st sind. Die Uebersiittigung ist aufgehohen, sobald yon den Mcdekiiien, die friiher mit derr Molekiilkomplexen
im Gleichge~vicht gestanden hatten, nur so
vieie iibrig sind, als der LOslichkeit des Keimes
entspricht 4).
4) !m-Prinzip ist diese Anschauung durehaas nicht
neu. Der Vorgang des Waehsens grol~ererTeilchen auf
Kosten der kleineren ist als O s t w a I d'scher Reiftmgsprozel3 vielfach beschrieben worden (Z- B. LfappoCramer u:R. E. t.iesegang, P.P.v.Weimarn usw.).
Die vorliegende Arbeit gelangte blo6 zu dem Befund.
dal~ dasselbe Oesetz auch fiir Molektilkomplexe Oeltung hat.
Vielfach ist nun eine beabsichtigte Aufhebung eiher UebersAtt/gung~durch einen kiinstlichen Keim gar nicht nStig. Es geniigen ja
die beschetdensten Konzentrationsunterschiede
innerhalb des L6sungsvolumens, um einen,
wenn auch sehr geringen Unterschied des
Assoziationsgrades tiervorzurufen. Da ist die
Begrenzung des L6sungsvolumens, insbesondere
d'er Meniskusrand und unvermeidliche Unebenheiten der GefliBw~nde der Sitz yon Oberfl~ichenspannungen, die dutch Adsorption lokale.
Konzentrationsverschiebungen zur Folge haben.
Es bestehen dann gr6flere Molektilkomplexe
veben kleineren, yon denen die gr0ileren die
Roile des kfinstlichen Keimes you vorhin tragen.
Der iangsame Verlauf tines solchen Ausl6sungsvorglinges ist nun durchaus begreiflich, wenn
man bedenkt, daft der L6slichkeitsunterschied
zwischen den verschieden groflen Motekiilkomplexen ein sehr geringer ist und es sehr
lange Zeit braucht, bis der Motekfilaustausch
den L6slichkeitsausgleich besorgt hat. Es liegt
in dieser Ansch~uung ein scheinbarer Widerspruch : beim L6slichkeitsausgleich zwischen zwei
verschieden groflen Molektilkomplexen beteiligen
sich. ungleich mehr L6sungsmolekfile als im
FaUe eines grobdispersen Keimes; .es miiflte
sich also der L~s]ichkeitsausgleich SchneUer
votlziehen als i:m letzteren Falle. DieserWiderspruch ~llt abet s0fort weg, wenn mart sich
nur vergegenw~irtigt, daB im Verlaufe des Aus.16sungsvorganges der gr6flere Molektilkomptex
in der Dispersit~ttsgradserie fortlauferld dem
grobdispersen Ende entgegenstrebt und auf
diesem ganzen Weg als Keim wirkt. Einem
kiinstlichen,-yon Haus aus grobdispersen Keim
bleibt dagegen dieser Weg erspart.
Schon aus diesen~ Ueberlegungen geht hervor, dab sich das gesamte Problem der Keimwtrkung einzig mit z w e i L 6 s u n g s g l e i c h gewichten
zu befassen hat, die durch das
Schema
"
Keim ~ _ C1 L6sungsmolekfile/~.
Molekiiikomplex ~
Ca
,
~t':~::>tSl
dargestellt sind. So lange C~ > C1 ist, werden
die Molekiilkomplexe der iibers~itigten L6sung
abgebaut, wfihrend das Plus an LOsungsmolekftlen (C_~-C1) yore Keim aufgenommen wird.
Welche Bedeutung hat nun der , Ue b e rs iittigun-gsgrad?"
dedenfaIls handelt es
slch um einen r e l a t i v e n Begriff. Aus dem
vorher'ehenden ist der Uebers~ittigungsgrad
e.mer vorliegenden L6sung einem grobdispersen
Keim teegen/iber gr6ger als einem hochdispersen Keim gegeniiber, da bei gleichem C2
257
this C1 im letzteren Fall gr6fier ist. Jecler Angabe des Uebers~ittigungsgrades sollte also.die
Angabe des Dispersit~tsgrades des Keimes beigeschlossen sein. lm allgemeinen verzichtet
man wohl auf diese notwendige Erg,~inzung
deshalb, well d~e Uebe~'~ttigung in bezug auf
die grobdisperse Phase gemein! wird:
Weitergehend kommen wit zu dergrunds~ifzlichen Erkenntnis, daft wir eine/~bers~ittigte
L6sung als eine ges~Ittigte LOsung anzusehen
haben, deren ,feste Phase'. die Molekfilkomplexe sind, und die eben mit einer viel gr6fieren
Menge yon L~sungsmo]ekiilen im Qleichgewicht
stehen, als eine grobdisperse Phase es rut. Wenn
wir also eine ilberS/ittigte L~)sung als ein unstabiles System ansehen, so ist dies unrichtig.
K6nnte man die unvermeidliehen Ursachen einet
lokalen Konzentrationsverschiedenheit innerhalb
des LOsungsvolumens ausschalten, so h~tte man
eine ebenso stabile L6sung vor sich, wie es
jede ges~ttigte L6sung ist. Zur ~bersiittigten
L6sung wird eine solche hochkonzentrierte L~5sung erst durch die Existenz grober disperser
und daher weniger 16slicher Teilchen.
.Was nun die Versuche betrifft, die zu den
hier entvcickelten Anschauungen geffihrt haben
und die ausffihrlich an anderer Stel~e ver6ffentlicht werden sol[en, so zeigte sich mit voUster
Klarheit die Bedeutung der L6slichkeit des ge16sten Stoffes, wie sie in der Theorie zum Ausdrttck k0mmt. Bei leicht 16slichen Stoffen ist
die Keimwirkung spontan, da sich eine groi~e
Menge yon L6sungsmolekfile~ am LOslichkeitsausgleich zwischen Keim und den Molekfilkomplexen beteiligt.
Bei schwer 16slichen
Stoffen dagegen ist die Keimwirkung sehr
tr/ige, so daft man Zeit genug hat, um den
Vorgang zeitlich zu verfolgen. Mit Hilfe einer
geeigneten Apparatur ist es auch gelungen, die
Bildung con Niederschlggen und die damit verquickten Fragen der Keimwlrkung zu studieren.
von den Uebersilttigungserscbeintmgcgt, die
dem Experimentator ja auf Schritt und Tritt
begegnen, sei noch der sogenann~e , S i e d e v e r z u g ". erwiihnt, dessen unangenehme Folgen
schon oft zum Nachdenken fiber die Ursachen
angeregt haben mag, ohne daft eine Erklarung
gefund,en werden, konnte. Aut/ empirisehem
Wege hat man den Siedeverzag dadurch umgehen k6nnen, indem man entweder.andauernd
Luftblasen in die erhitzte Fliissigkeit eintreten
I.~fit, oder mit Hilfe eines in das GerM5 ein 2
geschmolzenen Ptatindrahts eine lokal konzentrierte W~irmezufuhr er~eicht. Die Wirk'ungsweise dieser beiden Kunstgriffe wird nun sofort
yerst~indlich, wenn man sich vorstellt, daft das
beim Verdampfen der.Plfissigkeit gebildete Gas
primlir in derselben gelt~st wird und auch da
Uebersitttigungen im fiblichen Sinne auftreten
k6nnen: Diese Uebersattigungen werden dann
durch die als Keime wirksamen eingeleiteten
Gasblasen bzw. dutch den infolge der grol~en
Ueberhitzung mit tier Dampfhfille umkteideten
Platindraht aufgehoben.
lI. U n t e r k f i h l t e S c h m e l z e n . Dieselben
Gestchtspunkte, die fiir die Erk|tirung der Aus16sung fibersattigter LOsungen mat~gebend warerr,
geiten auch f/it u n t - e r k f i h l t e S c h m e l z e n .
Da wie dort handelt es sich um hochdisperse
Systeme mit Molekillkomplexen als disperse
Phase. Die Rolle, die vorhin die LSslichkeit
gespielt hat, wird im jetztgen Falle yon. der
S c h m elz b a rkeit fibernommen. Die Abh~aagigkeit des Schmelzpunktes yore Dispersitiitsgrad
ergibt eine. der L~Sslichkeitsfunktion voltkommen
analoge Kurve, so dat~ es kaum schwer fallen
wird, sich den Vorgang vorzustellen, der sich
zwischen Keim und den Molekfilkomplexen
einer unterklihlten Schmelze abspielt.
I[I. Die F r a g e d e r K a t a l y s e . Auf Gru~d
der angestellten Ueberlegungen fiber den Mechanismus der Keimwirkung kann nun die Frage
beantwortet werden, die anfangs often bleiben
mutate: Handelt es sich urn katalytisehe Vorgange oder nicht?. Nach dem, was wir gegenwitrtig v o n d e r Katalyse wissen, ist sie vor
allem dadurch gekennzeiehnet, dab der Katatysator wohl eine bestehende Reaktion zeitlich
zu beeinflussen vermag, abet niemals imstande
ist, eine Reakfion hervorzurufen. Welter betitigt
sich eirrKatalysator e ~ e r g e t i s e h an der Reaktton, indem er entweder ein Zwischenprodukt
aufbaut, das durch seinen Zerfall schneller zum
Endprodukt fflhrt als die nicht katalysierte Reaktion (homogene Katalyse) oder unter Zuhilfenahme der Oherflachenenergien auf ahnliche Weise wirkt, wie es bei der heterogenen
Katalyse der Fall ist.
Rile diese Kennzeiehen fehlen der Keimwirkung. Wit sind 2u der Erkenntnis gelangt;
daft fiber~i"ttigte L6sungen nichts anderes sind
als gesiittigte LSsungen, deren ,Bodenk6rper"
die Molekfilkomplexe sind und die sieher keine
Tendenz zeigen wt'irden, ihren Zustand zu verAndern, wean nicht unvermeidliche Keimwirkungen grOt~erer Molekfilkomplexe mitspielen
wf|rden. Es ist also gar ketn Vorgang.da, der
katalysiert werden k6nnte. Der Keim wirkt
welter nicht energetisch, wie die vorhin be-
258
zeichneten F~ille, sondern sehafft dutch sein~
Anwesenhait sozusagen ein Reservoir ffir jenes
Plus an "Liivungsmolekiilen, mit denen er ins
seiner geringeren LOslichkeit nicht im
Gleichgewicbt zu stehen yermag. Es handelt sich
einzig um das Zustreben auf ein Li~sungsgleich-gewieht mit einer hinzugekomrr~nen Phase. - Dies sind die Hauptgrfin6e, .die maggebend erscheinenj der Keimwirkung den vermeintlichen
Katalysencharakter abzusprechen.
Wien. On Dezember 1920.
Tedanische Hochsehule.
Nachtrag.
Dutch etne briefliche Aussprache mit Herrn
Prof. Wo. O s t w a l d wurde ieh angeregt, auf
folgendes niiher einzugehen. W i I h e I m O s tw a I d 5) konnte experimentell den Beweis ftihren,
dag zur Aufltebung einer Ueberstittigung, wenn
auch sehr .geringe, so doch gewisse Mengen
Keinisubstanz n6tig sind. lch versuchte naturgemag im Rahmen meiner Arbeiten dieses Ergebnis 2u .reprodazieren uhd kam zu dem Resuliat, dab diese Bedingung eines Keimminimums
wohl bei L6sungen leicht 15slicher Stoffe und
auch dann nur im ,stark fiberstittigten"~
experimentell festgesteltt werd~n kann, nicht aber
bei/ibersiittigten LSsungen schwer 16slicher Stoffe
oder in allen Fallen bei geringem Uebers~ittigungsgrad. Wenn e8 mix aueh nfcht gelungen ist,
diesem Umstand quantitafiv n~iher zu kommen;
so m~chte ieh iolgende Deutung in Betracht
ziehen. Die Ltistichkeit eines Stoffes ist nicht
5) Wilhelm Ostwatd, Lehrbuch der all~,emeinen
Chemie.
nur vom Dispersittitsgrad alleiu abhtlngig, sondern auch yon der Form des Ke~mteilchen~.
Es kommt sehr viel darauf an, ob ein unbesch~idigter Kristall oder Bruchsgicke eines Kristalles als Keimewirken. Unter derVoraussetzung,
dab in beiden F~iilen gleiche Gewichtsmengen
Keimsubstanz zur Anwendung gelangen, ist die
L6slichkeit der KristaUbruchstiicke wesentlich
gr6ger als die des unbeschiidigten Kristalles,
da die ersteren vor aUem Trehr und auch
schiiffere Kanten besitzen. Und die Kanten
sind doch der Sitz der hSheren L0slichkeit.
Wi. O s t w a I d hat nun zur Feststellung des
Keimminimums offensichtlich Mahiprodukte, also
Kristalltr/iramer verwendet. Da ka n n es nun
eintreten, daft beim Eintragen eines solchen
Bruchstfickes in eine stark fibersiittigte L6sung
diese gegeniiber dem Keim gar nicht fibers~ttigt ist. Es steht vielmehr der Keim infolge
seiner grogen L6slichkeit mit mehr L~Ssungsmolekiilen im Oleichgewicht als die MolekQlkomplexe. Das Verhiiltnis zwischen Keim und
Molekfilkomplexen ist dana gerade umgekehrt
als es in der Regel der Fall ist. Die Folge:
die Molekiilkomplexe zehren den Keim auf und
die hohe LOsungskonzentration bleibt bestehen.
In ~weniger i~bersiit~tigten" L6sungen wird man
dieses abfiormale Verh~hnis zwisehen Keim und
Molekfilkomplexen nichl beobachten k6nnen,
well die Motekfilkomplexe immer kleiner sein
Werden als ein w~igbarer Keim. In iibers~ittigten
L6sungen schwer 16slicher Stoffe ist die Diffusionsbewegung so tr~ige, daft derartige Versuche
unmSglich sind..
Wien, im Januar 1921.
Technische Hodzschule.
(Jeber die Lichtempfindlkhkeit des :Rrsentrisuifidsols,
Von H. F r e u n d l i c h a n d A. N a t h a n s o h n .
Dem einen yon uns (N.)schien es im Hinblick auf biologische Pragen wichtig ~zu sein,
photoehemische Vorg~inge in kollolden Gebilden
zu untersuchen..Wir gingen deshalb yon der
Beobachtung des anderen (FrX aus, dab das
AS2Sa-Sol offenbar stark lichtempfindlich ist;
ihm war es schon vor Jahren aufgefalleh, dag
sich vielfach ein A% Sa-Sol, alas sich im Dankeln
lange klar gehalten hatte, am Tageslicht trtibtel).
Es wutde deshalb eine Reihe yon Versuchen
derart angestellt, dag das Sol in Reagenz1) $iehe auch H. F r e u n d l i c h u. H Schuch,t,
Zelt~r f. ph~ik. Chem, 80, 5,65 (1912).
(lgingegaugenam 9. Mlirz |921.)
gltisern durch eine 3000-HK-Nitralampe, i n
30 .cm Abstand, unter Dazwischenschaltung
eines Wassertroges beleuchtet wurde. S.chon
nach etwa zehn Minuten zeigte sich eine Triibung des vorher' im durchfallenden Lichte ganz
klaren S01s, und bei ltingerem, stundenlangem
Beleuchten schied sich aus dem Sol Sehwefel
ats :fein'es, weiBes Pulver ab.
D~e zu den Versuchen benutzten Sole
wurden f01gendermaflen bereitet : 40 -- 100 ccm
eil~er kaltgestittigten L6sung yon Arsenkioxyd
w e r d e n ' a u / 2 0 0 ccm verdiinnt and mit 100 ccm
einer H~S ' Lssung, die 1 ccm ges~ittigtenSchwefel-
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https://zenodo.org/records/5180173/files/source.pdf
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English
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to the classification and identification of Weinmannia (Cunoniaceae) in Madagascar and the Comoro Islands
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Zenodo (CERN European Organization for Nuclear Research)
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MOTS CLÉS
Weinmannia,
Cunoniaceae,
cladistique,
classification,
clés taxonomiques,
inflorescence,
Madagascar,
Comores. KEY WORDS
Weinmannia,
Cunoniaceae,
cladistics,
classification,
taxonomic keys,
inflorescence,
Madagascar,
Comoro Islands. RÉSUMÉ Application d’une analyse cladistique à la classification et l’identification des
Weinmannia (Cunoniaceae) à Madagascar et aux Comores. g
Les espèces malgaches de Weinmannia sont actuellement difficiles à identifier
avec les publications disponibles en raison à la fois de la pauvreté des caractères
qualificatifs utilisés dans les clés d’identification et d’un nombre élevé d’espèces
non décrites. Cependant, des caractères morphologiques nouvellement recon-
nus, en particulier ceux de l’inflorescence, peuvent êre utilisés pour les dia-
gnoses des sections et des groupes d’espèces malgaches. Ces caractères,
employés précédemment pour une analyse cladistique du genre Weinmannia
(BRADFORD 1998) sont appliqués ici dans les diagnoses et une clé de détermi-
nation des sections et groupes d’espèces de Weinmannia à Madagascar. ADANSONIA, sér. 3 • 2001 • 23 (2) : 237-246
© Publications Scientifiques du Muséum national d’Histoire naturelle, Paris. The application of a cladistic analysis
to the classification and identification
of Weinmannia (Cunoniaceae)
in Madagascar and the Comoro Islands Jason C. BRADFORD
Missouri Botanical Garden, P.O. Box 299, St. Louis,
Missouri 63166-0299, U.S.A. bradford@mobot.org ABSTRACT Weinmannia species from Madagascar are currently difficult to identify with
available published accounts due to both a paucity of qualitative characters
used in identification keys, and a large number of undescribed species. However, newly recognized morphological characters, especially of the inflo-
rescence, can be used to diagnose Malagasy sect. and species-groups. These
characters, used previously for a comprehensive cladistic analysis of the genus
Weinmannia (BRADFORD 1998), are applied here in diagnoses and a key to
the resulting sections and species-groups of Weinmannia in Madagascar. ADANSONIA, sér. 3 • 2001 • 23 (2) : 237-246
© P bli
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S i
tifi
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l INTRODUCTION BARNES 2001), is represented in Madagascar
solely by its largest genus, Weinmannia L., with
c. 150 species worldwide. Most Weinmannia
species are trees and shrubs of tropical montane BARNES 2001), is represented in Madagascar
solely by its largest genus, Weinmannia L., with
c. 150 species worldwide. Most Weinmannia
species are trees and shrubs of tropical montane Cunoniaceae, a southern hemisphere family
with 26 genera and c. 300 species (BRADFORD & 237 Bradford J.C. forests, although tropical lowland species occur in
Madagascar, and temperate species in Chile,
Argentina, Australia, New Zealand and South
Africa. Cunonia L. and Platylophus D. Don, from
South Africa, are the only African members of the
family. As exemplified by the number of species found
during field work at Marojejy (BRADFORD &
MILLER 2001), Madagascar is one of the most
species-dense areas in the world for Weinmannia,
with levels of sympatry similar to that found in
Andean forests. Unlike other parts of the world,
however, the species richness of Malagasy
Weinmannia is accompanied by a diversity of
morphological characters that enable relatively
rapid identification of species. y
Cunoniaceae have been the subject of recent
phylogenetic and revisionary investigations, with
most attention given thus far to Weinmannia
(BRADFORD 1998; HOPKINS 1998a, 1998b,
1998c; HOPKINS & FLORENCE 1998). Based on
cladistic studies (discussed below), the infra-
generic classification of BERNARDI (1961, 1964)
and the placement of some species has been
slightly modified to make sections mono-
phyletic. BRADFORD (1998) recognized five sec-
tions of Weinmannia that circumscribe species
from the Americas plus two species from the
Mascarenes (sect. Weinmannia), Malesia and
Melanesia (sect. Fasciculata Bernardi), the South
Pacific (sect. Leiospermum D. Don), and
Madagascar and the Comores (sect. Spicata
Bernardi and Inspersa Bernardi; see also HOPKINS
1998a). Taxonomic changes affect a few
Malagasy species. p
p
Study of available herbarium specimens and
field work indicate that 35-40 Weinmannia
species occur in Madagascar, 15-20 more than
were known to BERNARDI (1965, 1969). Given
the large number of undescribed species and the
limited array of qualitative characters used by
BERNARDI in his keys, it is difficult to identify
Weinmannia species. INTRODUCTION The present paper addresses
this problem by: (1) describing and illustrating
previously overlooked qualitative character varia-
tion among Malagasy Weinmannia, (2) present-
ing a synoptic key to the sections and
species-groups of Weinmannia in Madagascar and
the Comores, (3) providing diagnostic features
for each section and species-group, and (4) listing
all presently described species in each species-
group. Because a number of Weinmannia species
remain to be described from Madagascar, it
would be premature to write a key to the species
level. Instead, the key given below aids species-
level identification by reducing the number of
species with which one must compare an uniden-
tified specimen. This synoptic key has proven
useful as the basis for a species-level key of
Weinmannia from the Marojejy massif
(BRADFORD & MILLER 2001), and could be
applied to other regions and eventually the entire
genus in Madagascar. Malagasy Weinmannia species were last treated
by BERNARDI (1964, 1965, 1969). He estab-
lished two endemic sections Spicata and Inspersa,
and placed a few species in the more wide-
spread sections Weinmannia and Leiospermum
(sect. Leiospermum has priority over BERNARDI’s
equivalent sect. Racemosa, see HOPKINS 1998a). However, BERNARDI’s assignment of some
Malagasy-Comoral species to sections normally
found outside of Madagascar has been proven
incorrect. Weinmannia baehniana Bernardi and
W. comorensis Tul., which BERNARDI placed in
sect. Leiospermum, belong instead in sect. Spicata, and the species he included in sect. Weinmannia, W. rutenbergii Engl. and W. venusta
Bernardi, belong in sect. Inspersa (see BRADFORD
1998 and below). Both a morphological cladistic
analysis of Weinmannia species-groups and sec-
tions (BRADFORD 1998) and molecular system-
atic work on the genus (BRADFORD 2000;
BRADFORD & BARNES 2001) support the mono-
phyly of sections Spicata and Inspersa as now cir-
cumscribed and provide evidence that they are
sister taxa. g
g
The key and diagnoses are largely based on a
recent morphological cladistic study (BRADFORD
1998) that found several characters not discussed
by BERNARDI (1964, 1965). In Madagascar, sev-
eral groups of species can be recognized that share
unique combinations of these qualitative charac-
ters. The key and diagnoses for the Malagasy
species-groups utilize inflorescence terminology
that may not be familiar. For this reason, figures,
illustrations and a discussion of important char-
acters precede the key. ADANSONIA, sér. 3 • 2001 • 23 (2) 238 Cladistic analysis of Weinmannia (Cunoniaceae) Fig. 1. INTRODUCTION — Cladogram of Weinmannia species-groups (with representative species) based on morphology (from BRADFORD 1998). The
main characters used in the key are plotted on the tree. Fig. 1. — Cladogram of Weinmannia species-groups (with representative species) based on morphology (from BRADFORD 1998). The
main characters used in the key are plotted on the tree. m of Weinmannia species-groups (with representative species) based on morphology (from BRADFORD 1998). Th
sed in the key are plotted on the tree. ADANSONIA, sér. 3 • 2001 • 23 (2) FROM CLADOGRAMS TO KEYS monophyletic. At present, not enough is known
about the phyletic relationships of Weinmannia
species-groups A-G to give them formal taxo-
nomic names and rank, which would violate the
principles of phylogenetic systematics and proba-
bly lead to taxonomic instability. Nevertheless, a
real need exists to communicate group informa-
tion on this complex genus, which can be done
by informally recognizing species-groups A-G for
the purpose of discussion. Such informal tax-
onomies have a long history (e.g. BENTHAM &
HOOKER 1862-1883) and are commonly used in
phylogenetic systematics when cladistic resolu-
tion is lacking or clades are poorly supported (e.g. Angiosperm Phylogeny Group 1998). By
acknowledging uncertainty, informal classifica-
tions may actually communicate information
more effectively than do some rigid systems that
require all individuals to be part of a formal taxon
at each hierarchical rank. Prior to conducting a cladistic analysis of
Weinmannia and the related genus Cunonia,
specimens representing all known species of these
genera were examined to find unique combina-
tions of qualitative morphological characters
among species (see vouchers in BRADFORD 1998). This process led to the recognition of species-
groups, each representing from one to many
species, that served as a terminal units in the
cladistic analysis. Seven species-groups were rec-
ognized in Madagascar and the Comores, five of
which have apparent autapomorphies, whereas
two lack clearly apomorphic features (see charac-
ter states in Fig. 1). Species-groups B, C, D, E,
and G (see below) may therefore be mono-
phyletic, whereas no characters have been found
that support the monophyly of species-groups A
and F. Classifications communicate grouping infor-
mation, but phylogenetic systematics also
requires that formally recognized groups be Cladograms can be applied towards taxonomic
identification by including in a standard dichoto-
mous key the qualitative characters of the data ADANSONIA, sér. 3 • 2001 • 23 (2) 239 matrix as plotted on a dichotomously branching
tree. The key presented below is organized using
character states as mapped on the cladogram
from more general characters to more specific
ones. In other words, synapomorphies of the
largest clades are used in the first order couplet,
followed by more exclusive synapomorphies in
second order couplets, and terminating with apo-
morphic or diagnostic characters for a species-
group (Fig. 1). often organized into compound structures borne
on a peduncle, and these are called Inflorescence
Modules (IMs). FROM CLADOGRAMS TO KEYS Among sections or species-
groups, IMs may have distinct patterns of organi-
zation along the main stem. The Total
Inflorescence (TI) is formed by the architectural
arrangement of the IMs and Racemes (Fig. 2). Figure 2 does not show the full range of TI varia-
tion among Malagasy species, but is sufficient to
diagnose species-groups. It may not always be practical to apply the
characters used in a cladistic analysis to taxo-
nomic keys if, for example, most of them are
anatomical and therefore difficult to use with
herbarium specimens or on material in the field. The case of Weinmannia provides a good exam-
ple. The first couplets use inflorescence and pedi-
cel characters that can readily be seen. Some
couplets also emphasize structural features of the
flower-bearing axis that can be used whether the
material is in bud, flower or fruit. By contrast, the
key in BERNARDI (1965) emphasizes the floral
nectary, the pubescence of the ovary, and the dis-
tribution of trichomes on seeds. Most of the species-groups in Madagascar (A,
B, F, G; see below) have similar architectural fea-
tures, in that IMs consist of a single metamer (i.e. an internode plus a node and associated organs)
with an opposite pair of Racemes and a terminal
vegetative bud. The number of IMs and their
position may vary somewhat among species. For
example, the TI may be reduced to a terminal IM
or a pair of lateral IMs (Fig. 2). Species-groups C and D can be recognized by a
terminal Raceme in the IM, although they are
variable and may have the more general IM form
(i.e. that found in A, B, F, G). Even when the
IMs are of the general form, members of groups
C and D can often be recognized by a prolific TI,
in which IMs occur at several subdistal nodes
along the main stem. Prolific TIs are especially
common in W. rutenbergii, which is indicated by
the broken lines and arrow in Fig. 2. Species in
group D have a branched IM, whereas branching
is rare in group C. In both groups C and D, the
TI is basitonic (i.e. IMs are larger at basal nodes
and smaller at terminal nodes). This paper provides detailed illustrations and
discussions of important features in Malagasy
species of Weinmannia. FROM CLADOGRAMS TO KEYS BRADFORD (1998) may
be referred to for a more general discussion of
characters in the genus and cladistic methods
used to study them. Figure 1 shows the inferred
plesiomorphic and derived conditions for several
qualitative traits that differ among Malagasy
species-groups. Figures 2-4 illustrate and com-
pare these characters (except for seed indumen-
tum), which are discussed below. Group E is easily recognized by the absence of
IMs. Racemes are borne from leaf axils along the
main stem. Architecture The two sections of Weinmannia in Mada-
gascar can be distinguished by whether the flow-
ers are subtended by a pedicel (sect. Inspersa) or
are sessile along the Raceme (sect. Spicata). BERNARDI (1964, 1965) instead emphasized the
form of the floral nectary to circumscribe sec-
tions, which is why he placed some Malagasy
species in sections otherwise from the Malesian-
Pacific region. However, nectary form is homo- Weinmannia inflorescences can be described at
three architectural levels: the Total Inflorescence,
the Inflorescence Module, and the Raceme
(Fig. 2) (BRADFORD 1998). The term Raceme
(with capital “R”) is used generally to describe the
unbranched, ultimate flower-bearing axes in
Weinmannia, which have also been called pseudo-
racemes or spikes (BERNARDI 1964). Racemes are ADANSONIA, sér. 3 • 2001 • 23 (2) 240 Cladistic analysis of Weinmannia (Cunoniaceae) Cladistic analysis of Weinmannia (Cunoniaceae)
Fig. 2. — Inflorescence architecture among Weinmannia species-groups in Madagascar. See main text for the discussion of
features and the list of species in each group. Fig. 2. — Inflorescence architecture among Weinmannia species-groups in Madagascar. See main text for the discussion of
features and the list of species in each group. plasious and varies within Malagasy sections
(BRADFORD 1998), although it can be useful to
diagnose Malagasy species-groups. the axil of a bract dissociate during elongation of
the flower-bearing axis, the relationship between
a flower and its bract can usually be traced by a
ridge or differently-colored linear zone between
them. Flowers are often clustered in groups of
four, and appear to develop from decussate pairs
of buds (see Fig. 4, Group E). When floral
groups dissociate during axis elongation, the pair
of flowers perpendicular to the floral bract is
spread apart more so than the pair of flowers
borne in the same plane as the bract (see Fig. 4,
Group F). The organization of flowers along the Raceme
varies in two important ways: (1) whether flow-
ers are initiated solitarily or in a group, and (2)
whether flowers remain near their point of initi-
ation or dissociate from it. Characters of the
Raceme are often easiest to see when flowers are
in bud, or when mature flowers or fruits are
removed to view clearly their positional relation-
ships (Figs. 3, 4). Even when flowers initiated in ADANSONIA, sér. 3 • 2001 • 23 (2) 241 Bradford J.C. Fig. 3. Architecture — The relationship between flowers and floral bracts as illustrated for Weinmannia sect. Inspersa species-groups:
Group B: Weinmannia lowryana with a single flower dissociating from a bract (Humbert 23057, MO 05006602); Group C:
Weinmannia rutenbergii for portion shown in bud (Bradford 699, MO 05079575), and for portion in fruit, racemes often in threes,
flowers mostly solitary and in the axil of a bract [shown in bud to left] or flowers grouped at the base of the medial raceme [shown in
fruit on right] (Bradford 701, MO 05079557); Group D: Weinmannia venusta, with Inflorescence modules usually branched (only
one portion shown), and flowers grouped in the axil of a bract (Service Forestier (Capuron) 27632, MO 05006598). Some flowers
have been removed to see pedicels clearly. Group B
Group D
Group C
5 mm
50 mm
5 mm
5 mm
5 mm
5 mm 5 mm 50 mm Group B 5 mm Fig. 3. — The relationship between flowers and floral bracts as illustrated for Weinmannia sect. Inspersa species-groups:
Group B: Weinmannia lowryana with a single flower dissociating from a bract (Humbert 23057, MO 05006602); Group C:
Weinmannia rutenbergii for portion shown in bud (Bradford 699, MO 05079575), and for portion in fruit, racemes often in threes,
flowers mostly solitary and in the axil of a bract [shown in bud to left] or flowers grouped at the base of the medial raceme [shown in
fruit on right] (Bradford 701, MO 05079557); Group D: Weinmannia venusta, with Inflorescence modules usually branched (only
one portion shown), and flowers grouped in the axil of a bract (Service Forestier (Capuron) 27632, MO 05006598). Some flowers
have been removed to see pedicels clearly. Fig. 3. — The relationship between flowers and floral bracts as illustrated for Weinmannia sect. Inspersa species-groups:
Group B: Weinmannia lowryana with a single flower dissociating from a bract (Humbert 23057, MO 05006602); Group C:
Weinmannia rutenbergii for portion shown in bud (Bradford 699, MO 05079575), and for portion in fruit, racemes often in threes,
flowers mostly solitary and in the axil of a bract [shown in bud to left] or flowers grouped at the base of the medial raceme [shown in
fruit on right] (Bradford 701, MO 05079557); Group D: Weinmannia venusta, with Inflorescence modules usually branched (only
one portion shown), and flowers grouped in the axil of a bract (Service Forestier (Capuron) 27632, MO 05006598). ADANSONIA, sér. 3 • 2001 • 23 (2) Architecture Some flowers
have been removed to see pedicels clearly. Bracts may fall at floral or fruit maturity, but
because bract scars are shaped differently than
floral scars the positional and numerical relation-
ships between these structures remain clear. Floral scars are round, with the vascular cylinder espe-
cially visible in species of sect. Spicata. Bract scars
are more difficult to see, but they may be cres-
cent-shaped, or small, irregular protrusions along ADANSONIA, sér. 3 • 2001 • 23 (2) 242 Cladistic analysis of Weinmannia (Cunoniaceae) Fig. 4. — The relationship between flowers and floral bracts as illustrated for Weinmannia sect. Spicata species-groups. A central
portion of a spike is shown: Group E: Weinmannia lucens (voucher unknown) with groups of flowers clustered near bract; Group F:
Weinmannia hildebrandii, with groups of flowers dissociating from a bract (Service Forestier (Capuron) 24417, MO 05012268);
Group G: Weinmannia arguta, with a single flower dissociated from a bract (Miller & Randrianasolo 4659, MO 3759761). Flowers
removed to see positional relationships more clearly, leaving a circular scar with a point in the middle. (Illustration of Weinmannia
lucens from BERNARDI 1965)
Group E
Group F
Group G
5 mm
5 mm
5 mm 5 mm Group E Group F Group G Fig. 4. — The relationship between flowers and floral bracts as illustrated for Weinmannia sect. Spicata species-groups. A central
portion of a spike is shown: Group E: Weinmannia lucens (voucher unknown) with groups of flowers clustered near bract; Group F:
Weinmannia hildebrandii, with groups of flowers dissociating from a bract (Service Forestier (Capuron) 24417, MO 05012268);
Group G: Weinmannia arguta, with a single flower dissociated from a bract (Miller & Randrianasolo 4659, MO 3759761). Flowers
removed to see positional relationships more clearly, leaving a circular scar with a point in the middle. (Illustration of Weinmannia
lucens from BERNARDI 1965) the Raceme. In sect. Inspersa, remaining bases of
pedicels show the locations of flowers. Comparing the position of flower clusters in
W. rutenbergii to that of lateral Racemes in a close
relative, W. venusta, suggests that they are homol-
ogous. Flowers along other parts of the Raceme
in W. rutenbergii are usually solitary, although
pairs of flowers do occur sometimes in many
specimens. Comparing the position of flower clusters in
W. rutenbergii to that of lateral Racemes in a close
relative, W. venusta, suggests that they are homol-
ogous. Architecture Flowers along other parts of the Raceme
in W. rutenbergii are usually solitary, although
pairs of flowers do occur sometimes in many
specimens. The structure of the central portion of the
flower-bearing axis is most consistent and useful
for identification. Often flowers will arise in clus-
ters at the very base of a Raceme but will other-
wise be solitary (e.g. Fig. 3, Group C illustration
on right). Usually, the distal portion of a flower-
bearing axis bears solitary flowers no matter what
the rest of the axis is like. The key below includes six new species
described and one new species-level combina-
tion made for Weinmannia from the Marojejy
massif (BRADFORD & MILLER 2001). These new
taxa represent three of the seven species groups,
with four of the novelties found in group G
alone. Because group G is so species-rich in
general, additional couplets are provided to sub-
divide it into more manageable groups, although
these are not based on cladistic characters
(Fig. 1). Authors of the recognized taxa are
given below following the diagnoses of each
species-group. One species, W. rutenbergii (Group C), may
sometimes key out to species-group D because of
variation in the number of flowers per bract
(although the IM rarely branches in members of
species-group C as it does in those of D, see
Fig. 2). Often in W. rutenbergii, at the base of the
terminal Raceme a large, stipular bract can be
seen that subtends clusters of flowers (Figs. 2, 3). Between this cluster of flowers and other flowers
on the axis a sterile gap can be observed. ADANSONIA, sér. 3 • 2001 • 23 (2) 243 Key to sections and species-groups of Malagasy Weinmannia Key to sections and species-groups of Malagasy Weinmannia
1. Pedicels distinct, easily visible and slender; seeds with surfaces more or less evenly covered with trichomes .... .......................................................................................................................................... 2, sect. Inspersa
1’. Pedicels lacking, very short, or thick as if an extension of the receptacle; seeds usually comose at ends only,
sometimes more or less covered with trichomes throughout ................................................ 5, sect. Spicata
2. Flowers positioned away from the floral bract at maturity; IMs equal in size; racemes borne in pairs from a
short internode with a vegetative bud between them ................................................................................ 3
2’. WEINMANNIA L. Trees and shrubs, sometimes hemiepiphytic
and strangling. Populations monoecious and
flowers bisexual, dioecious or polygamodioe-
cious. Leaves decussate in pairs or rarely whorls,
imparipinnate or unifoliolate, rachis alate or not,
leaf margins toothed or rarely mostly entire. Inflorescence racemose (including spikes and
pseudoracemes); borne solitary in leaf axils or in
compound units of various forms, compound
units borne axillary or terminal. Flowers initiated
solitarily or grouped in the axil of a small bract,
remaining near bract or dissociating from it dur-
ing elongation of the flower-bearing axis; pedicel-
late or sessile; bisexual or unisexual; perianth of
sepals and petals, 4-5-merous, hypogynous or
perigynous, imbricate, calyx lobes free or barely
fused basally, petals persistent or caducous; floral
nectary annular or segmented; androecium
diplostemonous (rarely one whorl of stamens),
filaments slender, equal to or exceeding petals,
anthers dorsifixed, bithecal with an apical con- MALAGASY CLADE OF WEINMANNIA (sections
Inspersa and Spicata) Flowers mostly pentamerous, hypogynous,
bisexual, petals persistent, calyx persistent, endo-
carp and exocarp usually separating in old fruits. DESCRIPTION OF WEINMANNIA AND
DIAGNOSTIC FEATURES OF MALAGASY
CLADE, SECTIONS AND SPECIES-GROUPS nective, longitudinally dehiscent; ovary bicarpel-
late, carpels fused to the level of the styles, locules
two, each with an axile placenta, ovules 2 to
numerous in two rows per locule, styles slender
and diverging, stigmas small and terminal. Fruits
capsular, carpels diverging and opening along
their ventral sutures; calyx persistent or caducous;
placenta often remaining upright between sepa-
rate carpels; endocarp and exocarp separating in
old fruits or remaining adherent; seeds comose or
trichomes more or less covering the surface, lack-
ing wings. WEINMANNIA L. Architecture Flowers positioned in the axil of the floral bract at maturity; IMs larger basally; racemes borne in threes or at
the ends of a decussate branching system with multiple internodes that usually terminate in a raceme, or
racemes in pairs with a vegetative bud between them ................................................................................ 4
3. Floral bracts subtending groups of flowers ........................................................................ species-group A
3’. Floral bracts subtending solitary flowers ............................................................................ species-group B
4. IM unbranched; floral bracts subtending solitary flowers or less often groups of flowers .... species-group C
4’. IM branched; floral bracts subtending groups of flowers .................................................. species-group D
5. Spikes borne solitarily in leaf axils; floral nectary composed of more or less distinct segments; flowers distrib-
uted near each other and the floral bract, borne in clusters of usually four ........................ species-group E
5’. Spikes borne in pairs from a short internode in leaf axils or terminally; floral nectary usually an entire,
membranous disc (rarely segmented); flowers not distributed in clusters, but spread in a line away from the
floral bract ................................................................................................................................................ 6
6. Floral bracts subtending multiple flowers; floral nectary sometimes easily broken into segments .................. .......................................................................................................................................... species-group F
6’. Floral bracts subtend a single flower; nectary usually entire and membranous .................... species-group G Acknowledgments BENTHAM G. & HOOKER J.D. 1862-1883. — Genera
plantarum: ad exemplaria imprimis in Herberii
Kewensibus servata definita. A. Black (v.1, pt. 1);
Reeve & Co. (v.1, pt. 2-v. 3), London. For financial support I thank the Mellon
Foundation, the Missouri Botanical Garden and
Washington University’s Division of Biology and
Biomedical Sciences. Most illustrations in Figs. 3 and
4 were kindly done by Kim MARTIN. The Muséum
National d’Histoire Naturelle, Laboratoire de
Phanérogamie kindly permitted the use of an illustra-
tion of W. lucens in Fig. 3 that originally appeared in
BERNARDI (1965, fig. 7b). For field work assistance I
thank my Malagasy collaborators at the Parc Bota-
nique et Zoologique de Tsimbazaza, especially Guy
RAFAMANTANANTSOA and Frank RAKOTONASOLO. p
BERNARDI L. 1964. — Revisio generis Weinmanniae
Pars III: Sect. III-IV-V-VI (verteris orbis). Bot. Jahrb. Syst. 83: 126-184. y
BERNARDI L. 1965. — Cunoniacées. Flore de
Madagascar et des Comores, in HUMBERT H. (ed.). Muséum National d’Histoire Naturelle, Laboratoire
de Phanérogamie, Paris. g
BERNARDI L. 1969. — Weinmanniae species nova, isoste-
mona, ex Madagascaria boreali. Candollea 24: 85-87. g
BRADFORD J.C. 1998. — A cladistic analysis of
species-groups in Weinmannia (Cunoniaceae) based
on morphology and inflorescence architecture. Ann. Missouri Bot. Gard. 85: 565-593. Weinmannia sect. Inspersa Bernardi Flowers pedicellate, floral nectary usually
annular and ribbed; seed surface with dense indu-
mentum throughout. ADANSONIA, sér. 3 • 2001 • 23 (2) 244 Cladistic analysis of Weinmannia (Cunoniaceae) Weinmannia sect. Spicata Bernardi Bradford, W. rakotomalazana J.C. Bradford. Spikes borne in IMs, flowers initiated in groups
that dissociate from bract, nectary annular and
thin or composed of more or less distinct segments. species-group G. — Species are listed within the
key given below. Spikes borne in IMs, flowers
borne solitarily and usually dissociate from axil
of bract, nectary usually annular and thin. 1. Leaves unifoliolate .................................................................................................................................... 2
1’. Leaves imparipinnate ................................................................................................................................ 3
2. Ovaries glabrous ................................................................................................ W. humbertiana Bernardi
2’. Ovaries pubescent ........................................................................................................................................ ......W. bojeriana Tul., W. mammea Bernardi, W. pauciflora, J.C. Bradford, W. integrifolia J.C. Bradford
3. Ovaries glabrous ................................................................................................................ W. decora Tul. 3’. Ovaries pubescent .................................................................................................................................... 4
4. Hairs on seeds restricted to ends ................................................................................ W. humblottii Baill.,
...................... W. sanguisugarum Bernardi, W. arguta (Bernardi) J.C. Bradford, W. venosa J.C. Bradford
4’. Hairs on seeds widely distributed .............................................. W. eriocarpa Tul., W. stenostachya Baker 1. Leaves unifoliolate .................................................................................................................................... 2
1’. Leaves imparipinnate ................................................................................................................................ 3
2. Ovaries glabrous ................................................................................................ W. humbertiana Bernardi
2’. Ovaries pubescent ........................................................................................................................................ ......W. bojeriana Tul., W. mammea Bernardi, W. pauciflora, J.C. Bradford, W. integrifolia J.C. Bradford
3. Ovaries glabrous ................................................................................................................ W. decora Tul. 3’. Ovaries pubescent .................................................................................................................................... 4
4. Hairs on seeds restricted to ends ................................................................................ W. humblottii Baill.,
...................... W. sanguisugarum Bernardi, W. arguta (Bernardi) J.C. Bradford, W. venosa J.C. Bradford
4’. Hairs on seeds widely distributed .............................................. W. eriocarpa Tul., W. stenostachya Baker Weinmannia sect. Spicata Bernardi species-group A. — Weinmannia henricorum
Bernardi, W. madagascariensis DC. ex Ser. species-group A. — Weinmannia henricorum
Bernardi, W. madagascariensis DC. ex Ser. TI isotonic, IM terminating in a bud, flowers
borne in clusters that dissociate from bract. Flowers sessile; seeds comose, only rarely seed
surface with indumentum throughout. species group E
Weinmannia lucens Baker y
y
surface with indumentum throughout. species-group E. — Weinmannia lucens Baker, species-group B. — Weinmannia commersonii
Bernardi, W. louveliana Bernardi, W. lowryana
J.C. Bradford. TI isotonic, IM terminates in a bud, flowers
borne solitarily and dissociate from bract. p
g
p
W. minutiflora Baker, W. comorensis Tul.,
W. baehniana Bernardi. Spikes borne in leaf axils, flowers borne in clus-
ters that remain in axil of bract, nectary com-
posed of more or less distinct segments. W. minutiflora Baker, W. comorensis Tul.,
W. baehniana Bernardi. Spikes borne in leaf axils, flowers borne in clus-
ters that remain in axil of bract, nectary com-
posed of more or less distinct segments. species-group C. — Weinmannia hepaticarum
Bernardi, W. rutenbergii Engl. TI basitonic, IM terminating in a raceme or a
bud, IM sometimes branched, flowers borne
solitarily or sometimes in small groups and
remaining in axil of bract. species-group C. — Weinmannia hepaticarum
Bernardi, W. rutenbergii Engl. TI basitonic, IM terminating in a raceme or a
bud, IM sometimes branched, flowers borne
solitarily or sometimes in small groups and
remaining in axil of bract. species-group F. — Weinmannia hildebrandtii Baill.,
W. icacifolia Bernardi, W. marojejyensis J.S. Mill. &
J.C. Bradford, W. rakotomalazana J.C. Bradford. Spikes borne in IMs, flowers initiated in groups
that dissociate from bract, nectary annular and
thin or composed of more or less distinct segments. species-group G. — Species are listed within the
key given below. Spikes borne in IMs, flowers
borne solitarily and usually dissociate from axil
of bract, nectary usually annular and thin. species-group F. — Weinmannia hildebrandtii Baill.,
W. icacifolia Bernardi, W. marojejyensis J.S. Mill. &
J.C. Bradford, W. rakotomalazana J.C. Bradford. Spikes borne in IMs, flowers initiated in groups
that dissociate from bract, nectary annular and
thin or composed of more or less distinct segments. species-group G. — Species are listed within the
key given below. Spikes borne in IMs, flowers
borne solitarily and usually dissociate from axil
of bract, nectary usually annular and thin. species-group F. — Weinmannia hildebrandtii Baill.,
W. icacifolia Bernardi, W. marojejyensis J.S. Mill. &
J.C. Manuscript received 14 February 2001;
revised version 19 September 2001. ADANSONIA, sér. 3 • 2001 • 23 (2) REFERENCES BRADFORD J.C. 2000. — Phylogenetic systematics of
Cunoniaceae (Oxalidales), with an emphasis on
species-groups and inflorescence evolution in
Weinmannia and related genera. Ph.D. thesis. ANGIOSPERM PHYLOGENY GROUP 1998. — An ordinal
classification for the families of Flowering Plants. Ann. Missouri Bot. Gard. 85: 531-553. ADANSONIA, sér. 3 • 2001 • 23 (2) 245 Bradford J.C. HOPKINS H.C.F. 1998b. — A revision of Weinmannia
(Cunoniaceae) in Malesia and the Pacific. 2. Sulawesi and the Philippines. Adansonia, sér. 3, 20:
43-66. Washington University, Department of Biology,
St. Louis. BRADFORD J.C. & BARNES R.W. 2001. —
Phylogenetics and classification of Cunoniaceae
(Oxalidales) using chloroplast DNA sequences and
morphology. Syst. Bot. 26: 354-385. HOPKINS H.C.F. 1998c. — A revision of Weinmannia
(Cunoniaceae) in Malesia and the Pacific. 3. New
Guinea, Solomon Islands, Vanuatu and Fiji, with
notes on the species of Samoa, Rarotonga, New
Caledonia and New Zealand. Adansonia, sér. 3, 20:
67-106. p
gy
y
BRADFORD J.C. & MILLER J.S. 2001. — New taxa and
nomenclatural notes on the flora of the Marojejy
massif, Madagascar. V. Cunoniaceae: Weinmannia. Adansonia, sér. 3, 23: 219-236. HOPKINS H.C.F. 1998a. — A revision of Weinmannia
(Cunoniaceae) in Malesia and the Pacific. 1. Introduction and an account of the species of
Western Malesia, the Lesser Sundas and the
Moluccas. Adansonia, sér. 3, 20: 5-41. HOPKINS H.C.F. & FLORENCE J. 1998. — A revision
of Weinmannia
(Cunoniaceae) in Malesia
and the Pacific. 4. The Society, Marquesas
and Austral Islands. Adansonia, sér. 3, 20:
107-130. Manuscript received 14 February 2001;
revised version 19 September 2001. ADANSONIA, sér. 3 • 2001 • 23 (2) 246
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Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata Pelajaran Bahasa Indonesia
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Jurnal Ilmiah Kampus Mengajar
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Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata…
80
Peningkatan Literasi Menulis Siswa SMA Prawira Marta
Melalui Pendampingan Mata Pelajaran Bahasa Indonesia
Bunga Lidinta Putri1*, Windi Dian Safitri2, Imfrianti Augtiah3, Romi Iriandi Putra4
1,2Fakultas Keguruan dan Ilmu Pendidikan, Universitas Muhammadiyah Surakarta,
Indonesia
3,4Fakultas Komunikasi dan Bisnis, Universitas Muhammadiyah Karanganyar,
Indonesia
*Korespondesi Penulis:
Bunga Lidinta Putri
Fakultas Keguruan dan Ilmu Pendidikan, Universitas Muhammadiyah Surakarta, Indonesia
Email: a310170214@student.ums.ac.id
doi: 10.56972/jikm.v2i2.44
Submit: 2 Agustus 2022 | Revisi: 16 Oktober 2022 | Diterima: 19 Oktober 2022
Dipublikasikan: 22 Oktober 2022 | Periode Terbit: Oktober 2022
Abstrak
Jurnal Ilmiah Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata…
80
Peningkatan Literasi Menulis Siswa SMA Prawira Marta
Melalui Pendampingan Mata Pelajaran Bahasa Indonesia
Bunga Lidinta Putri1*, Windi Dian Safitri2, Imfrianti Augtiah3, Romi Iriandi Putra4
1,2Fakultas Keguruan dan Ilmu Pendidikan, Universitas Muhammadiyah Surakarta,
Indonesia
3,4Fakultas Komunikasi dan Bisnis, Universitas Muhammadiyah Karanganyar,
Indonesia
*Korespondesi Penulis:
Bunga Lidinta Putri
Fakultas Keguruan dan Ilmu Pendidikan, Universitas Muhammadiyah Surakarta, Indonesia
Email: a310170214@student.ums.ac.id
doi: 10.56972/jikm.v2i2.44
Submit: 2 Agustus 2022 | Revisi: 16 Oktober 2022 | Diterima: 19 Oktober 2022
Dipublikasikan: 22 Oktober 2022 | Periode Terbit: Oktober 2022
Abstrak
Jurnal Ilmiah Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata…
80 Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata… Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata 80 Jurnal Ilmiah Peningkatan Literasi Menulis Siswa SMA Prawira Marta
Melalui Pendampingan Mata Pelajaran Bahasa Indonesia Bunga Lidinta Putri1*, Windi Dian Safitri2, Imfrianti Augtiah3, Romi Iriandi Putra4
1,2Fakultas Keguruan dan Ilmu Pendidikan, Universitas Muhammadiyah Surakarta,
Indonesia
3,4Fakultas Komunikasi dan Bisnis, Universitas Muhammadiyah Karanganyar,
Indonesia Bunga Lidinta Putri1 , Windi Dian Safitri2, Imfrianti Augtiah3, Romi Iriandi Putra4
1,2Fakultas Keguruan dan Ilmu Pendidikan, Universitas Muhammadiyah Surakarta,
Indonesia
3,4Fakultas Komunikasi dan Bisnis, Universitas Muhammadiyah Karanganyar,
Indonesia
*Korespondesi Penulis:
Bunga Lidinta Putri
Fakultas Keguruan dan Ilmu Pendidikan, Universitas Muhammadiyah Surakarta, Indonesia
Email: a310170214@student.ums.ac.id
doi: 10.56972/jikm.v2i2.44
Submit: 2 Agustus 2022 | Revisi: 16 Oktober 2022 | Diterima: 19 Oktober 2022
Dipublikasikan: 22 Oktober 2022 | Periode Terbit: Oktober 2022
Abstrak
Kondisi pembelajaran di dunia pendidikan saat ini harus merubah sistem pendidikannya karena me-
nyesuaikan dengan situasi dan kondisi selama masa pandemi Covid-19. Pembelajaran sekolah diadakan
secara daring karena tidak bisa dipungkiri, dalam proses pembelajaran terjadi kontak antar individu
dalam jumlah banyak. Motivasi siswa terhadap kegiatan belajar harus ditingkatkan melalui pembelaja-
ran yang menyenangkan. Salah satunya adalah peningkatan kemampuan literasi siswa selama masa
pandemi dengan didampingi oleh mahasiswa KKN-DIK Universitas Muhammadiyah Surakarta guna
meningkatkan kemampuan literasi menulis siswa SMA Prawira Marta. Metode yang dijalankan dalam
KKN-Dikmas ini adalah metode observasi dan metode dokumentasi. Hasil yang diperoleh berupa bukti
tugas-tugas yang berkaitan dengan kemampuan literasi menulis siswa yang berbeda-beda. Pentingnya
kegiatan peningkatan literasi ini perlu untuk dikuasai agar siswa menjadi seseorang dengan kemam-
puan bahasa yang baik. Kata Kunci: literasi menulis, media whatsapp doi: 10.56972/jikm.v2i2.44
Submit: 2 Agustus 2022 | Revisi: 16 Oktober 2022 | Diterima: 19 Oktober 2022
Dipublikasikan: 22 Oktober 2022 | Periode Terbit: Oktober 2022 Abstrak Kondisi pembelajaran di dunia pendidikan saat ini harus merubah sistem pendidikannya karena me-
nyesuaikan dengan situasi dan kondisi selama masa pandemi Covid-19. Pembelajaran sekolah diadakan
secara daring karena tidak bisa dipungkiri, dalam proses pembelajaran terjadi kontak antar individu
dalam jumlah banyak. Motivasi siswa terhadap kegiatan belajar harus ditingkatkan melalui pembelaja-
ran yang menyenangkan. Salah satunya adalah peningkatan kemampuan literasi siswa selama masa
pandemi dengan didampingi oleh mahasiswa KKN-DIK Universitas Muhammadiyah Surakarta guna
meningkatkan kemampuan literasi menulis siswa SMA Prawira Marta. Metode yang dijalankan dalam
KKN-Dikmas ini adalah metode observasi dan metode dokumentasi. Hasil yang diperoleh berupa bukti
tugas-tugas yang berkaitan dengan kemampuan literasi menulis siswa yang berbeda-beda. Pentingnya
kegiatan peningkatan literasi ini perlu untuk dikuasai agar siswa menjadi seseorang dengan kemam-
puan bahasa yang baik. Kata Kunci: literasi menulis, media whatsapp selama masa pandemi Covid-19. Penu-
laran yang sangat cepat melalui kontak an-
tar individu menyebabkan perkumpulan
dalam bentuk apapun dihindari. Hal ini 1.
Pendahuluan Dewasa ini dunia pendidikan harus
merubah sistem pendidikannya karena
menyesuaikan dengan situasi dan kondisi Vol (2) (2) (2022) 80-92 e-ISSN 2828-6375 Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata… 81 hampir terjadi di semua sektor kehidupan,
baik sektor kesehatan, sektor bisnis dan
perdagangan, sektor sosial, sektor politik,
sektor budaya, sektor perkantoran terma-
suk pada sektor Pendidikan (Sutopo et al.,
2021). Maka dari itu, sekolah diadakan
secara daring karena tidak bisa dipungkiri,
dalam proses pembelajaran terjadi kontak
antar individu dalam jumlah banyak. Pada
masa pandemi corona ini, semua proses
belajar mengajar dilakukan secara daring
sesuai tema (Wisaksono et al., 2020). Pem-
berlakuan praktik pendidikan dengan sis-
tem daring tersebut pada dasarnya meru-
pakan desain ideal untuk melaksanakan
kegiatan pengajaran di tengah masa pan-
demi saat ini (Lathifah & Utami, 2021). Peran teknologi sangat penting karena
hubungan antara pembelajaran jarak jauh
(PJJ) dengan pemanfaatan platform pem-
belajaran sangat berdampak positif karena
penggunaannya
sangat
praktis
(Khikmawati
et
al.,
2021). Pesatnya
perkembangan teknologi zaman sekarang
memberikan pengaruh yang begitu banyak
bagi kemajuan dunia pendidikan khu-
susnya dalam proses pembelajaran. Ada
beberapa unsur pergeseran dalam proses
pembelajaran, 1) dari pelatihan menjadi
penampilan, 2) dari pembelajaran bersifat
luring menjadi daring, 3) dari kertas men-
jadi online atau saluran, 4) fasilitas berupa
fisik menjadi fasilitas jaringan kerja, 5)
serta dari waktu siklus menjadi waktu
nyata (Suryadi, 2015). Selain itu kemajuan
teknologi juga mempunyai peran penting
dalam bidang pendidikan diantaranya: 1) hampir terjadi di semua sektor kehidupan,
baik sektor kesehatan, sektor bisnis dan
perdagangan, sektor sosial, sektor politik,
sektor budaya, sektor perkantoran terma-
suk pada sektor Pendidikan (Sutopo et al.,
2021). Maka dari itu, sekolah diadakan
secara daring karena tidak bisa dipungkiri,
dalam proses pembelajaran terjadi kontak
antar individu dalam jumlah banyak. Pada
masa pandemi corona ini, semua proses
belajar mengajar dilakukan secara daring
sesuai tema (Wisaksono et al., 2020). Pem-
berlakuan praktik pendidikan dengan sis-
tem daring tersebut pada dasarnya meru-
pakan desain ideal untuk melaksanakan
kegiatan pengajaran di tengah masa pan-
demi saat ini (Lathifah & Utami, 2021). Peran teknologi sangat penting karena
hubungan antara pembelajaran jarak jauh
(PJJ) dengan pemanfaatan platform pem-
belajaran sangat berdampak positif karena
penggunaannya
sangat
praktis
(Khikmawati
et
al.,
2021). Pesatnya
perkembangan teknologi zaman sekarang
memberikan pengaruh yang begitu banyak
bagi kemajuan dunia pendidikan khu-
susnya dalam proses pembelajaran. 1.
Pendahuluan Ada
beberapa unsur pergeseran dalam proses
pembelajaran, 1) dari pelatihan menjadi
penampilan, 2) dari pembelajaran bersifat
luring menjadi daring, 3) dari kertas men-
jadi online atau saluran, 4) fasilitas berupa
fisik menjadi fasilitas jaringan kerja, 5)
serta dari waktu siklus menjadi waktu
nyata (Suryadi, 2015). Selain itu kemajuan
teknologi juga mempunyai peran penting
dalam bidang pendidikan diantaranya: 1)
sebagai sumber ilmu dan pusat pendidi-
kan, 2) munculnya metode pembelajaran baru yang dapat memudahkan tenaga pen-
didik atau guru dalam proses pembelaja-
ran, 3) system pembelajaran tidak harus ta-
tap muka melainkan dengan daring atau
secara online (Effendi, 2019). Kondisi pembelajaran dalam situasi
daring seperti ini tentunya berdampak ter-
hadap semangat belajar siswa. Tanpa disa-
dari pelaksanaan pembelajaran dalam
jaringan (daring) telah berjalan lebih dari
satu tahun yang pengaruhnya amat besar
bagi dunia pendidikan, khususnya peru-
bahan perilaku anak yang dipaksa harus
bersentuhan dengan teknologi tanpa bekal
cukup mengontrol penggunaan gadget
bila tanpa pendampingan dari orang tua
secara maksimal (Nuroniyah, 2021). Selain
itu juga ada beberapa hambatan dalam
proses pembelajaran jarak jauh di masa
pandemic. Selain keterbatasan sarana pen-
dukung dan jaringan internet, faktor kesia-
pan dari sumber daya manusia merupakan
bagian penting dalam sebuah keberhasilan
dalam pelaksanaan pembelajaran secara
online (Latip, 2020). Namun,
Pendidikan
yang
dil-
aksanakan secara daring membuat peserta
didik mengalami penurunan dalam minat
dan keaktifan belajar (Ihwan et al., 2021). Pembelajaran secara daring dinilai kurang
berjalan efektif. Hal ini dilihat dari respon
siswa saat kegiatan pembelajaran daring
yang cenderung tidak merespon ketika
diskusi dan pengumpulan tugas (Choiru-
din & Rahmasari, 2021). Banyak siswa
yang
kurang
termotivasi
dalam
melaksanakan proses pembelajaran karena
merasa dirinya tidak diawasi sehingga rasa
tanggung jawab yang mereka miliki Vol (2) (2) (2022) 80-92 Vol (2) (2) (2022) 80-92 e-ISSN 2828-6375 e-ISSN 2828-6375 Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata… 82 menurun. Proses belajar mengajar yang
dilakukan secara online selayaknya tidak
dilaksanakan dalam durasi panjang dan
memakan waktu lama, hal ini dikarenakan
peserta didik akan mengalami kesulitan
dalam mempertahankan konsentrasi lebih
dari satu jam (Sumarsono et al., 2021). Ten-
tunya permasalahan tersebut menjadikan
Pendidikan tidak berjalan dengan seha-
rusnya dan berimbas kepada proses dan
hasil pembelajaran yang cacat (Sholihah et
al., 2021). Namun, pendidikan harus tetap
berjalan
hingga
tujuan
pendidikan
tercapai. Motivasi siswa terhadap kegiatan
belajar harus ditingkatkan melalui pem-
belajaran
yang
menyenangkan. 1.
Pendahuluan Salah
satunya adalah peningkatan kemampuan
literasi siswa selama masa pandemi
dengan didampingi oleh mahasiswa KKN-
DIK Universitas Muhammadiyah Sura-
karta. politik, dan juga meningkatkan kepekaan
terhadap lingkungan. Upaya pengem-
bangan literasi perlu dikembangkan secara
terintegrasi, terutama melalui pendidikan. Ini karena, Pendidikan memiliki fungsi
dan tujuan dalam memajukan peradaban
bangsa (Agustina et al., 2020). Literasi tidak
hanya sekadar kemampuan membaca dan
menulis, tetapi literasi bisa berarti melek
teknologi, politik, berpikir kritis, dan peka
terhadapi lingkungan sekitar (Sunanda et
al., 2020). Budaya literasi juga penting un-
tuk bangsa karena dapat menggerakan ide-
ide dan pengetahuan bangsa Indonesia,
khususnya para anak muda di Indonesia
(Ningrum et al., 2021). Mengajarkan lit-
erasi pada intinya menjadikan manusia
secara fungsional mampu berbaca-tulis,
terdidik, cerdas dan menunjukkan apre-
siasi terhadap sastra (Machromah et al.,
2020). Literasi merupakan suatu gerakan
yang bertujuan untuk mengatasi masalah
buta huruf pada suatu lingkungan pen-
didikan. Gerakan literasi sering dikaitkan
dengan kegiatan membaca buku apa saja
karena hal ini dapat meningkatkan moti-
vasi siswa terhadap kegiatan membaca. Kemampuan literasi membaca merupakan
aspek dasar yang harus dimiliki oleh siswa
yang digunakan untuk menyerap berbagai
sumber informasi yang diterima (Ma-
hardhani et al., 2021). Menurut Hartati
(2017) literasi merupakan kemampuan un-
tuk memahami, mengelola, dan memakai
informasi pada berbagai konteks. Literasi
tidak hanya memberikan manfaat di bi-
dang menulis dan membaca, namun juga
memberikan manfaat di bidang teknologi, Menurut kamus besar bahasa Indone-
sia (KBBI) yang dimaksud dengan literasi
yaitu kemampuan menulis dan membaca;
pengetahuan atau keterampilan pada bi-
dang atau kegiatan tertentu; kemampuan
individu untuk mengolah informasi serta
pengetahuan untuk kecakapan hidup. Lit-
erasi adalah istilah yang memiliki kaitan
erat dengan kegiatan membaca dan menu-
lis terutama dalam hal penggunaan bahasa
dan fungsi bahasa tersebut. Menurut Kern
(2000) dalam Firmansyah (2018: 12) literasi
merupakan penggunaan praktik- praktik
situasi sosial, dan historis, serta kultural
dalam mewujudkan dan menafsirkan
suatu makna melalui teks. Jadi dapat di-
jelaskan bahwa literasi merupakan sebuah
keahlian yang dimiliki oleh seseorang Vol (2) (2) (2022) 80-92 e-ISSN 2828-6375 Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata… 83 untuk
menuangkan
dan
menyajikan
situasi-situasi dan berbagai fenomena atau
kejadian melalui sebuah teks. Menurut
Buckingham (2007) dalam Suhaimi (2017)
literasi memiliki 4 elemen penting yakni
representasi, bahasa, kreasi, dan khalayak. Bahasa termasuk salah satu komponen
penting dalam literasi maka dari itu ke-
mampuan berbahasa dapat ditingkatkan
melalui sebuah literasi baik menulis atau
membaca. lancar membaca. 1.
Pendahuluan Hasil dari minggu kedua
adalah cara membaca dan minat baca
siswa bisa menjadi lebih lancar dan lebih
baik dari sebelumnya. Kemudian hasil
pada minggu ketiga peserta didik jadi lebih
percaya diri untuk terampil berbicara di
depan kelas dan kosa kata yang mereka
gunakan dalam berbicara lebih banyak
setelah pembiasaan budaya literasi dan
public speaking yang diterapkan oleh
peneliti. Dalam pelajaran Bahasa Indonesia,
kegiatan literasi sangat banyak peran-
annya karena literasi identik dengan mem-
baca dan menulis. Walaupun dalam kon-
disi daring seperti saat ini yaitu pembelaja-
ran dilakukan secara online jarak jauh, ke-
mampuan literasi siswa tetap perlu diasah
dan ditingkatkan karena membaca dan
menulis adalah sebuah kemampuan yang
penting untuk dikuasai agar menjadi
seseorang dengan kemampuan bahasa
yang baik. Walaupun demikian, pen-
guasaan literasi yang tinggi tentunya tidak
boleh mengabaikan aspek sosiokultural,
karena literasi merupakan bagian dari kul-
tur atau budaya manusia (Kurniawan et
al., 2019). Literasi yang dimiliki oleh siswa
SMA Prawira Marta tergolong dalam kon-
disi yang terbatas dikarenakan masih ban-
yak siswa yang tidak peduli dan termoti-
vasi untuk belajar menulis. Maka dari itu,
pada kegiatan KKN-DIK yang dilakukan
oleh mahasiswa Universitas Muhammadi-
yah Surakarta di SMA Prawira Marta akan
fokus
kepada
pendampingan
guna
meningkatkan kemampuan literasi menu-
lis siswa SMA Prawira Marta. 2.
Metode Metode yang dijalankan dalam KKN-
Dikmas ini ialah metode observasi dan
metode dokumentasi. Observasi merupa-
kan salah satu kegiatan ilmiah empiris
yang mendasarkan fakta-fakta lapangan
maupun teks, melalui pengalaman panca
indra tanpa menggunakan manipulasi
apapun (Hasanah, 2017). Observasi dil-
akukan dengan cara meninjau dan men-
catat secara sistematiks semua hal yang
ada di area SMA Prawira Marta. Pada
metode ini, penulis terjun ke lokasi untuk
meninjau secara langsung aktivitas-aktivi-
tas dan kejadian-kejadian yang ditemui se-
bagai pengaruh dari pelaksanaan KKN- Dalam kegiatan KKN yang dil-
aksanakan oleh Saiful Jihad dan kelompok-
nya pada tahun 2020 dengan judul
kegiatan “Meningkatkan Budaya Literasi
dan Public Speaking pada Kalangan Peserta
Didik di MI Muhammadiyah Ngancar”
menghasilkan beberapa hasil kegiatan tiap
minggunya. Hasil minggu pertama, minat
baca dari masing-masing dari tiap siswa
menunjukkan bahwa siswa MI Muham-
madiyah Ngancar banyak yang belum Vol (2) (2) (2022) 80-92 Vol (2) (2) (2022) 80-92 e-ISSN 2828-6375 84 Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata… Dikmas. Data yang dibutuhkan dalam
metode ini yakni meninjau secaralangsung
ke lokasi, proses pelaksanaan pembelaja-
ran daring, dan aktivitas warga SMA
Prawira Marta di situasi daring. Dikmas. Data yang dibutuhkan dalam
metode ini yakni meninjau secaralangsung
ke lokasi, proses pelaksanaan pembelaja-
ran daring, dan aktivitas warga SMA
Prawira Marta di situasi daring. Melaksanakan pendampingan belajar
melalui media whatsapp metode: men-
guraikan cara yang digunakan untuk me-
mecahkan masalah. 3.
Hasil dan Pembahasan Metode Dokumentasi adalah alat
pengumpulan data yang digunakan untuk
mencari, mengenal hal – hal atau variable
yang berupa catatan, transkip buku, surat
kabar, majalah, peraturan – peraturan,
notulen rapat dan sebagainya (U. Hasanah,
2017). Metode dokumentasi dilakukan me-
lalui cara menggabungkan sumber data
berwujud catatan dokumen, foto-foto, dan
video. Metode ini merupakan salah satu
wujud pengumpulan data yang paling se-
derhana, karena penulis hanya mengamati
benda mati dan bilamana terdapat kesala-
han mudah untuk memperbaikinya karena
sumber datanya tetap dan tidak berubah. Hasil penelitian diuraikan berdasar-
kan hasil pengamatan dalam proses
meningkatkan literasi menulis siswa SMA
Marta melalui pendampingan dalam pem-
belajaran Bahasa Indonesia yang dil-
akukan melalui aplikasi whatsapp chat
dan grup. Hasil data ini berdasarkan data
yang telah dikumpulkan melalui observasi
dan catatan selama pembelajaran daring
berlangsung. Dalam program meningkatkan lit-
erasi menulis siswa ini, siswa diarahkan
untuk menulis sebuah proposal kegiatan
dan karya ilmiah. Penulisan proposal
kegiatan dan karya ilmiah ini berdasarkan
materi pelajaran yang sedang diajarkan
pada pembelajaran Bahasa Indonesia. Selain dijadikan sebagai data program
kerja yang sedang dilakukan dalam KKN-
Dikmas, penulisan proposal kegiatan dan
karya ilmiah juga dijadikan sebagai bentuk
upaya
meningkatkan
literasi
menulis
siswa dijadikan sebagai nilai tugas pelaja-
ran Bahasa Indonesia. Strategi pelaksanaan peningkatan
rasa tanggung jawab siswa berkenaan tu-
gas yang diberikan oleh guru ini dilakukan
dalam
bentuk
pendampingan
dan
penyusunan bahan ajar melalui tahapan-
tahapan sebagai berikut: a)
Melakukan observasi pada ling-
kungan
pembelajaran
dan
mengamati proses pembelajaran dar-
ing. b)
Menyusun jadwal pertemuan dengan
guru mata pelajaran BahasaIndone-
sia. Saat pelaksanaan program tersebut
siswa diarahkan untuk menulis sebuah
proposal kegiatan dengan tema bebas dan
siswa bebas mencarinya referensi di inter-
net. Kemudian siswa menulis proposal
kegiatan sesuai dengan sistematika yang
telah ditentukan. Sistematika tersebut
sesuai dengan yang telah disampaikan
pada materi pelajaran Bahasa Indonesia. c)
Mempersiapkan konsep materi yang
akan disampaikan dalam kegiatan
sesuai dengan kebutuhan siswa. d)
Mengolah materi, pengetikan, dan
latihan-latihan soal yang akan diberi-
kan untuk siswa. Vol (2) (2) (2022) 80-92 e-ISSN 2828-6375 Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata… 85 Guru menyampaikan materi pelajaran me-
lalui whatsapp grup dengan mengirimkan
modul sesuai materi yang diajarkan. (Lembar Kerja Siswa). Untuk mengem-
bangkan baca siswa, perlu adanya pening-
katan
sumber
rujukan
yang
me-
nyenangkan dengan berbantuan modul
pembelajaran digital yang dibuat lebih
ringkas dan menarik untuk mengurangi
kejenuhan siswa dan ketergantungan pada
LKS. 3.
Hasil dan Pembahasan Berikut merupakan laporan hasil
pelaksanaan kegiatan yang dilaksanakan
selama program KKN-Dikmas berlang-
sung. Laporan hasil pelaksanaa KKN-Dik
tersaji dalam bentuk rincian kegiatan yang
dimulai pada tanggal 1 Februari 2021 sam-
pai 6 Maret 2021. Pembelajaran daring mata pelajaran
Bahasa Indonesia di SMA Prawira Marta
dilaksanakan setiap hari Rabu pukul 07.30
WIB hingga pukul 12.00 WIB. Penyusunan
materi pembelajaran yang berupa modul
digital tersebut dirancang setiap hari Senin
dan Selasa, dan diberikan kepada siswa
SMA Prawira Marta di hari Rabu melalui
media grup whatsapp. Berikut adalah be-
berapa contoh modul pembelajaran yang
diberikan ke siswa: c. Materi pelajaran dengan berbantuan
modul digital tersampaikan kepada se-
luruh siswa SMA Prawira Marta dengan
lancar. Akan tetapi, masih ada sebagian
siswa yang merespon dengan lambat
bahkan tidak mau membaca modulnya
sama sekali sehingga harus ditegur terus
menerus. Untuk mengatasi masalah terse-
but, pendidik selalu mengingatkan dan
menghimbau siswa agar mereka mau
bertekad meningkatkan kemampuan lit-
erasi minat bacanya sehingga saat me-
masuki tahap literasi menulis tidak akan
mengalami kendala. Modul digital memiliki keunggulan
dapat
menampilkan
beberapa
materi
dengan menggunakan media interaktif
(Aprilliyah & Wahjudi, 2014; Sugianto, Ab-
dullah, Elvyanti & Muladi, 2013; Sujanem
Suswandi & Ganesha, 2012). Tujuan pem-
buatan modul digital yakni untuk memu-
dahkan siswa menguasai materi pelajaran
yang diajarkan guru. Selain itu, modul digital dapat
dipadukan dengan model pembelajaran
inovatif yang dipandang mampu mening-
katkan hasil belajar. Dalam modul digital,
proyek bisa dijadikan sebagai salah satu
teknik penilaian yang digunakan. Teknik
ini mampu mendorong pembelajaran yang
menantang dan meningkatkan kemam-
puan siswa dalam memecahkan masalah
(Winatha, Suharsono & Agustin, 2018). Oleh karena itu, pengembangan modul
digital ini dalam bentuk proyek, dipan-
dang penting untuk meningkatkan moti-
vasi, pemahaman konsep, kemampuan
siswa. a.
Hasil Program berupa Modul Pem-
belajaran Untuk mengembangkan kemampuan
literasi menulis siswa dapat dimulai dari
pengembangan minat baca siswa. Dalam
tahap ini, pembelajaran Bahasa Indonesia
di SMA Marta hanya mengacu pada variasi
teks yang tersedia dalam format LKS Gambar 1. Tampilan Awal
Modul Kelas XII
Gambar 2. Tampilan Materi
Modul Kelas XI
Gambar 3. Tampilan Materi
Modul Kelas X Gambar 1. Tampilan Awal
Modul Kelas XII
Gambar 2. Tampilan Materi
Modul Kelas XI Gambar 1. Tampilan Awal
Modul Kelas XII Gambar 3. Tampilan Materi
Modul Kelas X Gambar 3. Tampilan Materi
Modul Kelas X Gambar 1. Tampilan Awal
Modul Kelas XII Gambar 2. Tampilan Materi
Modul Kelas XI Vol (2) (2) (2022) 80-92 Vol (2) (2) (2022) 80-92 e-ISSN 2828-6375 e-ISSN 2828-6375 Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata… 86 b.
Pembahasan Hasil Program Literasi
Menulis (Modul Pembelajaran Dig-
ital) dikembangkan memiliki dua manfaat
yakni sebagai alat bantu belajar siswa di
rumah dan bisa digunakan oleh guru se-
bagai alat bantu mengajar di kelas. Jadi
pembuatan
modul
digital
tersebut
dirancang dengan menarik serta materi
yang dibuat lebih ringkas, supaya siswa se-
mangat untuk membacanya dan akan
mengembangkan
kemampuan
literasi
lanjut. National Center For Vocational Educa-
tion
Research
Ltd
(Prastowo,
2014)
mendefinisikan materi ajar sebagai segala
bentuk materi yang dipakai untuk mem-
bantu pendidik atau instruktur saat
melaksanakan proses pembelajaran di ke-
las. Salah satu bentuk materi ajar adalah
modul (Tania & Susilo wibowo, 2017). Saat
ini, modul terbagi menjadi dua kategori,
yakni modul yang cetak dan modul digital. d.
Hasil Program Literasi Menulis
(Latihan Soal) Untuk mengembangkan kemampuan
literasi menulis siswa di tahap berikutnya
adalah dengan cara memberikan latihan
soal. Semakin
sering
siswa
berlatih
mengerjakan latihan soal, maka kemam-
puan literasi menulisnya pun akan se-
makin meningkat. Dalam tahap ini, latihan
soal pada pembelajaran Bahasa Indonesia
di SMA Prawira Marta dituangkan dalam
modul digital pada bagian akhir. Hasil Menguasai dan mengamalkan suatu
draf tertentu (Zulhaini, 2016). Modul yang Vol (2) (2) (2022) 80-92 e-ISSN 2828-6375 Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata… 87 jawabannya dituangkan kedalamselem-
barkertasdandifotokanlalu dikirimkan ke
nomor whatsapp pendidik sebagai bukti
bahwa siswa telah selesai mengerjakan. kebutuhan dan kemampuan siswa. Jumlah
soal-soal latihannya antara 3-6 butir soal
saja, sehingga siswa tidak akan merasa
terbebani apabila mendapatkan banyak
butir soal. Berikut adalah beberapa contoh
latihan soal yang diberikan ke siswa: Latihan soal dibuat dan dirancang
dengan
sederhana
sesuai
dengan Gambar 4. Hasil Latihan Soal
Kelas X
Gambar 5. Hasil Latihan Soal
Kelas XI
Gambar 6. Hasil Latihan Soal
Kelas XI Gambar 6. Hasil Latihan Soal
Kelas XI Gambar 5. Hasil Latihan Soal
Kelas XI Gambar 4. Hasil Latihan Soal
Kelas X Gambar 4. Hasil Latihan Soal
Kelas X Gambar 5. Hasil Latihan Soal
Kelas XI Gambar 5. Hasil Latihan Soal
Kelas XI Gambar 4. Hasil Latihan Soal
Kelas X Gambar 6. Hasil Latihan Soal
Kelas XI Dengan menulis dapat melatih kita untuk
berpikir kritis dan logis dalam memeca-
hkan masalah (Ati and Widiyarto, 2020). e. Pembahasan Hasil Program Literasi g.
Hasil Program Literasi Menulis
(Pengayaan
dan
Penambahan
Waktu) g. Hasil Program Literasi Menulis
(Pengayaan
dan
Penambahan
Waktu) Berdasarkan teori di atas, kemahiran
pemecahan masalah adalah kemampuan
untuk mengamalkan pemahaman yang di-
miliki sebelumnya ke dalam kondisi baru
yang mengaitkan proses berpikir tingkat
tinggi. Kemahiran pemecahan masalah
(latihan soal) siswa SMA Prawira Marta
ditinjau dari motivasi belajarnya. Perihal
tersebut bertujuan supaya pendidik dapat
menunjang motivasi belajar, memperbaiki
kesalahan dalam pemecahan masalah (lati-
han soal), dan menangani kesulitan yang
dialami oleh siswa. Jadi, semakin sering
siswa berlatih mengerjakan latihan soal,
maka semakin banyak kesempatan siswa
melatih manajemen waktu pengerjaannya. Semakin rutin mengerjakan latihan soal,
maka
siswa
akan
semakin
siap
menghadapi ujian-ujian kedepannya. Untuk mengembangkan kemampuan
literasi menulis siswa pada tahap terakhir
adalah dengan cara memberikan penga-
yaan dan penambahan waktu untuk siswa
yang kemampuan literasi menulisnya ku-
rang. Pemberian pengayaan dan penamba-
han waktu tersebut sebagai bentuk kesem-
patan kepada siswa yang mungkin belum
sempat membaca modul dan mengerjakan
latihan soal karena suatu halangan ter-
tentu. Dalam tahap ini, pemberian penga-
yaan dan penambahan waktu pada siswa
SMA Prawira Marta yang kemampuan lit-
erasi menulisnya kurang dibatasi dengan
waktu yang telah ditentukan. Biasanya
pemberian penambahan waktu pada siswa
berkisar antara satu sampai tujuh hari, ter-
gantung tingkat kesulitan tiap-tiap soal. Berikut adalah beberapa contoh bukti
penambahanwaktu
yang
diberikan
kesiswa yang kemampuan literasi menu-
lisnya kurang:
Si
A e.
Pembahasan Hasil Program Literasi e. Pembahasan Hasil Program Literasi
Menulis (Latihan Soal) Kemahiran
dalam pemecahan masalah (latihan soal)
merupakan salah satu kemampuan pent-
ing yang harus dikuasai siswa. Menurut
Anderson (2009), pemecahan masalah ada-
lah keahlian hidup yang melibatkan teknik
menelaah, menguraikan, melogika, mem-
prediksi, menilai, dan merefleksikan. Ren-
dahnya kemampuan pemecahan masalah
siswa salah satunya disebabkan oleh moti-
vasi belajar. Selain itu, menulis juga meru-
pakan suatu aktivitas komunikasi yang
menggunakan bahasa sebagai medianya. Muzaki (2010) menemukan hasil
bahwa kreativitas dan motivasi belajar
siswa berdampak penting pada keahlian
siswa
dalam
mengatasi
persoalan. Menurut Sihombing (2021) siswa yang
menguasai konsep dapat mengindetifikasi-
kan dan mengerjakan soal baru yang lebih
bervariasi. Selain itu, apabila anak me-
mahami suatu konsep maka ia dapat
menggeneralisasikan suatu objek dalam
berbagai situasi lain yang tidak digunakan Vol (2) (2) (2022) 80-92 Vol (2) (2) (2022) 80-92 e-ISSN 2828-6375 Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata… 88 kesempatan bagi siswa yang belum sempat
mengerjakan latihan soal. dalam situasi belajar. Hal ini menyimpul-
kan bahwa apabila siswa mempunyai kre-
ativitas dan motivasi belajar yang tinggi
maka kemahiran siswa dalam memeca-
hkan persoalan juga akan baik. f.
Evaluasi dan Tindak Lanjut Latihan-latihan soal yang dituangkan
dalam modul digital pada bagian akhir ter-
sampaikan kepada seluruh siswa SMA
Prawira Marta dengan baik dan beberapa
siswa
mampu
merespon
serta
mengerjakan dengan baik pula. Namun,
ada pula siswa yang lambat bahkan ada
yang tidak mengerjakannya sama sekali. Untuk mengatasi masalah tersebut, pen-
didik selalu menghimbau dan memotivasi
siswa supaya mau berusaha mengerjakan
latihan soal semampunya dan memberikan Siswa B
Siswa C Siswa C Vol (2) (2) (2022) 80-92 e-ISSN 2828-6375 Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata… 89 Berdasarkan pemaparan di atas, ke-
mampuan literasi menulis siswa SMA
Prawira Marta berbeda-beda. Hal ini ter-
bukti dari siswa yang mengumpulkan tu-
gas ke guru tiap minggunya hanya sedikit. Bahkan, rata-rata siswanya hampir semua
memiliki kemampuan literasi menulis
yang kurang. Dengan demikian, pem-
berian pengayaan dan penambahan waktu
sangat penting dan dibutuhkan siswa un-
tuk menyeimbangkan siswa satu dengan
yang
lain,
serta
meningkatkan
ke-
percayaan diri siswa-siswa yang lit-
erasinya kurang supaya mereka yakin
bahwa mereka bisa meningkatkan kemam-
puannya seperti siswa lain yang kemam-
puan literasinya baik. h. Pembahasan Hasil Program Literasi
Menulis (Pengayaan dan Penamba-
han Waktu) h.
Pembahasan Hasil Program Literasi
Menulis (Pengayaan dan Penamba-
han Waktu) Kegiatan pengayaan yakni aktivitas
yang diberikan kepada siswa agar mereka
mampu
mengembangkan
potensinya
secara optimal dengan memanfaatkan sisa
waktu yang dimilikinya. Aktivitas tersebut
bertujuan untuk memberikan kesempatan
bagi siswa untuk memperdalam pema-
hamannya tentang materi pelajaran yang
bertautan dengan tugas yang sedang dik-
erjakan, sehingga dapat mencapai tingkat
perkembangan yang baik dan optimal. Jadi, dapat dikatakan bahwa pengayaan
merupakan aktivitas yang diberikan pada
siswa supaya mereka menjadi lebih kaya
pengetahuan dan kemahirannya serta bisa
mendalami materi pembelajaran yang se-
dang mereka pelajari. i.
Evaluasi dan Tindak Lanjut Pemberian pengayaan dan penamba-
han waktu yang diberikankepada siswa
SMA Prawira Marta yang memiliki ke-
mampuan literasi menulisnya kurang
dapat terlaksana dengan baik. Beberapa
siswa mampu memanfaatkan kesempatan
tersebut untuk mengerjakan tugas-tugas
yang tertunda. Namun, masih ada pula
siswa yang tidak memanfaatkan kesem-
patan tersebut dengan baik. Untuk menga-
tasi masalah tersebut, pendidik selalu
memotivasi dan mendorong semangat
siswa supaya mereka mampu memanfaat-
kan kesempatan tambahan waktu tersebut
dengan sebaik-baiknya untuk menutup
nilai-nilai yang kosong dan dengan hara-
pan mampu meningkatkan kemampuan
literasi menulis mereka. Ketika guru merancang tugas yang
akan diberikan kepada siswa untuk dik-
erjakan di rumah, maka guru harus dapat
memperhitungkan jangka waktu pengum-
pulan tugas tersebut sesuai tingkat kesu-
litan soal dan kemampuan masing- masing
siswa. Jika tugas tersebut diselesaikan da-
lam waktu 3 hari, maka guru dapat mem-
berikan tambahan waktu sekitar 1-7 hari
kepada siswa yang kemampuan literasi
menulisnya kurang. Dengan begitu, siswa
yang kemampuan literasi menulisnya ku-
rang akan mampu mengimbangi siswa lain
yang kemampuan literasi menulisnya baik. Bahkan, rata-rata siswanya hampir
semua
memiliki
kemampuan
literasi Vol (2) (2) (2022) 80-92 Vol (2) (2) (2022) 80-92 e-ISSN 2828-6375 Peningkatan Literasi Menulis Siswa SMA Prawira Marta Melalui Pendampingan Mata… 90 Y., Agung, W., & Sholihah, I. (2020). Revitalisasi
Perpustakaan
untuk
Meningkatkan Minat Literasi Siswa
di SD Muhammadiyah Nurul Ilmi,
Klaten. Buletin KKN Pendidikan, 1(2),
97–105. Y., Agung, W., & Sholihah, I. (2020). Revitalisasi
Perpustakaan
untuk
Meningkatkan Minat Literasi Siswa
di SD Muhammadiyah Nurul Ilmi,
Klaten. Buletin KKN Pendidikan, 1(2),
97–105. menulis yang kurang. Dengan demikian,
pemberian pengayaan dan penambahan
waktu sangat penting dan dibutuhkan
siswa untuk menyeimbangkan siswa satu
dengan yang lain, serta meningkatkan ke-
percayaan diri siswa-siswa yang lit-
erasinya kurang supaya mereka yakin
bahwa mereka bisa meningkatkan kemam-
puannya seperti siswa lain yang kemam-
puan literasinya baik. Ati, A.P. and Widiyarto, S. (2020) ‘Literasi
Bahasa Dalam Meningkatkan Minat
Baca Dan Menulis Pada Siswa Smp
Kota Bekasi’, Basastra, 9(1), p. 105. Choirudin, F. M., & Rahmasari, S. N. (2021). Tingkat Cakap Tanggap Pe-
serta Didik dalam Perangkat Google
Classroom Selama Pembelajaran Dar-
ing. Jurnal Ilmiah Kampus Mengajar,
62-69. 5.
Daftar Pustaka
Agustina, L., Arffianto, A., Khalishah, S.
H., Indarwati, L., Putri, D. R., El-
Majid, S. E., Rahayu, K. S., Nurleli, D. 4.
Simpulan Kegiatan KKN-Dik di SMA Prawira
Marta berjalan dengan lancar sesuai
dengan apa yang direncanakan dalam pro-
gram kerja. Adapun kesimpulan dari
pelaksanaan program kerja KKN-Dik Te-
matik ini yaitu: (1) Pelaksanaan kegiatan
KKN-Dikmas di SMA Prawira Marta dim-
ulai tanggal 1 Februari 2021 sampai dengan
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https://figshare.com/articles/journal_contribution/Supplementary_Figure_2_from_A_Fully_Human_Insulin-Like_Growth_Factor-I_Receptor_Antibody_SCH_717454_Robatumumab_Has_Antitumor_Activity_as_a_Single_Agent_and_in_Combination_with_Cytotoxics_in_Pediatric_Tumor_Xenografts/22485270/1/files/39936825.pdf
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Supplementary Figure 3 from A Fully Human Insulin-Like Growth Factor-I Receptor Antibody SCH 717454 (Robatumumab) Has Antitumor Activity as a Single Agent and in Combination with Cytotoxics in Pediatric Tumor Xenografts
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https://www.cambridge.org/engage/api-gateway/coe/assets/orp/resource/item/61134722abc9e20d306ae1d0/original/robin-criterion-on-divisibility.pdf
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Robin Criterion on Divisibility
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Mathematics Subject Classification (2010) MSC 11M26 · MSC 11A41 · MSC
11A25 Mathematics Subject Classification (2010) MSC 11M26 · MSC 11A41 · MSC
11A25 Noname manuscript No.
(will be inserted by the editor) Noname manuscript No. (will be inserted by the editor) Noname manuscript No. (will be inserted by the editor) F. Vega
CopSonic, 1471 Route de Saint-Nauphary 82000 Montauban, France
ORCiD: 0000-0001-8210-4126
E-mail: vega.frank@gmail.com Robin Criterion on Divisibility Frank Vega the date of receipt and acceptance should be inserted later Abstract Robin criterion states that the Riemann Hypothesis is true if and only if the
inequality σ(n) < eγ ×n×loglogn holds for all n > 5040, where σ(n) is the sum-of-
divisors function and γ ≈0.57721 is the Euler-Mascheroni constant. We show that the
Robin inequality is true for all n > 5040 which are not divisible by any prime number
between 2 and 953. We prove that the Robin inequality holds when π2
6 ×loglogn′ ≤
loglogn for some n > 5040 where n′ is the square free kernel of the natural number
n. The possible smallest counterexample n > 5040 of the Robin inequality complies
that necessarily (logn)β < 1.2592 × log(Nm), where Nm = ∏m
i=1 qi is the primorial
number of order m and β = ∏m
i=1
qai+1
i
qai+1
i
−1 when n is an Hardy-Ramanujan integer of
the form ∏m
i=1 qai
i . the form ∏m
i=1 qai
i . Keywords Riemann hypothesis · Robin inequality · sum-of-divisors function ·
prime numbers Theorem 1.4 The smallest counterexample of the Robin inequality greater than 5040
must be a superabundant number [1]. On the other hand, suppose that n > 5040 might be the possible smallest coun-
terexample of the Robin inequality, then we demonstrate that necessarily (logn)β <
1.2592 × log(Nm), where Nm = ∏m
i=1 qi is the primorial number of order m and β =
qai+1
ai ∏m
i=1
q i
i
qai+1
i
−1 when n is an Hardy-Ramanujan integer of the form ∏m
i=1 qai
i . ∏m
i=1
qi
qai+1
i
−1 when n is an Hardy-Ramanujan integer of the form ∏m
i=1 qai
i . 1 Introduction In mathematics, the Riemann Hypothesis is a conjecture that the Riemann zeta func-
tion has its zeros only at the negative even integers and complex numbers with real
part 1
2 [6]. As usual σ(n) is the sum-of-divisors function of n [3]: ∑
d|n
d 2 F. Vega F. Vega where d | n means the integer d divides to n and d ∤n means the integer d does not
divide to n. Define f(n) to be σ(n)
n . Say Robins(n) holds provided where d | n means the integer d divides to n and d ∤n means the integer d does not
divide to n. Define f(n) to be σ(n)
n . Say Robins(n) holds provided f(n) < eγ ×loglogn. f(n) < eγ ×loglogn. The constant γ ≈0.57721 is the Euler-Mascheroni constant, and log is the natural
logarithm. The importance of this property is: Theorem 1.1 Robins(n) holds for all n > 5040 if and only if the Riemann Hypothesis
is true [6]. Theorem 1.1 Robins(n) holds for all n > 5040 if and only if the Riemann Hypothesis
is true [6]. It is known that Robins(n) holds for many classes of numbers n. Theorem 1.2 Robins(n) holds for all n > 5040 that are not divisible by 2 [3]. Theorem 1.2 Robins(n) holds for all n > 5040 that are not divisible by 2 [3]. On the one hand, we prove that Robins(n) holds for all n > 5040 that are not divisible
by any prime number between 3 and 953. We recall that an integer n is said to be
square free if for every prime divisor q of n we have q2 ∤n [3]. Robins(n) holds for all
n > 5040 that are square free [3]. In addition, we show that Robins(n) holds for some
n > 5040 when π2
6 ×loglogn′ ≤loglogn such that n′ is the square free kernel of the
natural number n. Let q1 = 2,q2 = 3,...,qm denote the first m consecutive primes,
then an integer of the form ∏m
i=1 qai
i with a1 ≥a2 ≥··· ≥am ≥0 is called an Hardy-
Ramanujan integer [3]. A natural number n is called superabundant precisely when,
for all m < n f(m) < f(n). Theorem 1.3 If n is superabundant, then n is an Hardy-Ramanujan integer [2]. Theorem 1.3 If n is superabundant, then n is an Hardy-Ramanujan integer [2]. Theorem 1.4 The smallest counterexample of the Robin inequality greater than 5040
must be a superabundant number [1]. 2 A Central Lemma These are known results: These are known results: These are known results: Lemma 2.1 [3]. For n > 1: Lemma 2.1 [3]. For n > 1: f(n) < ∏
q|n
q
q−1. (2.1) (2.1) Lemma 2.2 [4]. Lemma 2.2 [4]. ∞
∏
k=1
1
1−1
q2
k
= ζ(2) = π2
6 . (2.2) (2.2) 3 3 Robin Criterion on Divisibility The following is a key lemma. It gives an upper bound on f(n) that holds for all n. The bound is too weak to prove Robins(n) directly, but is critical because it holds for
all n. Further the bound only uses the primes that divide n and not how many times
they divide n. Lemma 2.3 Let n > 1 and let all its prime divisors be q1 < ··· < qm. Then, f(n) < π2
6 ×
m
∏
i=1
qi +1
qi
. Proof We use that lemma 2.1: Proof We use that lemma 2.1: Proof We use that lemma 2.1: Now for q > 1, So So Then by lemma 2.2, Then by lemma 2.2, q
Putting this together yields the proof: f(n) <
m
∏
i=1
qi
qi −1
≤
m
∏
i=1
1
1−1
q2
i
× qi +1
qi
< π2
6 ×
m
∏
i=1
qi +1
qi
. f(n) <
m
∏
i=1
qi
qi −1
≤
m
∏
i=1
1
1−1
q2
i
× qi +1
qi
< π2
6 ×
m
∏
i=1
qi +1
qi
. f(n) <
m
∏
i=1
qi
qi −1
≤
m
∏
i=1
1
1−1
q2
i
× qi +1
qi
< π2
6 ×
m
∏
i=1
qi +1
qi
. 3 About the p-adic order In basic number theory, for a given prime number p, the p-adic order of a natural
number n is the highest exponent νp ≥1 such that pνp divides n. This is a known
result: Lemma 3.1 In general, we know that Robins(n) holds for a natural number n > 5040
that satisfies either ν2(n) ≤19, ν3(n) ≤12 or ν7(n) ≤6, where νp(n) is the p-adic
order of n [5]. F. Vega 4 We know the following lemmas: We know the following lemmas: Lemma 3.2 [5]. Let ∏m
i=1 qai
i be the representation of n as a product of primes q1 <
··· < qm with natural numbers as exponents a1,...,am. Then, f(n) =
m
∏
i=1
qi
qi −1
! ×
m
∏
i=1
1−
1
qai+1
i
! . Lemma 3.3
[5]. Let n > ee23.762143 and let all its prime divisors be q1 < ··· < qm,
then m
∏
i=1
qi
qi −1
! < 1771561
1771560 ×eγ ×loglogn. Lemma 3.4 Robins(n) holds for all 101010 ≥n > 5040 [5]. since 923520
923521 × 1771561
1771560 < 1. In light of lemma 3.4 and the fact that ee23.762143 < 101010,
we then conclude that Robins(n) holds for n > 5040 when ν31(n) ≤3. Lemma 3.4 Robins(n) holds for all 101010 ≥n > 5040 [5]. Putting together all these results, then we obtain that Putting together all these results, then we obtain that Lemma 3.5 Robins(n) holds for n > 5040 when ν31(n) ≤3. Lemma 3.5 Robins(n) holds for n > 5040 when ν31(n) ≤3. Lemma 3.5 Robins(n) holds for n > 5040 when ν31(n) ≤3. Proof From lemma 3.2, we note that Proof From lemma 3.2, we note that f(n) =
m
∏
i=1
qi
qi −1
! ×
m
∏
i=1
1−
1
qai+1
i
! ≤
m
∏
i=1
qi
qi −1
! ×
1−
1
31ν31(n)+1
when ν31(n) ≤3. We only need to look at the case where ν31(n) = 3 since the weaker
cases follow because when ν31(n) ≤3. We only need to look at the case where ν31(n) = 3 since the weaker
cases follow because
1−
1
311+1
<
1−
1
312+1
<
1−
1
313+1
. In this way, we obtain that In this way, we obtain that f(n) ≤
m
∏
i=1
qi
qi −1
! ×
1−
1
313+1
= 923520
923521 ×
m
∏
i=1
qi
qi −1
! when ν31(n) ≤3. With lemma 3.3, we have for n > ee23.762143 923520
923521 ×
m
∏
i=1
qi
qi −1
! < 923520
923521 × 1771561
1771560 ×eγ ×loglogn < eγ ×loglogn since 923520
923521 × 1771561
1771560 < 1. In light of lemma 3.4 and the fact that ee23.762143 < 101010,
we then conclude that Robins(n) holds for n > 5040 when ν31(n) ≤3. since 923520
923521 × 1771561
1771560 < 1. In light of lemma 3.4 and the fact that ee23.762143 < 101010,
we then conclude that Robins(n) holds for n > 5040 when ν31(n) ≤3. 5 Robin Criterion on Divisibility A Particular Case 4 A Particular Case 4 A Particular Case 4 A Particular Case We can easily prove that Robins(n) is true for certain kind of numbers: Lemma 4.1 Robins(n) holds for n > 5040 when q ≤7, where q is the largest prime
divisor of n. Proof Let n > 5040 and let all its prime divisors be q1 < ··· < qm ≤5, then we need
to prove Proof Let n > 5040 and let all its prime divisors be q1 < ··· < qm ≤5, then we need
to prove f(n) < eγ ×loglogn f(n) < eγ ×loglogn that is true when that is true when
m
∏
i=1
qi
qi −1 ≤eγ ×loglogn
according to the lemma 2.1. For q1 < ··· < qm ≤5, m
∏
i=1
qi
qi −1 ≤eγ ×loglogn
. For q1 < ··· < qm ≤5, m
∏
i=1
qi
qi −1 ≤eγ ×loglogn according to the lemma 2.1. For q1 < ··· < qm ≤5, m
∏
i=1
qi
qi −1 ≤2×3×5
1×2×4 = 3.75 < eγ ×loglog(5040) ≈3.81. However, we know for n > 5040 However, we know for n > 5040 eγ ×loglog(5040) < eγ ×loglogn and therefore, the proof is complete when q1 < ··· < qm ≤5. The remaining case
is for n > 5040 when all its prime divisors are q1 < ··· < qm ≤7. Robins(n) holds
for n > 5040 when ν7(n) ≤6 according to the lemma 3.1 [5]. Hence, it is enough to
prove this for those natural numbers n > 5040 when 77 | n. For q1 < ··· < qm ≤7, m
∏
i=1
qi
qi −1 ≤2×3×5×7
1×2×4×6 = 4.375 < eγ ×loglog(77) ≈4.65. However, for n > 5040 and 77 | n, we know that However, for n > 5040 and 77 | n, we know that eγ ×loglog(77) ≤eγ ×loglogn and as a consequence, the proof is complete when q1 < ··· < qm ≤7. and as a consequence, the proof is complete when q1 < ··· < qm ≤7. 5 A Better Bound This is a known result: This is a known result: Lemma 5.1 [7]. For x > 1:
∑
q≤x
1
q < loglogx+B+
1
log2 x
(5.1)
where
B = 0.2614972128···
denotes the (Meissel )Mertens constant [7] a 5.1 [7]. For x > 1:
∑
q≤x
1
q < loglogx+B+
1
log2 x
(5.1) ∑
q≤x
1
q < loglogx+B+
1
log2 x
(5.1) (5.1) where where
B = 0.2614972128··· denotes the (Meissel-)Mertens constant [7]. denotes the (Meissel-)Mertens constant [7]. F. Vega 6 We show a better result: We show a better result: Lemma 5.2 For x ≥11, we have ∑
q≤x
1
q < loglogx+γ −0.12. ∑
q≤x
1
q < loglogx+γ −0.12. Proof Let’s define H = γ −B. The lemma 5.1 is the same as Proof Let’s define H = γ −B. The lemma 5.1 is the same as Proof Let s define H = γ −B. The lemma 5.1 is the same as
∑
q≤x
1
q < loglogx+γ −(H −
1
log2 x). For x ≥11,
(H −
1
log2 x) > (0.31−
1
log2 11) > 0.12
and thus,
∑
q≤x
1
q < loglogx+γ −(H −
1
log2 x) < loglogx+γ −0.1 ∑
q≤x
1
q < loglogx+γ −(H −
1
log2 x). (H −
1
log2 x) > (0.31−
1
log2 11) > 0.12 ∑
q≤x
1
q < loglogx+γ −(H −
1
log2 x). For x ≥11, and thus, ∑
q≤x
1
q < loglogx+γ −(H −
1
log2 x) < loglogx+γ −0.12. 6 On a Square Free Number We know the following results: We know the following results: We know the following results: Lemma 6.1 [3]. For 0 < a < b: Lemma 6.1 [3]. For 0 < a < b: Lemma 6.1 [3]. For 0 < a < b: Lemma 6.1 [3]. For 0 < a < b: logb−loga
b−a
=
1
(b−a)
Z b
a
dt
t > 1
b. (6.1) (6.1) Lemma 6.2 [3]. For q > 0: log(q+1)−logq =
Z q+1
q
dt
t < 1
q. (6.2) (6.2) We recall that an integer n is said to be square free if for every prime divisor q of
n we have q2 ∤n [3]. Robins(n) holds for all n > 5040 that are square free [3]. Lemma 6.3 For a square free number Lemma 6.3 For a square free number n = q1 ×···×qm such that q1 < q2 < ··· < qm are odd prime numbers, qm ≥11 and 3 ∤n, then: π2
6 × 3
2 ×σ(n) ≤eγ ×n×loglog(219 ×n). 7 Robin Criterion on Divisibility Proof By induction with respect to ω(n), that is the number of distinct prime factors
of n [3]. Put ω(n) = m [3]. We need to prove the assertion for those integers with
m = 1. From a square free number n, we obtain σ(n) = (q1 +1)×(q2 +1)×···×(qm +1)
(6.3) (6.3) when n = q1 × q2 × ··· × qm [3]. In this way, for every prime number qi ≥11, then
we need to prove π2
6 × 3
2 ×(1+ 1
qi
) ≤eγ ×loglog(219 ×qi). (6.4) (6.4) For qi = 11, we have π2
6 × 3
2 ×(1+ 1
11) ≤eγ ×loglog(219 ×11) is actually true. For another prime number qi > 11, we have is actually true. For another prime number qi > 11, we have (1+ 1
qi
) < (1+ 1
11) and loglog(219 ×11) < loglog(219 ×qi) which clearly implies that the inequality (6.4) is true for every prime number qi ≥11. Now, suppose it is true for m−1, with m ≥2 and let us consider the assertion for those
square free n with ω(n) = m [3]. So let n = q1 ×···×qm be a square free number and
assume that q1 < ··· < qm for qm ≥11. 6 On a Square Free Number q1
qm
qm ≥
Case 1: qm ≥log(219 ×q1 ×···×qm−1 ×qm) = lo q1
qm
qm
Case 1: qm ≥log(219 ×q1 ×···×qm−1 ×qm) = log(219 ×n). q1
qm
qm
Case 1: qm ≥log(219 ×q1 ×···×qm−1 ×qm) = log(219 ×n). (
By the induction hypothesis we have π2
6 × 3
2 ×(q1+1)×···×(qm−1+1) ≤eγ ×q1×···×qm−1×loglog(219×q1×···×qm−1) and hence and hence π2
6 × 3
2 ×(q1 +1)×···×(qm−1 +1)×(qm +1) ≤
eγ ×q1 ×···×qm−1 ×(qm +1)×loglog(219 ×q1 ×···×qm−1) when we multiply the both sides of the inequality by (qm +1). We want to show when we multiply the both sides of the inequality by (qm +1). We want to show eγ ×q1 ×···×qm−1 ×(qm +1)×loglog(219 ×q1 ×···×qm−1) ≤
eγ ×q1×···×qm−1×qm×loglog(219×q1×···×qm−1×qm) = eγ ×n×loglog(219×n). Indeed the previous inequality is equivalent with
qm ×loglog(219 ×q1 ×···×qm−1 ×qm) ≥(qm +1)×loglog(219 ×q1 ×···×qm−1)
or alternatively eγ ×q1 ×···×qm−1 ×(qm +1)×loglog(219 ×q1 ×···×qm−1) ≤
eγ ×q1×···×qm−1×qm×loglog(219×q1×···×qm−1×qm) = eγ ×n×loglog(219×n). I d
d h
i
i
li
i
i
l
i h Indeed the previous inequality is equivalent with qm ×loglog(219 ×q1 ×···×qm−1 ×qm) ≥(qm +1)×loglog(219 ×q1 ×···×qm−1)
or alternatively qm ×(loglog(219 ×q1 ×···×qm−1 ×qm)−loglog(219 ×q1 ×···×qm−1))
logqm
≥ logqm F. Vega 8 We can apply the inequality in lemma 6.1 just using b = log(219 ×q1 ×···×qm−1 ×
qm) and a = log(219 ×q1 ×···×qm−1). Certainly, we have We can apply the inequality in lemma 6.1 just using b = log(219 ×q1 ×···×qm−1 ×
qm) and a = log(219 ×q1 ×···×qm−1). Certainly, we have We can apply the inequality in lemma 6.1 just using b = log(21
qm) and a = log(219 ×q1 ×···×qm−1). Certainly, we have log(219 ×q1 ×···×qm−1 ×qm)−log(219 ×q1 ×···×qm−1) =
log 219 ×q1 ×···×qm−1 ×qm
219 ×q1 ×···×qm−1
= logqm. In this way, we obtain In this way, we obtain qm ×(loglog(219 ×q1 ×···×qm−1 ×qm)−loglog(219 ×q1 ×···×qm−1))
logqm
>
qm
log(219 ×q1 ×···×qm). qm ×(loglog(219 ×q1 ×···×qm−1 ×qm)−loglog(219 ×q1 ×···×qm−1))
logqm
> Using this result we infer that the original inequality is certainly satisfied if the next
inequality is satisfied qm
log(219 ×q1 ×···×qm) ≥loglog(219 ×q1 ×···×qm−1)
logqm which is trivially true for qm ≥log(219 ×q1 ×···×qm−1 ×qm) [3]. Case 2: qm < log(219 ×q1 ×···×qm−1 ×qm) = log(219 ×n). We need to prove π2
6 × 3
2 × σ(n)
n
≤eγ ×loglog(219 ×n). We know 3
2 < 1.503 <
4
2.66. 6 On a Square Free Number Nevertheless, we could have We know 3
2 < 1.503 <
4
2.66. Nevertheless, we could have 3
2 × σ(n)
n
× π2
6 < 4×σ(n)
3×n
×
π2
2×2.66 and therefore, we only need to prove and therefore, we only need to prove σ(3×n)
3×n
× π2
5.32 ≤eγ ×loglog(219 ×n) where this is possible because of 3 ∤n. If we apply the logarithm to the both sides of
the inequality, then we obtain log( π2
5.32)+(log(3+1)−log3)+
m
∑
i=1
(log(qi +1)−logqi) ≤γ +logloglog(219 ×n). In addition, note that log( π2
5.32) < 1
2 +0.12. However, we know 2) < 1
2 +0.12. However, we know In addition, note that log( π2
5.32) < 1
2 +0.12. However, we know In addition, note that log( π2
5.32) < 1
2 +0.12. However, we know In addition, note that log( π2
5.32) < 1
2 +0.12. However, we know
γ +loglogqm < γ +logloglog(219 ×n) In addition, note that log( π2
5.32) < 1
2 +0.12. However, we know
γ +loglogqm < γ +logloglog(219 ×n) γ +loglogqm < γ +logloglog(219 ×n) 9 Robin Criterion on Divisibility since qm < log(219 ×n). We use that lemma 6.2 for each term log(q+1)−logq and
thus, since qm < log(219 ×n). We use that lemma 6.2 for each term log(q+1)−logq and
thus 0.12+ 1
2 + 1
3 + 1
q1
+···+ 1
qm
≤0.12+ ∑
q≤qm
1
q ≤γ +loglogqm where qm ≥11. Hence, it is enough to prove where qm ≥11. Hence, it is enough to prove ∑
q≤qm
1
q ≤γ +loglogqm −0.12 ∑
q≤qm
1
q ≤γ +loglogqm −0.12 but this is true according to the lemma 5.2 for qm ≥11. In this way, we finally show
the lemma is indeed satisfied. but this is true according to the lemma 5.2 for qm ≥11. In this way, we finally show
the lemma is indeed satisfied. 7 Robin on Divisibility Robins(n) holds for every n > 5040 that is not divisible by 2 [3]. We extend this
property to other prime numbers: Lemma 7.1 Robins(n) holds for all n > 5040 when 3 ∤n. More precisely: every pos-
sible counterexample n > 5040 of the Robin inequality must comply with (220 ×313) |
n. Proof We will check the Robin inequality is true for every natural number n =
qa1
1 × qa2
2 × ··· × qam
m > 5040 such that q1,q2,··· ,qm are distinct prime numbers,
a1,a2,··· ,am are natural numbers and 3 ∤n. We know this is true when the great-
est prime divisor of n > 5040 is lesser than or equal to 7 according to the lemma 4.1. Therefore, the remaining case is when the greatest prime divisor of n > 5040 is greater
than or equal to 11. We need to prove f(n) < eγ ×loglogn that is true when that is true when π2
6 ×
m
∏
i=1
qi +1
qi
≤eγ ×loglogn according to the lemma 2.3. Using the formula (6.3) for the square free numbers, then
we obtain that is equivalent to π2
6 × σ(n′)
n′
≤eγ ×loglogn where n′ = q1 × ··· × qm is the square free kernel of the natural number n [3]. The
Robin inequality has been proved for all integers n not divisible by 2 (which are
bigger than 10) [3]. Hence, we only need to prove the Robin inequality is true when
2 | n′. In addition, we know that Robins(n) holds for every n > 5040 when ν2(n) ≤19
according to the lemma 3.1 [5]. Consequently, we only need to prove that Robins(n)
holds for n > 5040 when 220 | n and thus, eγ ×n′ ×loglog(219 × n′
2 ) ≤eγ ×n′ ×loglogn F. Vega 10 because of 219 × n′
2 ≤n where 220 | n and 2 | n′. So, because of 219 × n′
2 ≤n where 220 | n and 2 | n′. So, because of 219 × n′
2 ≤n where 220 | n and 2 | n′. So, π2
6 ×σ(n′) ≤eγ ×n′ ×loglog(219 × n′
2 ). 7 Robin on Divisibility According to the formula (6.3) for the square free numbers and 2 | n′, then, According to the formula (6.3) for the square free numbers and 2 | n′, then, π2
6 ×3×σ(n′
2 ) ≤eγ ×2× n′
2 ×loglog(219 × n′
2 ) which is the same as which is the same as π2
6 × 3
2 ×σ(n′
2 ) ≤eγ × n′
2 ×loglog(219 × n′
2 ) where this is true according to the lemma 6.3 when 3 ∤n′
2 . In addition, we know that
Robins(n) holds for every n > 5040 when ν3(n) ≤12 according to the lemma 3.1 [5]. Hence, we only need to prove that Robins(n) holds for every n > 5040 when 220 | n
and 313 | n. To sum up, the proof is complete. Lemma 7.2 Robins(n) holds for all n > 5040 when 5 ∤n or 7 ∤n. Lemma 7.2 Robins(n) holds for all n > 5040 when 5 ∤n or 7 ∤n. Proof We need to prove Proof We need to prove f(n) < eγ ×loglogn when (220 ×313) | n. Suppose that n = 2a ×3b ×m, where a ≥20, b ≥13, 2 ∤m, 3 ∤m
and 5 ∤m or 7 ∤m. Therefore, we need to prove f(2a ×3b ×m) < eγ ×loglog(2a ×3b ×m). We know Lemma 7.3 Robins(n) holds for all n > 5040 when a prime number 11 ≤q ≤4
complies with q ∤n. Proof We know that Robins(n) holds for every n > 5040 when ν7(n) ≤6 according
to the lemma 3.1 [5]. We need to prove Proof We know that Robins(n) holds for every n > 5040 when ν7(n) ≤6 according
to the lemma 3.1 [5]. We need to prove f(n) < eγ ×loglogn when (220 ×313 ×77) | n. Suppose that n = 2a ×3b ×7c ×m, where a ≥20, b ≥13,
c ≥7, 2 ∤m, 3 ∤m, 7 ∤m, q ∤m and 11 ≤q ≤47. Therefore, we need to prove when (220 ×313 ×77) | n. Suppose that n = 2a ×3b ×7c ×m, where a ≥20, b ≥13,
c ≥7, 2 ∤m, 3 ∤m, 7 ∤m, q ∤m and 11 ≤q ≤47. Therefore, we need to prove f(2a ×3b ×7c ×m) < eγ ×loglog(2a ×3b ×7c ×m). f(2a ×3b ×7c ×m) < eγ ×loglog(2a ×3b ×7c ×m). We know We know f(2a ×3b ×m) = f(3b)× f(2a ×m) since f is multiplicative [3]. In addition, we know f(3b) < 3
2 for every natural number
b [3]. In this way, we have f(3b)× f(2a ×m) < 3
2 × f(2a ×m). However, that would be equivalent to However, that would be equivalent to 3
2 × f(2a ×m) = 9
8 × f(3)× f(2a ×m) = 9
8 × f(2a ×3×m) where f(3) = 4
3 since f is multiplicative [3]. Nevertheless, we have where f(3) = 4
3 since f is multiplicative [3]. Nevertheless, we have 9
8 × f(2a ×3×m) < f(5)× f(2a ×3×m) = f(2a ×3×5×m) 9
8 × f(2a ×3×m) < f(5)× f(2a ×3×m) = f(2a ×3×5×m) and 9
8 × f(2a ×3×m) < f(7)× f(2a ×3×m) = f(2a ×3×7×m) 11 Robin Criterion on Divisibility where 5 ∤m or 7 ∤m, f(5) = 6
5 and f(7) = 8
7. We know the Robin inequality is true
for 2a ×3×5×m and 2a ×3×7×m when a ≥20, since this is true for every natural
number n > 5040 when ν3(n) ≤12 according to the lemma 3.1 [5]. Hence, we would
h f(2a ×3×7×m) < eγ ×loglog(2a ×3×7×m) < eγ ×loglog(2a ×3b ×m)
when b ≥13. Lemma 7.3 Robins(n) holds for all n > 5040 when a prime number 11 ≤q ≤47
complies with q ∤n. f(n) < eγ ×loglogn f(n) < eγ ×loglogn when (220 ×313 ×314) | n. Suppose that n = 2a ×3b ×31c ×m, where a ≥20, b ≥13,
c ≥4, 2 ∤m, 3 ∤m, 31 ∤m, q ∤m and 53 ≤q ≤953. Therefore, we need to prove that f(2a ×3b ×31c ×m) < eγ ×loglog(2a ×3b ×31c ×m). f(2a ×3b ×31c ×m) < eγ ×loglog(2a ×3b ×31c ×m). We know that We know f(2a ×3b ×7c ×m) = f(7c)× f(2a ×3b ×m) f(2a ×3b ×7c ×m) = f(7c)× f(2a ×3b ×m) since f is multiplicative [3]. In addition, we know f(7c) < 7
6 for every natural number
c [3]. In this way, we have f(7c)× f(2a ×3b ×m) < 7
6 × f(2a ×3b ×m). However, that would be equivalent to However, that would be equivalent to 7
6 × f(2a ×3b ×m) = 49
48 × f(7)× f(2a ×3b ×m) = 49
48 × f(2a ×3b ×7×m)
where f(7) = 8
7 since f is multiplicative [3] In addition we know where f(7) = 8
7 since f is multiplicative [3]. In addition, we know 49
48 × f(2a ×3b ×7×m) < f(q)× f(2a ×3b ×7×m) = f(2a ×3b ×7×q×m) 49
48 × f(2a ×3b ×7×m) < f(q)× f(2a ×3b ×7×m) = f(2a ×3b ×7×q×m) where q ∤m, f(q) = q+1
q and 11 ≤q ≤47. Nevertheless, we know the Robin inequality
is true for 2a × 3b × 7 × q × m when a ≥20 and b ≥13, since this is true for every
natural number n > 5040 when ν7(n) ≤6 according to the lemma 3.1 [5]. Hence, we
would have where q ∤m, f(q) = q+1
q and 11 ≤q ≤47. Nevertheless, we know the Robin inequality
is true for 2a × 3b × 7 × q × m when a ≥20 and b ≥13, since this is true for every
natural number n > 5040 when ν7(n) ≤6 according to the lemma 3.1 [5]. Hence, we
would have f(2a ×3b ×7×q×m) < eγ ×loglog(2a ×3b ×7×q×m)
< eγ ×loglog(2a ×3b ×7c ×m) when c ≥7 and 11 ≤q ≤47. when c ≥7 and 11 ≤q ≤47. 12 F. Vega F. Vega Lemma 7.4 Robins(n) holds for all n > 5040 when a prime number 53 ≤q ≤953
complies with q ∤n. Lemma 7.4 Robins(n) holds for all n > 5040 when a prime number 53 ≤q ≤953
complies with q ∤n. Lemma 7.4 Robins(n) holds for all n > 5040 when a prime number 53 ≤q ≤953
complies with q ∤n. Proof We know that Robins(n) holds for every n > 5040 when ν31(n) ≤3 according
to the lemma 3.5. We need to prove that We know that f(2a ×3b ×31c ×m) = f(31c)× f(2a ×3b ×m) since f is multiplicative [3]. In addition, we know that f(31c) < 31
30 for every natural
number c [3]. In this way, we have that f(31c)× f(2a ×3b ×m) < 31
30 × f(2a ×3b ×m). However, that would be equivalent to 31
30 × f(2a ×3b ×m) = 961
960 × f(31)× f(2a ×3b ×m) = 961
960 × f(2a ×3b ×31×m) where f(31) = 32
31 since f is multiplicative [3]. In addition, we know that 961
960 × f(2a ×3b ×31×m) < f(q)× f(2a ×3b ×31×m) = f(2a ×3b ×31×q×m) where q ∤m, f(q) = q+1
q
and 53 ≤q ≤953. Nevertheless, we know the Robin in-
equality is true for 2a ×3b ×31×q×m when a ≥20 and b ≥13, since this is true for
every natural number n > 5040 when ν31(n) ≤3 according to the lemma 3.5. Hence,
we would have that f(2a ×3b ×31×q×m) < eγ ×loglog(2a ×3b ×31×q×m)
< eγ ×loglog(2a ×3b ×31c ×m) when c ≥4 and 53 ≤q ≤953. 13 Robin Criterion on Divisibility 13 8 Helpful Lemmas For every prime number pn > 2, we define sequence Yn = e
1
2×log(pn)
(1−
1
log(pn) ). Lemma 8.1 For every prime number pn > 2, the sequence Yn is strictly decreasing. Proof For every real value x ≥3, we state the function Proof For every real value x ≥3, we state the function f(x) =
e
1
2×log(x)
(1−
1
log(x)) which is equivalent to f(x) = g(x)×h(u) where g(x) = e
1
2×log(x) and h(u) =
u
u−1 for u = log(x). We know that g(x) decreases as
x ≥3 increases, Moreover, we note that h(u) decreases as u > 1 increases where u =
log(x) > 1 for x ≥3. In conclusion, we can see that the function f(x) is monotonically
decreasing for every real value x ≥3 and therefore, the sequence Yn is monotonically
decreasing as well. In addition, Yn is essentially a strictly decreasing sequence, since
there is not any natural number n > 1 such that Yn = Yn+1. where g(x) = e
1
2×log(x) and h(u) =
u
u−1 for u = log(x). We know that g(x) decreases as
x ≥3 increases, Moreover, we note that h(u) decreases as u > 1 increases where u =
log(x) > 1 for x ≥3. In conclusion, we can see that the function f(x) is monotonically
decreasing for every real value x ≥3 and therefore, the sequence Yn is monotonically
decreasing as well. In addition, Yn is essentially a strictly decreasing sequence, since
there is not any natural number n > 1 such that Yn = Yn+1. In mathematics, the Chebyshev function θ(x) is given by Due to the theorem 8.3, we prove that Due to the theorem 8.3, we prove that m
∏
i=1
qi
qi −1 < eγ ×(logqm +
1
2×log(qm)) < eγ ×log(Ym ×θ(qm)) m
∏
i=1
qi
qi −1 < eγ ×(logqm +
1
2×log(qm)) < eγ ×log(Ym ×θ(qm)) when qm > 286. when qm > 286. In mathematics, the Chebyshev function θ(x) is given by In mathematics, the Chebyshev function θ(x) is given by θ(x) = ∑
p≤x
log p where p ≤x means all the prime numbers p that are less than or equal to x. Lemma 8.2 [7]. For x ≥41: Lemma 8.2 [7]. For x ≥41: θ(x) > (1−
1
log(x))×x. Besides, we know that Besides, we know that Lemma 8.3 [7]. For x ≥286: Lemma 8.3 [7]. For x ≥286:
∏
q≤x
q
q−1 < eγ ×(logx+
1
2×log(x)). ∏
q≤x
q
q−1 < eγ ×(logx+
1
2×log(x)). We will prove another important inequality: We will prove another important inequality: Theorem 8.4 Let q1,q2,...,qm denote the first m consecutive primes such that q1 <
q2 < ··· < qm and qm > 286. Then Theorem 8.4 Let q1,q2,...,qm denote the first m consecutive primes such that q1 <
q2 < ··· < qm and qm > 286. Then m
∏
i=1
qi
qi −1 < eγ ×log(Ym ×θ(qm)). F. Vega 14 Proof From the theorem 8.2, we know that Proof From the theorem 8.2, we know that Proof From the theorem 8.2, we know that
θ(qm) > (1−
1
log(qm))×qm. In this way, we can show that
log(Ym ×θ(qm)) > log
Ym ×(1−
1
log(qm))×qm
= logqm +log
Ym ×(1−
1
log(qm))
. We know that
log
Ym ×(1−
1
log(qm))
= log
e
1
2×log(qm)
(1−
1
log(qm)) ×(1−
1
log(qm))
= log
e
1
2×log(qm)
=
1
2×log(qm). Consequently, we obtain that
logqm +log
Ym ×(1−
1
log(qm))
≥(logqm +
1
2×log(qm)). Due to the theorem 8.3, we prove that
m
∏
i=1
qi
qi −1 < eγ ×(logqm +
1
2×log(qm)) < eγ ×log(Ym ×θ(qm))
when qm > 286. θ(qm) > (1−
1
log(qm))×qm. In this way, we can show that log(Ym ×θ(qm)) > log
Ym ×(1−
1
log(qm))×qm
= logqm +log
Ym ×(1−
1
log(qm))
. We know that log
Ym ×(1−
1
log(qm))
= log
e
1
2×log(qm)
(1−
1
log(qm)) ×(1−
1
log(qm))
= log
e
1
2×log(qm)
=
1
2×log(qm). Consequently, we obtain that Consequently, we obtain that logqm +log
Ym ×(1−
1
log(qm))
≥(logqm +
1
2×log(qm)). 9 Proof of Main Theorems Theorem 9.1 Robins(n) holds for all n > 5040 when a prime number q ≤953 com-
plies with q ∤n. Proof This is a compendium of the results from the theorem 1.2 and the lemmas 7.1,
7.2, 7.3 and 7.4. Proof This is a compendium of the results from the theorem 1.2 and the lemmas 7.1,
7.2, 7.3 and 7.4. Theorem 9.2 Let π2
6 × loglogn′ ≤loglogn for some n > 5040 such that n′ is the
square free kernel of the natural number n. Then Robins(n) holds. 15 Robin Criterion on Divisibility Proof Let n′ be the square free kernel of the natural number n. Let n′ be the product
of the distinct primes q1,...,qm. By assumption we have that π2
6 ×loglogn′ ≤loglogn. π2
6 ×loglogn′ ≤loglogn. π2
6 ×loglogn′ ≤loglogn. For all square free n′ ≤5040, Robins(n′) holds if and only if n′ /∈{2,3,5,6,10,30} [3]. However, Robins(n) holds for all n > 5040 when n′ ∈{2,3,5,6,10,15,30} due to the
lemma 4.1. When n′ > 5040, we know that Robins(n′) holds and so f(n′) < eγ ×loglogn′. By the previous lemma 2.3: f(n) < π2
6 ×
m
∏
i=1
qi +1
qi
. f(n) < π2
6 ×
m
∏
i=1
qi +1
qi
. Suppose by way of contradiction that Robins(n) fails. Then Suppose by way of contradiction that Robins(n) fails. Then f(n) ≥eγ ×loglogn. f(n) ≥eγ ×loglogn. f(n) ≥eγ ×loglogn. We claim that We claim that π2
6 ×
m
∏
i=1
qi +1
qi
> eγ ×loglogn. Since otherwise we would have a contradiction. This shows that π2
6 ×
m
∏
i=1
qi +1
qi
> π2
6 ×eγ ×loglogn′. Thus Thus
m
∏
i=1
qi +1
qi
> eγ ×loglogn′, m
∏
i=1
qi +1
qi
> eγ ×loglogn′, and and
m
∏
i=1
qi +1
qi
> f(n′), m
∏
i=1
qi +1
qi
> f(n′), This is a contradiction since f(n′) is equal to This is a contradiction since f(n′) is equal to (q1 +1)×···×(qm +1)
q1 ×···×qm according to the formula (6.3) for the square free numbers. according to the formula (6.3) for the square free numbers. Theorem 9.3 Let ∏m
i=1 qai
i
be the representation of n as a product of the first m
consecutive primes q1 < ··· < qm with natural numbers as exponents a1,...,am. 9 Proof of Main Theorems If
n > 5040 is the smallest integer such that Robins(n) does not hold, then (logn)β <
Ym × log(Nm), where Nm = ∏m
i=1 qi is the primorial number of order m and β =
∏m
i=1
qai+1
i
qai+1
1. Theorem 9.3 Let ∏m
i=1 qai
i
be the representation of n as a product of the first m
consecutive primes q1 < ··· < qm with natural numbers as exponents a1,...,am. If
n > 5040 is the smallest integer such that Robins(n) does not hold, then (logn)β <
Ym × log(Nm), where Nm = ∏m
i=1 qi is the primorial number of order m and β =
∏m
i 1
qai+1
i F. Vega F. Vega 16 Proof According to the theorems 1.3 and 1.4, the primes q1 < ··· < qm must be the
first m consecutive primes since n > 5040 should be an Hardy-Ramanujan integer. From the theorem 9.1, we know that necessarily qm ≥953. From the lemma 3.2, we
note that f(n) =
m
∏
i=1
qi
qi −1
! ×
m
∏
i=1
1−
1
qai+1
i
! . However, we know that m
∏
i=1
qi
qi −1 < eγ ×log(Ym ×log(Nm)) because of the lemma 8.4 when qm ≥953. If we multiply by ∏m
i=1
1−
1
qai+1
i
the
both sides of the previous inequality, then we obtain that f(n) < eγ ×log(Ym ×log(Nm))×
m
∏
i=1
1−
1
qai+1
i
! . If n is the smallest integer exceeding 5040 that does not satisfy the Robin inequality,
then eγ ×loglogn < eγ ×log(Ym ×log(Nm))×
m
∏
i=1
1−
1
qai+1
i
! because of eγ ×loglogn ≤f(n). eγ ×loglogn ≤f(n). That is the same as m
∏
i=1
qai+1
i
qai+1
i
−1
×loglogn < log(Ym ×log(Nm)) which is equivalent to which is equivalent to (logn)β < Ym ×log(Nm) where β = ∏m
i=1
qai+1
i
qai+1
i
−1. Therefore, the proof is done. where β = ∏m
i=1
qai+1
i
qai+1
i
−1. Therefore, the proof is done. Theorem 9.4 Let ∏m
i=1 qai
i
be the representation of n as a product of the first m
consecutive primes q1 < ··· < qm with natural numbers as exponents a1,...,am. I thank Richard J. Lipton for helpful comments. I thank Richard J. Lipton for helpful comments. 9 Proof of Main Theorems If
n > 5040 is the smallest integer such that Robins(n) does not hold, then (logn)β <
1.2592 × log(Nm), where Nm = ∏m
i=1 qi is the primorial number of order m and
β = ∏m
i=1
qai+1
i
ai+1
1. β = ∏m
i=1
qai+1
i
qai+1
i
−1. Proof From the theorem 9.1, we know that necessarily qm ≥953. Using the theorem
9.3, we obtain that
β Proof From the theorem 9.1, we know that necessarily qm ≥953. Using the theorem
9.3, we obtain that
β (logn)β < 1.2592×log(Nm) because of the lemma 8.1 since we have that Ym < 1.2592 when qm ≥953. because of the lemma 8.1 since we have that Ym < 1.2592 when qm ≥953. 17 Robin Criterion on Divisibility Acknowledgments I thank Richard J. Lipton for helpful comments. References 1. Akbary, A., Friggstad, Z.: Superabundant numbers and the Riemann hypothesis. The American Math-
ematical Monthly 116(3), 273–275 (2009). DOI doi:10.4169/193009709X470128
2. Alaoglu, L., Erd˝os, P.: On highly composite and similar numbers. Transactions of the American Math-
ematical Society 56(3), 448–469 (1944). DOI doi:10.2307/1990319
3. Choie, Y., Lichiardopol, N., Moree, P., Sol´e, P.: On Robin’s criterion for the Riemann hypothesis. Journal de Th´eorie des Nombres de Bordeaux 19(2), 357–372 (2007). DOI doi:10.5802/jtnb.591
4. Edwards, H.M.: Riemann’s Zeta Function. Dover Publications (2001)
5. Hertlein, A.: Robin’s Inequality for New Families of Integers. Integers 18 (2018)
6. Robin, G.: Grandes valeurs de la fonction somme des diviseurs et hypoth`ese de Riemann. J. Math. pures appl 63(2), 187–213 (1984)
7. Rosser, J.B., Schoenfeld, L.: Approximate Formulas for Some Functions of Prime Numbers. Illinois
Journal of Mathematics 6(1), 64–94 (1962). DOI doi:10.1215/ijm/1255631807 1. Akbary, A., Friggstad, Z.: Superabundant numbers and the Riemann hypothesis. The American Math-
ematical Monthly 116(3), 273–275 (2009). DOI doi:10.4169/193009709X470128 1. Akbary, A., Friggstad, Z.: Superabundant numbers and the Riemann hypothesis. The American Math-
ematical Monthly 116(3), 273–275 (2009). DOI doi:10.4169/193009709X470128 2. Alaoglu, L., Erd˝os, P.: On highly composite and similar numbers. Transactions of the American Math-
ematical Society 56(3), 448–469 (1944). DOI doi:10.2307/1990319 y
3. Choie, Y., Lichiardopol, N., Moree, P., Sol´e, P.: On Robin’s criterion for the Riemann hypothesis. Journal de Th´eorie des Nombres de Bordeaux 19(2), 357–372 (2007). DOI doi:10.5802/jtnb.591 4. Edwards, H.M.: Riemann’s Zeta Function. Dover Publications (2001) 5. Hertlein, A.: Robin’s Inequality for New Families of Integers. Integers 18 (2018) 5. Hertlein, A.: Robin’s Inequality for New Families of Integers. Integers 18 (2018) 6. Robin, G.: Grandes valeurs de la fonction somme des diviseurs et hypoth`ese de Riemann. J. Math. pures appl 63(2), 187–213 (1984) p
pp
7. Rosser, J.B., Schoenfeld, L.: Approximate Formulas for Some Functions of Prime Numbers. Illinois
Journal of Mathematics 6(1), 64–94 (1962). DOI doi:10.1215/ijm/1255631807
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Nursing care time and quality indicators for adult intensive care: correlation analysis
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Revista latino-americana de enfermagem
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Corresponding Author:
Paulo Carlos Garcia
Universidade de São Paulo. Hospital Universitário
Av. Professor Lineu Prestes, 2565
Bairro: Butantã
CEP: 05508-000, São Paulo, SP, Brasil
E-mail: paulogarcia@usp.br 1 Paper extracted from Master’s Thesis “Tempo de assistência de enfermagem em UTI e indicadores de qualidade assistencial: análise
correlacional” presented to Escola de Enfermagem, Universidade de São Paulo, Brazil.
2 RN, MSc, Hospital Universitário, Universidade de São Paulo, Brazil.
3 PhD, Associate Professor, Escola de Enfermagem, Universidade de São Paulo, Brazil. Original Article Original Article Original Article Rev. Latino-Am. Enfermagem
2012 July-Aug.;20(4):651-8
www.eerp.usp.br/rlae Rev. Latino-Am. Enfermagem
2012 July-Aug.;20(4):651-8
www.eerp.usp.br/rlae Rev. Latino-Am. Enfermagem
2012 July-Aug.;20(4):651-8
www.eerp.usp.br/rlae Tempo de assistência de enfermagem em unidade de terapia intensiva
adulto e indicadores de qualidade assistencial: análise correlacional Trata-se de studo de abordagem quantitativa, correlacional e descritivo, elaborado com
o objetivo de analisar o tempo utilizado pela equipe de Enfermagem para assistir aos
pacientes internados em unidade de terapia intensiva adulto, bem como verificar sua
correlação com os indicadores de qualidade assistencial. A identificação do tempo médio
de assistência despendido e dos indicadores de qualidade assistencial foi efetivada por
meio de consulta aos instrumentos de gestão, utilizados pela chefia de Enfermagem
da unidade. O tempo médio de assistência despendido com os pacientes manteve-se
equilibrado, porém, inferior aos indicados pelos órgãos oficiais brasileiros. A correlacão
entre o tempo de assistência de Enfermagem despendido por enfermeiros e o indicador
incidência de extubação acidental indicou que a incidência de extubação acidental
diminui, à medida que aumenta o tempo de assistência de Enfermagem despendido por
enfermeiros. Os resultados desta investigação demonstram a influência do tempo de
assistência de Enfermagem, provido por enfermeiros, no resultado do cuidado ministrado. Descritores: Enfermagem; Administração de Recursos Humanos em Enfermagem; Carga
de Trabalho; Indicadores de Qualidade em Assistência à Saúde. Nursing care time and quality indicators for adult intensive care:
correlation analysis1 Paulo Carlos Garcia2
Fernanda Maria Togeiro Fugulin3 The objective of this quantitative, correlational and descriptive study was to analyze the
time the nursing staff spends to assist patients in Adult Intensive Care Units, as well as to
verify its correlation with quality care indicators. The average length of time spent on care
and the quality care indicators were identified by consulting management instruments the
nursing head of the Unit employs. The average hours of nursing care delivered to patients
remained stable, but lower than official Brazilian agencies’ indications. The correlation
between time of nursing care and the incidence of accidental extubation indicator indicated
that it decreases with increasing nursing care delivered by nurses. The results of this
investigation showed the influence of nursing care time, provided by nurses, in the outcome
of care delivery. Descriptors: Nursing; Nursing Administration of Human Resources; Workload; Quality
Indicators in Health Care. 652 Tiempo de atención de enfermería en la unidad de cuidados intensivos
de adultos y los indicadores de atención de calidad: análisis de
correlación. Un estudio de enfoque cuantitativo, de correlación y descriptiva, elaborado con objetivo
de analizar el tiempo empleado por equipo de enfermería para asistir pacientes
hospitalizados en Unidad de Cuidados Intensivos de Adultos y verificar su correlación
con indicadores de calidad de la atención. La identificación del promedio de tiempo de
cuidados y de indicadores de calidad asistencial fue realizada por medio de consulta a las
herramientas de gestión utilizadas por el jefe de enfermería de la Unidad. El promedio de
tiempo de cuidado ministrado a los pacientes se mantuvo equilibrado, pero inferior a los
indicados por organismos oficiales en Brasil. La correlación entre tiempo de cuidados de
enfermería e indicador de incidencia extubación accidental indicó que esta disminuye con
aumento de tiempo de atención de enfermería utilizado por enfermeras. Los resultados
de esta investigación demuestran la influencia del tiempo de atención de enfermería por
las enfermeras, en los resultados de la atención prestada. Descriptores: Enfermería; Gestión de Recursos Humanos en la Carga de Trabajo de
Enfermería; Los Indicadores de Calidad en la Atención de la Salud. www.eerp.usp.br/rlae Method A systematic literature review(4) appoints that
concerns with patient safety and care quality are
determining the accomplishment of research on clinical
practice and the cost-effectiveness relation of health
interventions are care, including the distribution of
human resources. This is particularly important in
intensive care, where a large part of hospitals’ financial
resources are consumed and the nursing staff represents
the main cost item. A quantitative and descriptive correlation study
was developed at the AICU of the HU-USP. The AICU offers 12 beds. Four of these are
structured and preferably reserved for the hospitalization
of patients who need isolation. It should be highlighted
that the Unit elaborates monthly reports, as a
management instrument, which are forwarded to the
Nursing Department (ND), informing on the number
of care hours delivered to hospitalized patients during
the period, with a view to supporting human resource
management decisions, so as to maintain quality care. Another literature review(5) indicates that research
done in recent decades shows an association between
the nursing staff and patient care outcomes. Most of
these studies were developed at intensive care units
and picture the direct relation between nursing staff and
increased infection rates, postoperative complications,
mortality and accidental extubation, also described as
unplanned extubation. The nursing time spent is calculated with the help
of an electronic worksheet called “Worksheet to calculate
the mean nursing care time spent”(8). Another characteristic of the Unit where the
research was accomplished is the use of care quality
indicators. The selected indicators, according to the
Institution’s Group of Quality Indicators, are: Incidence
of nasogastric tube loss for nutrition; Incidence of
central venous catheter loss; Incidence of accidental
extubation; Incidence of pressure ulcer; Incidence of
fall; Incidence of medication error. The author of that research(5) concluded that the
analysis of nursing staff needs at intensive care units is
important, mainly for the managers of these units, to
the extent that they have the power to make decisions
and distribute resources. Thus, the challenge for intensive care nurses is
to develop and quantify evidence to demonstrate that
a larger proportion of nurses positively influences the
outcomes of care delivery to patients and their families,
through studies that relate staff number with quality
care indicators(6). These indicators represent care practice at the
Institution and are validated and recommended in
Brazilian(9) and international literature(10). Method One exception
is the central venous catheter loss indicator, which was
developed according to the need evidenced at HU-USP. In addition, these indicators are frequently used in the
Brazilian hospital network, permitting intra and extra-
institutional comparability. In this perspective, the use of quality care indicators
represents
a
powerful
management
instrument
for nurses, as it permits showing the relevance of
quantitative and qualitative staff dimensioning with
a view to the promotion of excellent care delivery to
health service users. Introduction Alliance, with a view to promoting and developing patient
safety health practices and policies at global level(2). In the global context, the challenge of improving
the safety and quality of patient care delivery in health
services is not new. A document the International Council of Nurses
published also considers that the improvement of nursing
practice can guarantee patient safety and constitutes a
global priority goal(3). According to the campaign launched by the World
Health Organization(1), we are in the “Age of Safety” and,
since 2002, the organization has addressed this theme
as a priority issue. The program was strengthened in
2004, through the creation of the Global Patient Safety The same document(3) highlights that organizational
culture and management directly influence the results www.eerp.usp.br/rlae 653 Garcia PC, Fugulin FMT. rigor, they permit comparative analyses among health
services, with a view to planning quality improvements
in care delivery to patients/service users(7). reached in health institutions. Quality in the nursing
work environment, in turn, interferes in the quality and
safety of patient care and is influenced by the size of the
nursing staff, the number of professionals according to
professional category and the nursing/patient rate. The analysis of Brazilian literature, however, shows
a lack of studies that demonstrate the relation between
nursing staff conformity and care indicators. In that
sense, the aim of this study is to analyze the time the
nursing team uses to see to patients/clients hospitalized
at the Adult Intensive Care Unit (AICU) of the University
of São Paulo Teaching Hospital (HU-USP), as well as to
check its correlation quality care indicators. In that context, it is verified that issues related to
the nursing staff dimensioning process gain relevance
and are under investigation to produce technical and
scientific evidence to enhance awareness of the meaning
of health staff that sees not only to patients and health
institutions’ needs, but also to patients and nursing
team professionals’ safety. www.eerp.usp.br/rlae Data collection procedures Data were collected monthly from the management
instruments the Unit nursing head uses: “Worksheet
to calculate the mean nursing care time spent”, and
“Worksheet to obtain nursing quality indicators, AICU The use of clinical or quality indicators to assess
the care offered represents an important approach to
document attendance. In addition, when the indicators
are validated and tested with transparency and scientific Rev. Latino-Am. Enfermagem 2012 July-Aug.;20(4):651-8. 654 pk = mean productivity of professional category k (85%);
tk = work journey of professional category k (six hours);
n = mean daily number of patients attended. Unit”, with the approval of the HU-USP Research Ethics
Committee (CEP) (registration CEP-HU/USP: 982/10 –
Registration SISNEP – CAAE: 0009.0.198.196-10). Unit”, with the approval of the HU-USP Research Ethics
Committee (CEP) (registration CEP-HU/USP: 982/10 –
Registration SISNEP – CAAE: 0009.0.198.196-10). Unit”, with the approval of the HU-USP Research Ethics
Committee (CEP) (registration CEP-HU/USP: 982/10 –
Registration SISNEP – CAAE: 0009.0.198.196-10). No. of central venous catheter losses
No. of patients with central venous catheter/day
x 100 Spearman’s correlation coefficient, while Pearson’s
correlation coefficient was used for the other quality
indicators. Data treatment and analysis The results were presented through tables with
central trend and variability measures. To correlate the
mean care time spent on AICU patients with the quality
care indicators, initially, the Kolmogorov-Smirnov test
was applied to assess the normality distribution. It
was ascertained that the central venous catheter loss
indicator showed no normal distribution. Thus, for
this variable, correlation analysis was developed using Survey of quality care indicators at the AICU of the
HU-USP The “Worksheets to calculate the mean nursing
care time spent”, which are part the Unit head nurse’s
monthly reports, were consulted to identify the mean
monthly care time spent on patients hospitalized at
the Adult ICU of the HU-USP, between 01/01/2008 and
12/31/2009. The choice of the quality indicators used in this
research was based on the criteria recommended in
literature(7) and on data accessibility at the Unit. Among the indicators assessed at the HU-USP,
the decision was made to use those inherent in the
intensive care context: incidence of nasogastric tube
loss for nutrition; incidence of central venous catheter
loss, incidence of accidental extubation and incidence of
pressure ulcer. The mean nursing care time was calculated
electronically,
using
the
worksheet
developed
in
Microsoft Excel®, with the help of equation (1)(11): qk.pk.tk
n
hk=
(1) qk.pk.tk
n
hk=
(1) Thus, the quality care indicators were collected
monthly from the “Worksheet to obtain nursing quality
indicator data, AICU Unit”, which the Unit nursing head
uses to make calculations according to the equations
indicated in the Manual of Nursing Indicators(9). One
exception is the central venous loss indicator, whose
equation the institution developed by itself. Where: hk = mean nursing care time per patient, spend
by workers from professional category k; k = professional category; k = professional category; qk= mean number of nursing staff members in
professional category k; Incidence of central venous catheter loss =
No. of central venous catheter losses
No. of patients with central venous catheter/day Results C.(%)
3.49
8.13
3.79
5.22
5.44
5.50 As for the distribution of nursing care time between
professional categories, the mean care hours both
nurses and nursing technicians spent remained the
same in 2008 and 2009 (4.3 and 4.4 hours, respectively,
for nurses and 9.6 for technicians). On average, the
proportion of nurses’ care time corresponded to 31%
and that of nursing technicians to 69%. Slight variations were observed in the time spent
during the study period (minimum 13.3 hours and
maximum 14.6 hours in 2008 and minimum 13.1 hours
and maximum 15.4 hours in 2009). The mean number of
nursing care hours spent on the patients during the two
years studied is practically equivalent (13.9 h/patient/
day in 2008 and 14.1 h/patient/day in 2009), with a
difference of only 0.2 hours, i.e. 12 minutes. Table 2 – Mean scores of Nursing quality care indicators at AICU of HU-USP between Jan/2008 and Dec/2009. São
Paulo, SP, Brazil, 2011
Variables
2008
2009
Incidence of
Accidental
Extubation
Incidence of
NGT loss
Incidence of
CVC loss
Incidence
of PU
Incidence of
Accidental
Extubation
Incidence of
NGT loss
Incidence of
CVC loss
Incidence
of PU
January
1.14
0.00
0.00
18.60
0.00
4.69
0.00
3.85
February
1.26
1.51
1.08
10.20
0.00
4.61
1.65
5.77
March
0.62
4.05
0.00
5.26
0.43
1.79
0.00
14.58
April
0.74
1.88
1.37
6.25
0.53
3.13
0.00
6.67
May
0.48
2.78
1.20
12.82
1.18
1.06
0.00
2.38
June
0.72
1.44
0.00
7.27
0.00
1.72
0.00
7.50
July
1.88
3.32
0.00
16.67
0.00
1.03
0.00
6.56
August
1.09
0.87
1.17
10.71
0.00
2.97
0.00
0.00
September
0.00
4.48
0.79
12.50
1.75
6.19
0.00
5.56
October
0.00
0.87
0.39
3.85
0.97
0.83
0.37
7.94
November
0.00
4.52
0.00
0.00
0.60
1.06
0.43
2.22
December
0.83
2.25
0.50
12.50
0.00
1.32
0.00
17.65
Mean
0.73
2.33
0.54
9.72
0.46
2.53
0.20
6.72
sd
0.57
1.51
0.55
5.40
0.58
1.79
0.48
5.03
VAR. C.(%)
0.33
2.27
0.31
29.12
0.34
3.19
0.23
25.28 ores of Nursing quality care indicators at AICU of HU-USP between Jan/2008 and Dec/2009. São Table 2 – Mean scores of Nursing quality care indicators at AICU of HU-USP between Jan/2008 and Dec/2009. São
Paulo, SP, Brazil, 2011 Table 2 shows the performance of nursing quality
care indicators used at the AICU of the HU-USP during
the study period. Results Table 1, presented next, demonstrates the mean
care time spent on patients hospitalized at the Adult ICU
of the HU-USP between 01/01/2008 and 12/31/2009. Table 1 – Mean Nursing Care Time spend on AICU patients at HU-USP, between Jan/2008 and Dec/2009. São Paulo,
SP Brazil 2011 Variables
Mean Time (2008)
Mean Time (2009)
Total
Nurses
Nursing
Technician
Total
Nurses
Nursing
Technician
(h)
(%)
(h)
(%)
(h)
(%)
(h)
(%)
(h)
(%)
(h)
(%)
January
14.5
100
4.2
28.9
10.4
71.1
13.5
100
4.2
31.1
9.3
68.9
February
13.8
100
3.9
28.2
9.8
71.8
15.1
100
4.7
31.1
10.4
68.9
March
13.5
100
3.9
28.8
9.6
71.2
14.7
100
4.6
31.3
10.1
68.7
April
13.3
100
4.1
30.8
9.2
69.2
13.1
100
4.1
31.3
9.1
68.7
May
13.3
100
3.7
27.8
9.5
72.2
13.4
100
4.2
31.3
9.3
68.7
Table 1 – Mean Nursing Care Time spend on AICU patients at HU-USP, between Jan/2008 and Dec/2009. São Paulo,
SP, Brazil, 2011
(continue...) ursing Care Time spend on AICU patients at HU-USP, between Jan/2008 and Dec/2009. São Paulo, Table 1 – Mean Nursing Care Time spend on AICU patients at HU-USP, between Jan/2008 and Dec/
SP, Brazil, 2011 Table 1 – Mean Nursing Care Time spend on AICU patients at HU-USP, between Jan/2008 and Dec/2009. São Paulo,
SP, Brazil, 2011 655 Garcia PC, Fugulin FMT. Table 1 - (continuation) Variables
Mean Time (2008)
Mean Time (2009)
Total
Nurses
Nursing
Technician
Total
Nurses
Nursing
Technician
(h)
(%)
(h)
(%)
(h)
(%)
(h)
(%)
(h)
(%)
(h)
(%)
June
14.0
100
4.4
31.4
9.6
68.6
13.3
100
4.3
32.3
9.0
67.7
July
13.3
100
4.1
30.8
9.2
69.2
13.9
100
4.5
32.4
9.4
67.6
August
14.5
100
4.6
31.7
9.9
68.3
15.4
100
4.8
31.1
10.6
68.9
September
13.8
100
4.4
31.8
9.3
68.2
14.0
100
4.3
30.7
9.7
69.3
October
13.6
100
4.6
33.8
9.0
66.2
14.0
100
4.3
30.7
9.7
69.3
November
14.6
100
4.9
33.5
9.6
66.5
14.7
100
4.7
32.0
10.0
68.0
December
14.1
100
4.4
31.2
9.7
68.8
13.7
100
4.6
33.5
9.2
66.5
Mean
13.9
100
4.3
30.9
9.6
69.1
14.1
100
4.4
31.2
9.6
68.9
sd
0.48
0.35
0.36
0.73
0.24
0.53
VAR. www.eerp.usp.br/rlae Discussion The mean care time spent on patients hospitalized
at the AICU of the HU-USP between 01/01/2008 and
12/31/2009 corresponded to approximately 14 hours. Based on the comparison between the present research
results and those of a study(12) that identified the mean
nursing care hours at the AICU of the HU-USP between
2001 and 2005, it was ascertained that less nursing
care hours were spent on AICU patients in 2008 and
2009 than in the years the author analyzed (mean 15.4
hours), although the percentage distribution the same
researcher found did correspond (30% for nurses and
70% for nursing technicians)(12). The percentage of nursing care time AICU nurses
spent, however, is higher than that identified in RDC(14)
No. 7, proposed by the Joint Board of ANVISA(14) (20%). The percentage of nursing care time AICU nurses
spent, however, is higher than that identified in RDC(14)
No. 7, proposed by the Joint Board of ANVISA(14) (20%). In that sense, studies available in international
literature demonstrate an inverse relation between the
number of nurses and the occurrence of adverse events
in patients, correlating the number of care hours these
professionals spend with the quality of care delivery,
concluding that a larger number of care hours delivered
by nurses is associated with a drop in mortality and
adverse event rates (urinary tract infection, pressure
ulcer, hospital pneumonia, wound infections, central
venous
access
complications,
shock,
thrombosis,
medication errors, postoperative complications), as well
as with a decrease in mortality rates deriving from these
events(18-20). Concerning the times established by the Federal
Nursing Council(13) (COFEN) (17.9 h) and the National
Health Surveillance Agency(14) (ANVISA) (15 h), the
mean nursing care hours spent on patients hospitalized
at the AICU of HU-USP, except in February and August
2009, remained below these entities’ indications. This result suggests the possibility that the
nursing team is overburdened and evidences the need
to revise the Unit’s existing staff. Nevertheless, the
workload patients demand should also be assessed
through the application of specific instruments(15-16), as
the care time patients demand can differ from official
entities’ indications and their use in putting in practice
dimensioning methods could cause an over or under-
dimensioning of nursing staff. This
scenario
evidences
that
improving
the
percentage distribution of care hours attributed to
nurses represents not only a perspective for the AICU at
HU-USP, but also a challenge for Brazilian nursing. Results The CVC loss incidence indicator
showed the smallest variation coefficient (0.31% in 2008
and 0.23% in 2009), while the PU incidence indicator
showed the highest coefficient (29.12% in 2008 and
25.28% in 2009). Data in Table 3 demonstrate that the nursing care
time nurses spent and the quality indicator incidence of
accidental extubation showed a Pearson’s correlation
coefficient of (r =- 0.454), with p=0.026. For the other
variables, no statistically significant correlations were
found. 656 Rev. Latino-Am. Enfermagem 2012 July-Aug.;20(4):651-8. Table 3 - Correlation between mean care time spent, according to professional category, and quality care indicators
at the Adult ICU of HU-USP, between January 1st 2008 and December 31st 2009. HU-USP, São Paulo, Brazil, 2011 Table 3 - Correlation between mean care time spent, according to professional category, and quality care indicators
at the Adult ICU of HU-USP, between January 1st 2008 and December 31st 2009. HU-USP, São Paulo, Brazil, 2011
Variables
Incidence of NGT loss
Incidence of CVC loss*
Incidence of accidental
extubation
Incidence of PU
Pearson’s
correlation
p
Spearman’s
Correlation
p
Pearson’s
correlation
p
Pearson’s
correlation
p
Time spent
-0.015
0.944
0.033
0.878
-0.174
0.416
-0.162
0.450
Time spent, nurses
-0.003
0.987
-0.013
0.953
-0.454
0.026†
-0.306
0.146
Time spent, tech./aux. -0.019
0.931
0.208
0.330
0.067
0.756
-0.017
0.939
* The CVC loss incidence variable shows no normal distribution, so that Spearman’s non-parametric correlation was used. † Significance level for p < 0.05. * The CVC loss incidence variable shows no normal distribution, so that Spearman’s non-parametric correlation was used. † Significance level for p < 0.05. an official reference for nursing staff dimensioning
at hospital institutions, found that, at the six ICUs
studies, the proportion of hours attributed to the
nurses was smaller than that attributed to nursing
technicians and auxiliaries, remaining far below COFEN
recommendations(13). www.eerp.usp.br/rlae Discussion The analysis of AICU indicators suggests improved
care quality at the Unit in 2009, when compared with
2008, as the mean incidence levels of accidental
extubation, CVC loss and PU dropped. Due to the absence
of Brazilian research and the lack of international studies,
however, which used the same indicators and method
as adopted in the present research, AICU/HU-USP data
could not be confronted with those of other services. As for the percentage distribution of care time
between the professional categories who are part of
the nursing team, it is observed that the proportion of
time AICU nurses spent remains below the minimum
percentage recommended in COFEN Resolution(13)
293/2004 (52%). Concerning the analysis of the correlation between
nursing care time spent by nurses and the incidence of
accidental extubation quality indicator, it can be inferred
that the incidence of accidental extubation decreases
as the nursing care time spent by nurses increases. No An exploratory and descriptive study(17) developed
in fourteen hospitalization units of seven hospitals
in São Paulo City, aimed at assessing the parameters
recommended in COFEN Resolution(13) 293/04, as www.eerp.usp.br/rlae 657 Garcia PC, Fugulin FMT. 31st
2009
remained
below
COFEN
and
ANVISA
recommendations. studies were found with similar methods to the present
study. Some studies describe the influence of the nursing
staff variable on the incidence of unplanned extubation
and support the results found. The nursing care time spent by nurses and the
incidence of accidental extubation quality indicator
showed a negative Pearson’s correlation coefficient,
indicating that the incidence of accidental extubation
drops as the number of nursing care hours spent
by nurses increases. Concerning the other quality
indicators assessed in this study, i.e. the incidence of
nasogastric tube loss for feeding, incidence of central
venous catheter loss and incidence of pressure ulcer, no
statistically significant correlations were found. A study(21) that assessed the association between
the nursing workload and the probability of unplanned
extubations at a Pediatric Intensive Care Unit appointed
that the logistic regression model revealed positive
associations between unplanned extubations and the
patient/nurse ratio. In the same study, it was concluded
that the number of unplanned extubations may increase
in situations when the patient/nurse ratio is higher. References 1. World Health Organization (WHO). World Alliance for
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2. Pedreira MLG, MJCS Harada. Enfermagem dia a dia:
segurança do paciente. São Caetano do Sul: Yendis
Editora; 2009. Thus, the present research results demonstrate the
influence of nursing care time, provided by nurses, on
the outcomes of care delivered to patients attended at
AICU. 3. Braumann
A. Positive
practice
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Stud. 2009;46:993-1011. Discussion A research(22) aimed at exploring the effects of
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unplanned tracheal extubation at adult Intensive Care
Units reported that self-extubations were more frequent
at night and under routine care of nurses with less
professional experience. Those authors concluded that
an adequate patient/nurse ratio can generate better
work and safety conditions during nursing procedures. The limitations of the present research, i.e. the
fact that it was accomplished at a single unit of a single
institution, put constraints on its generalization. It does
offer support and equips nurses though, through the
presentation of a systematic method and the indicator
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no studies have been published that correlate these
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for unplanned extubation and determine professionals’
beliefs on perceived unplanned extubation risks revealed
that 60% of the interviewed professionals believe that
the nursing staff or the patient/nurse ratio are closely
related with accidental extubation cases(23). The accomplishment of this study also contributes
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staff on care outcomes and patient safety, supporting
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factors and outcomes after unplanned extubation at ICU
indicated that non-accidental extubation is a frequent
event during mechanical ventilation in critically ill
patients and can be associated with increased morbidity
and mortality. In the same study, the incidence of
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patients under mechanical ventilation and 0.4% per
ventilation day), due to the high nurse/patient ratio in
the units where the field research was accomplished. www.eerp.usp.br/rlae Conclusion As a result of this study, the mean nursing care time
spent on patients hospitalized at the AICU of the HU-USP
could be identified and analyzed, between 01/01/2008
and 12/31/2009, and its correlation with quality care
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JH. Educational Levels of Hospital Nurses and Surgical
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CL, Stevens SR, Harris M. Nurse Staffing and Inpatient
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JH. Educational Levels of Hospital Nurses and Surgical
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Zelevinsky K. Nurse-staffing levels and the quality of
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CL, Stevens SR, Harris M. Nurse Staffing and Inpatient
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de
ferramentas
tecnológicas
no
processo
de
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de
ferramentas
tecnológicas
no
processo
de
dimensionamento de pessoal. In: Prado C, Peres HHC,
Leite MMJ. Tecnologia da informação e comunicação na
enfermagem. São Paulo: Atheneu; 2011. p. 35-43. Leite MMJ. Tecnologia da informação e comunicação na
enfermagem. São Paulo: Atheneu; 2011. p. 35-43. 9. CQH. Programa de Qualidade Hospitalar: manual de
indicadores de enfermagem NAGEH. São Paulo: APM/
CREMESP; 2006. A systematic review and best practice recommendations. Pediatr Crit Care Med. 2010;11(2):287-94. A systematic review and best practice recommendations. Pediatr Crit Care Med. 2010;11(2):287-94. Pediatr Crit Care Med. 2010;11(2):287-94. 22. Yeh SH, Lee LN, Ho TH, Chiang MC, Lin LW. Implications of nursing care in the occurrence and
consequences of unplanned extubation in adult intensive
care units. Int J Nurs Stud. 2004;41:255-62. 10. American Nurses Association. National Database
of Nursing Quality Indicators (NDQI 2003-2011)
.[acesso 13 jan 2012]. Disponível em: https://www. nursingquality.org/ 22. Yeh SH, Lee LN, Ho TH, Chiang MC, Lin LW. Implications of nursing care in the occurrence and
consequences of unplanned extubation in adult intensive
care units. Int J Nurs Stud. 2004;41:255-62. 10. American Nurses Association. National Database
of Nursing Quality Indicators (NDQI 2003-2011)
.[acesso 13 jan 2012]. Disponível em: https://www. nursingquality.org/ 23. Tanios MA, Epstein SK, Livelo J, Teres D. Can We
Identify Patients at High Risk for Unplanned Extubation? A Large-Scale Multidisciplinary Survey. Respir Care. 2010;55(5):561-8. 11. Gaidzinski RR, Fugulin FMT. Dimensionamento
de pessoal de enfermagem em unidade de terapia
intensiva. In: Associação Brasileira de Enfermagem. Programa de atualização em Enfermagem: Saúde do
adulto (PROENF) – Ciclo 3 – Módulo 3. Porto Alegre:
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NMB. Tempo de assistência de enfermagem em
instituição hospitalar de ensino. Rev Esc Enferm USP. 2011;45(1):223-9. 23. Tanios MA, Epstein SK, Livelo J, Teres D. Can We
Identify Patients at High Risk for Unplanned Extubation? A Large-Scale Multidisciplinary Survey. Respir Care. 2010;55(5):561-8. 11. Gaidzinski RR, Fugulin FMT. Dimensionamento
de pessoal de enfermagem em unidade de terapia
intensiva. In: Associação Brasileira de Enfermagem. Programa de atualização em Enfermagem: Saúde do
adulto (PROENF) – Ciclo 3 – Módulo 3. Porto Alegre:
Artmed/Panamericana; 2008. p. 65-96. 11. Gaidzinski RR, Fugulin FMT. Conclusion Dimensionamento
de pessoal de enfermagem em unidade de terapia
intensiva. In: Associação Brasileira de Enfermagem. Programa de atualização em Enfermagem: Saúde do
adulto (PROENF) – Ciclo 3 – Módulo 3. Porto Alegre:
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Arbous MS. Risk factors and outcomes after unplanned
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Arbous MS. Risk factors and outcomes after unplanned
extubations on the ICU: a case-control study. Crit Care. 2011;15(19):1-9. 12. Rogenski KE, Fugulin FMT, Gaidzinski RR, Rogenski
NMB. Tempo de assistência de enfermagem em
instituição hospitalar de ensino. Rev Esc Enferm USP. 2011;45(1):223-9. 12. Rogenski KE, Fugulin FMT, Gaidzinski RR, Rogenski
NMB. Tempo de assistência de enfermagem em
instituição hospitalar de ensino. Rev Esc Enferm USP. 2011;45(1):223-9. 13. Conselho Federal de Enfermagem (BR). Resolução
n. 293, de 21 de setembro de 2004. Fixa e Estabelece
parâmetros para Dimensionamento do Quadro de
Profissionais de enfermagem nas Unidades Assistenciais
das Instituições de Saúde e Assemelhados [Internet]. [acesso 13 jan 2012]; Rio de Janeiro; 2004. Disponível
em: http://corensp.org.br/072005/ 14. Agência Nacional de Vigilância Sanitária (BR). RDC nº 7 de 24 de fevereiro de 2010. [acesso 13 jan
2012]; Disponível em: http://www.brasilsus.com.br/
legislacoes/rdc/102985-7.html 14. Agência Nacional de Vigilância Sanitária (BR). RDC nº 7 de 24 de fevereiro de 2010. [acesso 13 jan
2012]; Disponível em: http://www.brasilsus.com.br/
legislacoes/rdc/102985-7.html 15. Miranda DR, Rijk AD, Schaufeli W, Iapichino G. Nursing
activities score. Crit Care Med. 2003;31(2):374-82. 16. Panunto MR, Guirardello EB. Nursing workload at
a gastroenterology unit. Rev. Latino-Am. Enfermagem. 2009;17(6):1009-14. 17. Fugulin
FMT. Parâmetros
oficiais
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o
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293/04. [tese Livre-docência]. São Paulo : Escola de
Enfermagem da Universidade de São Paulo; 2010. Received: May 5th 2011
Accepted: Apr. 3th 2012 www.eerp.usp.br/rlae
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https://openalex.org/W3140257988
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https://pub.epsilon.slu.se/26230/1/schwochow_d_et_al_211129.pdf
|
English
| null |
The feather pattern <i>autosomal barring</i> in chicken is strongly associated with segregation at the <i>MC1R</i> locus
|
Pigment cell & melanoma research
| 2,021
|
cc-by
| 12,024
|
j
INTERNATIONAL FEDERATION OF PIGMENT CELL SOCIETIES · SOCIETY FOR MELANOMA RESEARCH PIGMENT CELL & MELANOMA
Research The feather pattern autosomal barring in
chicken is strongly associated with
segregation at the MC1R locus
Doreen Schwochow | Susanne Bornelöv | Tingxing Jiang |
Jingyi Li | David Gourichon | Bertrand Bed’Hom |
Ben J. Dorshorst | Cheng-Ming Chuong |
Michèle Tixier-Boichard| Leif Andersson If you wish to order reprints of this article,
please see the guidelines here Supporting Information for this article is freely available here K E Y W O R D S chicken, feather patterning, genetics, MC1R, pigmentation, SOX10 O R I G I N A L A R T I C L E Doreen Schwochow1,2
| Susanne Bornelöv3 | Tingxing Jiang4
| Jingyi Li5,6
|
David Gourichon7 | Bertrand Bed’Hom2
| Ben J. Dorshorst5 | Cheng-Ming Chuong4
|
Michèle Tixier-Boichard2
| Leif Andersson1,3,6 Doreen Schwochow1,2
| Susanne Bornelöv3 | Tingxing Jiang4
| Jingyi Li5,6
|
David Gourichon7 | Bertrand Bed’Hom2
| Ben J. Dorshorst5 | Cheng-Ming Chuong4
|
Michèle Tixier-Boichard2
| Leif Andersson1,3,6 Doreen Schwochow1,2
| Susanne Bornelöv3 | Tingxing Jiang4
| Jingyi Li5,6
|
David Gourichon7 | Bertrand Bed’Hom2
| Ben J. Dorshorst5 | Cheng-Ming Chuong4
|
Michèle Tixier-Boichard2
| Leif Andersson1,3,6 1Department of Animal Breeding and
Genetics, Swedish University of Agricultural
Sciences, Uppsala, Sweden
2Université Paris-Saclay, INRAE,
AgroParisTech, GABI, Jouy-en-Josas, France
3Science for Life Laboratory, Department
of Medical Biochemistry and Microbiology,
Uppsala University, Uppsala, Sweden
4Department of Pathology, University of
Southern California, Los Angeles, CS, USA
5Department of Animal and Poultry
Sciences, Virginia Tech, Blacksburg, VA, USA
6Department of Veterinary Integrative
Biosciences, College of Veterinary Medicine
and Biomedical Sciences, Texas A&M
University, College Station, TX, USA
7INRAE, PEAT, Nouzilly, France Abstract bst act
Color patterns within individual feathers are common in birds but little is known about
the genetic mechanisms causing such patterns. Here, we investigate the genetic basis
for autosomal barring in chicken, a horizontal striping pattern on individual feath-
ers. Using an informative backcross, we demonstrate that the MC1R locus is strongly
associated with this phenotype. A deletion at SOX10, underlying the dark brown
phenotype on its own, affects the manifestation of the barring pattern. The coding
variant L133Q in MC1R is the most likely causal mutation for autosomal barring in
this pedigree. Furthermore, a genetic screen across six different breeds showing dif-
ferent patterning phenotypes revealed that the most striking shared characteristics
among these breeds were that they all carried the MC1R alleles Birchen or brown. Our data suggest that the presence of activating MC1R mutations enhancing pigment
synthesis is an important mechanism underlying pigmentation patterns on individual
feathers in chicken. We propose that MC1R and its antagonist ASIP play a critical role
for determining within-feather pigmentation patterns in birds by acting as activator
and inhibitor possibly in a Turing reaction–diffusion model. Correspondence
Leif Andersson, Department of Medical
Biochemistry and Microbiology, Uppsala
University, Uppsala, Sweden. Email: leif.andersson@imbim.uu.se EMAIL ALERTS Subscribe to PCMR and stay up-to-date with the only journal committed to publishing
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More information about Pigment Cell & Melanoma Research at www.pigment.org Received: 18 February 2021 | Accepted: 26 March 2021
DOI: 10.1111/pcmr.12975 Significance Within-feather patterns are an important part of the stunning diversity of plumage color in
birds. Barring is a common feather pigmentation pattern present, for instance, in hawks and
owls. This study dissects the genetic basis for autosomal barring in the domestic chicken using
pedigree analysis and molecular characterization. We show that the autosomal barring pheno-
type co-segregates with a missense mutation L133Q in the melanocortin 1 receptor (MC1R)
gene. A general observation across breeds was that various feather pigmentation patterns in
chicken are associated with missense mutations in the MC1R gene. Pigment Cell Melanoma Res. 2021;34:1015–1028. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in
any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
© 2021 The Authors. Pigment Cell & Melanoma Research published by John Wiley & Sons Ltd. 1016 |
1 | INTRODUCTION 1016 SCHWOCHOW et al. located on chromosome 1 in close linkage with Db and Melanotic (Ml)
(Carefoot, 1999; Moore & Smyth, 1972). Pigmentation variation among birds is astonishing and is a conse-
quence of the ability to create complex patterns through varied pig-
ment deposition across the body as well as on individual feathers. Due to the usual homogeneous plumage appearance within species,
it is challenging to pinpoint the underlying genetic cause for the ob-
served phenotype diversity among species. The domestic chicken
is a widely used model for genetic studies of the bewildering pig-
ment variation in birds and has been used successfully so in the
past (Andersson et al., 2020). Chickens exhibit a variety of within-
feather pigmentation patterns of which five have been described to
be the consequence of the interaction between a proposed major
locus called Patterning (Pg) and other loci (Figure 1; Figure S1) (Smyth
Jr, 1990). Two of these interacting loci, Extension (E) and Dark brown
(Db), have been identified at the molecular level. E corresponds to the
melanocortin 1 receptor gene (MC1R), one of the major pigmentation
genes in vertebrates (see below). Db is caused by a 8.3 kb deletion
14 kb upstream of exon 1 in SOX10 on chromosome 1 (Gunnarsson
et al., 2011). Pg is assumed to affect the distribution of dark pigment
on individual feathers and to underlie penciling, which is character-
ized by elliptic bands of eumelanin on the feather (Figure 1b). Pg
has been described as an incompletely dominant, autosomal gene There are two distinct types of barring patterns in chicken, au-
tosomal barring, and sex-linked barring. While sex-linked barring is
adding a white bar on a pigmented background, autosomal barring
is adding a black bar on a gold or silver background on individual
feathers (Smyth Jr, 1990). Sex-linked barring is caused by the com-
bined effect of regulatory mutation(s) and missense mutations in the
tumor suppressor gene CDKN2A (Hellström et al., 2010; Schwochow
Thalmann et al., 2017). Autosomal barring on the other hand has been
suggested to be caused by the combined effect of the three autoso-
mal loci Db, E, and Pg (Andersson et al., 2020; Carefoot, 1984,1999;
Moore & Smyth, 1972) and is the trademark of some chicken breeds
such as Fayoumi, Campine, and Westfälische Totleger. Funding information Pigment Cell Melanoma Res. 2021;34:1015–1028.
| 1015
wileyonlinelibrary.com/journal/pcmr
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in
any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2021 The Authors. Pigment Cell & Melanoma Research published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in
any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2021 The Authors. Pigment Cell & Melanoma Research published by John Wiley & Sons Ltd. | 1015
wileyonlinelibrary.com/journal/pcmr | 101
wileyonlinelibrary.com/journal/pcmr Pigment Cell Melanoma Res. 2021;34:1015–1028. (eumelanin), whereas low or no activity leads to the default pro-
duction of red pigment (pheomelanin) (Garcia-Borron et al., 2005). In birds, associations of MC1R variants with pigmentation traits
have been found both in domesticated species (Davila et al., 2014;
Kerje et al., 2003; Nadeau et al., 2006; Ran et al., 2016; Takeuchi
et al., 1996) as well as in wild birds (Baiao & Parker, 2012; Johnson
et al., 2012; Lamichhaney et al., 2016; Mundy et al., 2004; San-Jose
et al., 2015; Theron et al., 2001). There are at least six phenotypically
distinct Extension/MC1R alleles described in chicken (Andersson
et al., 2020), which are here listed according to dominance and de-
creasing amount of eumelanin: Extended black (E*E), Birchen (E*R),
wild-type (E*N), brown (E*B), buttercup (E*BC), and wheaten (E*WH
or E*Y depending on dominance). These alleles determine the dis-
tribution of black eumelanin across the body and MC1R is therefore
considered to be involved in primary pattern formation (Andersson
et al., 2020; Smyth Jr, 1990). Birds carrying the dominant E and R
alleles usually exhibit very dark to black adult plumage while the re-
maining alleles create patterns of brown, salmon and wheat color in
different body regions. In adult males, only two alleles (E and R) can
be distinguished from wild-type, while the other variant alleles only
cause distinct phenotypic effects in female plumage (Andersson
et al., 2020; Smyth Jr, 1990). with complete genotype and phenotype data, except that sex re-
cordings were missing for two individuals. The chicks were phe-
notyped at hatch and at 12 weeks of age as well as genotyped for
Db/SOX10 and the E*R(Fay) allele. This protocol received the permit
02410.02 delivered by the French authority for animal experiments,
after advice from the INRA Val de Loire ethical committee for animal
experimentation. Feather samples for gene expression were collected after sacri-
ficing the animals, using electronarcosis followed by bleeding. The
feather shafts were frozen in liquid nitrogen and stored at −80°C
until further processing. DNA samples for genotyping were obtained
from blood samples. Blood samples were taken from the wing vein
and submerged with EDTA as an anti-clotting agent. Blood samples
from other breeds showing various patterning phenotypes were ob-
tained from fancy breeders. 2.2 | Whole-genome resequencing of the
Fayoumi backcross For the Fayoumi backcross, between 71 and 102 DNA samples
per phenotype category were pooled in equimolar quantities and
sequenced to 45× coverage on Illumina HiSeq2500 with 2 × 125
paired-end reads. In the present study, we generated an informative backcross
involving the Fayoumi breed showing autosomal barring (expected
genotype Pg/Pg, Db/Db, and R/R at the E/MC1R locus) and an in-
bred line of Light Brown Leghorn (LBL) showing no feather pattern
and assumed to be wild-type at all the major loci known to influence
plumage color. We used pooled sequencing to map the loci affecting
feather pigmentation patterns. Our data show that the major locus
controlling the segregation of autosomal barring in this cross is lo-
cated at the distal end of chromosome 11 where the E/MC1R locus is
located. We also show that Db is not required for autosomal barring
but that it affects the manifestation of this phenotype, and we did
not find any evidence for the segregation at the proposed Pg locus. Sequencing adapters and low-quality bases were removed using
Trim Galore! (with “--stringency 6 -q 15”). The trimmed reads were
aligned to the chicken reference genome (GRCg6a/Galgal6) using
bwa mem with the “-M” option. PCR duplicates were identified using
Picard MarkDuplicates, and variant calling was done using the GATK
toolkit (McKenna et al., 2010) v4.1.1.0. First, base quality scores
were recalibrated using known variants in dbSNP150. Next, the
HaplotypeCaller was used to call variants in gVCF format. All gVCF
files were combined and the raw variants were scored and filtered
by the VariantRecalibrator module using dbSNP150 as a training set
(--truth-sensitivity-filter-level 90.0), followed by a hard filter which
excluded SNPs with QD <2.0, FS >60.0, MQ <40.0, MQRankSum
< −12.5, or ReadPosRankSum < −8.0 and indels with QD <2.0, FS
>200.0 or ReadPosRankSum < −20.0. 2.1 | Animals Variants used in the linkage analysis were selected based on an
earlier version of the analysis, which was performed as described
above with the following differences: alignments were done against
Galgal4 using bwa aln followed by bwa sampe, the gatk toolkit v3.2.2
was used, an IndelRealigner step was included, all recalibration steps
were done using dbSNP140, and “--ts_filter_level 95.0” was used for
the VariantRecalibrator. All genomic coordinates from this analysis
have been updated to Galgal6 using the liftOver tool. The Fayoumi population used in this study has been kept at an ex-
perimental farm over several decades and has shown none or little
deviation from the characteristic pigmentation phenotype described
for this breed. Feather samples and phenotype data were obtained
from a Fayoumi backcross which was produced at the PEAT Poultry
Experimental Facility (INRAE, Nouzilly; https://doi.org/10.15454/
1.5572326250887292E12). Five silver autosomal barred Fayoumi
dams (presumed genotype: Pg/Pg, Db/Db, S/W) were crossed with
two Light Brown Leghorn males (wild-type genotype for all feather
color loci to the best of our knowledge). From the F1 generation,
twelve gold autosomal barred females (presumed genotype Pg/pg+,
Db/db+, s+/W) were selected and crossed with another Light Brown
Leghorn male to generate 365 backcross progenies in three batches 1016 |
1 | INTRODUCTION MC1R is a key locus in pigmentation biology, and MC1R muta-
tions are underlying pigmentation polymorphisms in many ver-
tebrates. It codes for a G-protein-coupled receptor located in the
plasma membrane of melanocytes. Upon activation by its agonist
α-melanocyte-stimulating hormone (αMSH), MC1R goes through
a conformation change triggering a signaling cascade, which even-
tually leads to an increase of intracellular cAMP levels, which in
turn activates transcription of genes involved in pigment produc-
tion. MC1R signaling promotes production of black/brown pigment FI G U R E 1 Phenotypic
characterization. (a) Pigmentation
phenotypes of the breeds used to
generate the intercross, Light Brown
Leghorn (left) showing wild-type color
and Fayoumi (right) showing autosomal
barring. Photograph credit: Laurence
Verrier for the LBL male and David
Gourichon for the Fayoumi female. (b)
Within-feather pigmentation pattern in
chickens. The proposed major Patterning
(Pg) locus interacts with several other
loci causing complex variation in melanin
distribution across individual feathers. Wild-type alleles are indicated by “+.” E/
MC1R—Extension/Melanocortin-1 receptor,
Ml—Melanotic, Co—Columbian, Db—Dark
Brown/SOX10. Modified after Andersson
et al (2020) FI G U R E 1 Phenotypic
characterization. (a) Pigmentation
phenotypes of the breeds used to
generate the intercross, Light Brown
Leghorn (left) showing wild-type color
and Fayoumi (right) showing autosomal
barring. Photograph credit: Laurence
Verrier for the LBL male and David
Gourichon for the Fayoumi female. (b)
Within-feather pigmentation pattern in
chickens. The proposed major Patterning
(Pg) locus interacts with several other
loci causing complex variation in melanin
distribution across individual feathers. Wild-type alleles are indicated by “+.” E/
MC1R—Extension/Melanocortin-1 receptor,
Ml—Melanotic, Co—Columbian, Db—Dark
Brown/SOX10. Modified after Andersson
et al. (2020) FI G U R E 1 Phenotypic
characterization. (a) Pigmentation
phenotypes of the breeds used to
generate the intercross, Light Brown
Leghorn (left) showing wild-type color
and Fayoumi (right) showing autosomal
barring. Photograph credit: Laurence
Verrier for the LBL male and David
Gourichon for the Fayoumi female. (b)
Within-feather pigmentation pattern in
chickens. The proposed major Patterning
(Pg) locus interacts with several other
loci causing complex variation in melanin
distribution across individual feathers. Wild-type alleles are indicated by “+.” E/
MC1R—Extension/Melanocortin-1 receptor,
Ml—Melanotic, Co—Columbian, Db—Dark
Brown/SOX10. Modified after Andersson
et al. (2020) SCHWOCHOW et al. 1017 2.5 | Linkage analysis Linkage analysis was performed using the cri-map 2.504 software
and 76 SNPs obtained through the custom-made genotyping assay
as well as genotype data at MC1R and Db/SOX10 (Tables S2 and
S3). The initial analyses were carried out using the entire pedigree. Genetic studies have predicted Pg to be located on chromosome
1 (Carefoot, 1999; Moore & Smyth, 1972), but evaluation of phe-
notype and genotype at Db in our cross suggested that Db is not
required for autosomal barring. We therefore eliminated backcross
individuals with wild-type plumage and only used those that showed
pattern (autosomal barring or unclear) and carried the Fayoumi allele
at MC1R as those could reveal a potential-associated locus on chro-
mosome 1 if there was any. We also noticed a strong sex bias within
the phenotype group “unclear” (Table 2), which was dominated by 2.4 | Genotyping and Sanger sequencing of
MC1R and Db/SOX10 RNA extraction from growing feather follicles, primer design, primer
testing, gene expression assays as well as allelic imbalance testing
was performed as described (Schwochow Thalmann et al., 2017). All primers used for expression analysis are provided in Table S10. Relative gene expression levels of target genes MC1R, NQO1, CDH1,
and WWP2 were calculated using the ΔΔCt method and normal-
ized with two housekeeping genes: eukaryotic translation elonga-
tion factor (EEF2) and β-actin (Schwochow Thalmann et al., 2017). The following sequence variants were used for the allelic imbalance
assays: MC1R (located at chr11:18,841,043), NQO1 (T to C change;
chr11:19,037,574), and CDH1 (A to T change; chr11:18,874,575). The entire Fayoumi pedigree was genotyped for Db, an 8.4 kb dele-
tion upstream of SOX10, by using primers and PCR conditions de-
scribed elsewhere (Gunnarsson et al., 2011). The MC1R genotype
was first evaluated by using an allele discrimination assay on a 7900
HT Fast Real-Time PCR System machine (LifeTechnologies). In brief,
10 ng of DNA was amplified in a reaction using 1× TaqMan Universal
PCR mastermix (LifeTechnologies) with 1× Assay mix containing
probe and primers and filled up with water to a total reaction vol-
ume of 5 μl/sample. The reaction mix was subjected to 40 cycles as
follows: 15 s at 92°C and 1 min at 60°C, preceded by 1 × 10 min at
95°C. The data were analyzed using the software sds 2.3. Custom-
made probes for the L133Q mutation were obtained from Thermo
Fisher. females. We therefore performed part of the analysis by only using
E*R(Fay)/E*N backcross females. excluding regions with a very low number of variants. The scores for
each region were transformed to a Z score, and regions with a score
corresponding to p < .05 after Bonferroni correction were reported. 2.7 | Immunohistochemistry and in situ
hybridization In order to select genetic markers for fine mapping, we used the
resequencing data and extracted SNPs located between position
40,155,407 and 59,843,680 bp on chromosome 1, which occurred
at a frequency of 0.4 to 0.6 in the “autosomal barring” pool and <0.2
frequency in the “wild-type” pool. Each SNP had to be covered by
at least 20 reads. The same procedure was used for retrieving SNPs
from chromosome 11 located between 15,539,481 and 19,932,279. Some high FST SNPs (FST > 0.35) between 6,258,804 bp and
71,048,324 on chromosome 2 were also included. The final com-
position of SNPs was chosen randomly by keeping the distance be-
tween neighboring SNPs in the range 189–322 kb on chromosomes
1 and 11. For chromosome 2, the SNPs were spread out across the
chromosome, as there was no distinct haplotype. The genotyping
service was provided by Neogen Europe Ltd, Geneseek. The haplo-
type composition was evaluated using Excel. For section immunostaining and in situ hybridization, fixed skin
tissue was embedded in paraffin and sectioned at 6–7 µm. After
de-paraffination, sections were processed for immunohistochem-
istry or in situ hybridization. The MITF antibody was from Abcam
(ab12039, 1:200 dilution). The peroxidase staining was used after
primary antibody treatment as described (Jiang & Chuong, 1992). Non-radioactive in situ hybridization was performed as described
(Chuong et al., 1996). Briefly, the sections were treated with pro-
teinase K (10 µg/ml in PBS) for 20 min, re-fixed with 0.2% glutar-
aldehyde/4% paraformaldehyde, and rinsed with PBT. The sections
were then prehybridized in hybridization buffer (containing 50%
formamide, 5× sodium citrate/sodium chloride buffer, 1% sodium
dodecyl sulfate, 50 µg/ml heparin, 50 µg/ml tRNA) at 65°C for 1 hr. After prehybridization, sections were placed in new prehybridiza-
tion buffer containing 1–3 µg/ml digoxigenin-labeled riboprobes
and hybridized overnight at 65°C. Finally, sections were incubated
with alkaline phosphatase-conjugated anti-digoxigenin Fab (Roche,
Indianapolis, IN) overnight. Positive signals were detected by incu-
bating the specimens with NBT (nitro-blue tetrazolium)/BCIP (5-b
romo-4-chloro-3'-indolyphosphate) substrates (Promega, Madison). 2.3 | Calculation of the fixation index (FST) FST was estimated for each called variant as FST = (πbetween –
πwithin) / πbetween. Variant-level FST values were then averaged across
30 kb sliding windows and visualized as a Manhattan plot after 1018 SCHWOCHOW et al. Variants were called with HaplotypeCaller from the gatk toolkit (v3.8)
followed by filtering using the VariantRecalibrator and hard filters as
described above. Variants were called with HaplotypeCaller from the gatk toolkit (v3.8)
followed by filtering using the VariantRecalibrator and hard filters as
described above. 21 males initially phenotyped as AB at hatch and 7, which exhib-
ited an irregular pattern, the pattern had completely disappeared at
12 weeks of age (Table S1, Figure S2), and instead, they developed
a reddish taint in the belly region and were termed “red belly” (RB). This group also includes four males, which were scored as wild-type
at hatch but developed the red belly phenotype by 12 weeks. These
28 + 4 male progenies classified as red belly were excluded from
most analyses. Pooled heterozygosity was calculated as described (Rubin
et al., 2010). For each observed SNP, the read count of the
major allele, nMAJ, and the minor allele, nMIN, was calculated
across all breeds. A pooled heterozygosity, Hp, was calculated as
Hp = 2 * ∑(nMAJ) * ∑(nMIN ) / (∑(nMAJ) + ∑(nMIN ))2 across all variants
within a 30 kb window and then transformed to a Z score according
to ZHp = (Hp − μ) / σ. Negative Z-scores correspond to regions with
less than expected heterozygosity, indicating that they may be iden-
tical by descent (IDB). Windows with Z-scores less than −4.70 were
considered significant, which correspond to a p-value less than .05
after Bonferroni correction. The resulting Z score was visualized in
Manhattan plot-style, omitting windows with too few variants. According to the assumed inheritance pattern of autosomal bar-
ring, we expected that 25%–50% of the backcross progeny should
be heterozygous carriers of Pg and Db and show this phenotype, the
reason for this range in expected frequency is because Pg has been
reported to be linked to Db but no precise estimate of the recom-
bination rate is available. We observed 102 AB versus 201 WT, 28
birds with unclear phenotype classification, and 32 with the red belly
phenotype (Table 1). By genotyping, we confirmed homozygosity
for the causal mutation at Db/SOX10 (Gunnarsson et al., 2011) in all
Fayoumi founder females (Db/Db) and all F1 females were heterozy-
gous Db/N as expected. 3.1 | Segregation of plumage color in a Fayoumi
backcross population We crossed five Fayoumi females (presumed genotype Pg/Pg, Db/
Db) displaying the typical black and white autosomal barring pattern
with two Light Brown Leghorn males showing no patterning (wild-
type at the Pg and Db loci) (Figure 1a). Twelve F1 females (heterozy-
gous carriers of Pg and Db) showing the typical autosomal barring
pattern of the Fayoumi breed were backcrossed with another Light
Brown Leghorn male generating a total of 365 backcross progeny
with complete phenotype information that were used for the ge-
netic analysis. Of these 365, 102 (43 males and 59 females) chick-
ens exhibited the typical autosomal barring pattern (AB), whereas
203 (99 males, 102 females, and 2 unrecorded sex) were classified
as wild type (WT) for patterning (Table 1, Figure S2). A total of 60
birds were more difficult to categorize, 28 (9 males and 19 females)
were neither plain nor did they have the typical autosomal barring
pattern and were classified as “unclear” (Figure 2a). Furthermore, for Surprisingly, not all of the autosomal barred
backcross progeny were genotyped as carriers of Db based on the
presence of the deletion upstream of SOX10 (66 heterozygous car-
riers of Db and 36 non-carriers) whereas the “unclear” group was
almost entirely (27 out of 32 chickens) wild-type for the SOX10 dele-
tion (Table 2). The WT group contained an almost equal distribution
with 100 heterozygous carriers of Db and 103 non-carriers. Thus,
the segregation and genotyping data question the critical role of the
SOX10 deletion for autosomal barring as well as the linkage between
Pg and Db. For the visualization of all haplotypes near MC1R (Figure 5), we
extracted all SNPs (including low-confidence ones) ±5kb (n = 105) or
±50kb (n = 1,424) of the MC1R gene. aSex recordings missing. 2.8 | Whole-genome resequencing of chicken
pools representing different within-feather
patterning phenotypes Six samples with pooled DNA from five individuals each of the
Brahma, Buttercup, Fayoumi, Hamburg, Plymouth Rock, and
Sebright breeds were sequenced to about 10× coverage on Illumina
HiSeq 2,500 with 2 × 100 bp paired-end reads. Genome align-
ment and variant calling were done against Galgal6 using bwa mem. SCHWOCHOW et al. 1019 Abbreviations: AB, autosomal barring; RB, red belly; unclear, irregular
pattern; WT, wild-type. TA B LE 2 Phenotype—genotype associations among backcross
progenies of the Fayoumi/Light Brown Leghorn cross TA B LE 2 Phenotype—genotype associations among backcross
progenies of the Fayoumi/Light Brown Leghorn cross MC1R haplotype (E*R(Fay)) inherited from the Fayoumi founder fe-
males. Linkage analysis revealed highly significant LOD scores for
loci located at the distal end of chromosome 11 (LOD scores = 28.4–
69.1), whereas only weak associations between autosomal barring
and some markers on chromosome 1 were observed, irrespective of
which subset of backcross progeny was used (Tables S4–S6). Genotype
Phenotype
AB
WT
unclear
RB
Total
SOX10
Db*N/N
36
103
25
27
191
Db*Db/N
66
100
3
5
174
MC1R
E*N/N
0
203
0
3
206
E*R(Fay)/N
102
0
28
29
159
Total
102
203
28
32
365
Abbreviations: AB, autosomal barring; RB, red belly; unclear, irregular
pattern; WT, wild-type. The 1 Mb interval on chromosome 11 showing a complete asso-
ciation with patterning contains 29 genes including MC1R (Table S7). We used our resequencing data to extract SNPs that differed be-
tween autosomal barred and wild-type backcross progeny within the
non-recombining region. The L133Q mutation was not extracted,
most likely because it did not meet the coverage requirements of at
least 20 reads/SNP. The obtained variant SNPs were analyzed using
the online tool UCSC Variant Annotation Integrator (VAI), which re-
sulted in a total of 6,627 variants. As expected, the great majority
of those variants were located in non-coding regions such as inter-
genic regions (2,342), introns (2,298) as well as down- or upstream
of genes (967 and 852, respectively; Figure S4), 19 variants were
detected as potential splice variants and 149 as exonic variants. Of
the 149 exonic variants, 107 were synonymous while 41 were non-
synonymous (Figure S4). Ten of the genes in the interval showing
no recombination harbored non-synonymous changes (Table S8). To further predict the possible effect of the missense mutations
on protein function, we used PROVEAN (http://provean.jcvi.org/
index.php), an online tool, which generates a score indicating how
likely it is that a missense mutation affects protein function (Choi &
Chan, 2015; Choi et al., 2012). Except for the known L133Q mutation
in the MC1R E*R(Fay) allele, none of the other missense mutations as-
sociated with the Fayoumi haplotype from this region was predicted
as having a deleterious effect on protein function (Table S8). Abbreviations: AB, autosomal barring; RB, red belly; unclear, irregular
pattern; WT, wild-type. TA B LE 2 Phenotype—genotype associations among backcross
progenies of the Fayoumi/Light Brown Leghorn cross covered the region 18.399–19.527 Mb (near the chromosome end)
with the highest signal around 18.702–19.185 Mb, which included
MC1R located at 18,840,646–18,841,590 bp. The signal on chromo-
some 11 was detected both when the “autosomal barring” pool and
the “unclear” pool were compared with either one or both of the
wild-type pools (Figure 2b). 3.2 | A major locus underlying autosomal barring
maps to a region on chromosome 11 Based on the phenotype data and the genotype data at the
Db/SOX10 locus described above, we set up four pools for whole-
genome resequencing using Illumina HiSeq technology in an at-
tempt to map the Pg locus. We used two different wild-type pools
but with different genotypes at Db/SOX10 (heterozygous carrier of
Db, n = 99 or wild-type, n = 102) whereas the two other pools either
contained clearly autosomal barred chickens (heterozygous carrier
of Db, n = 71) or chickens with a less clear barring phenotype includ-
ing all “unclear” progenies as well as some with a less pronounced
autosomal barring pattern (wild-type at Db, n = 72). The pools were
constructed taking into account the genotype at the Db/SOX10 locus
in order to maximize the chance to detect other loci affecting the
patterning phenotype. All four pools were compared to each other,
and regions of high differentiation between pools were determined
using the Fixation index (FST) in 30 kb sliding windows (Figure 2b;
Figure S3). Depending on which pools were compared, two regions
with high FST, one on chromosome 1 and one on chromosome 11,
were detected. The signal on chromosome 1 encompassing the Db/
SOX10 locus was expected since we constructed the pools on the
basis of the genotypes at this locus. The signal on chromosome 11 TA B LE 1 Segregation of four different phenotypes in the
Fayoumi/Light Brown Leghorn backcross Sex
Phenotype at 12 weeks of age
Total
AB
WT
unclear
RB
Males
43
99
9
32
183
Females
59
102
19
0
180
Unknowna
0
2
0
0
2
Total
102
203
28
32
365
Abbreviations: AB, autosomal barring; RB, red belly; unclear, irregular
pattern; WT, wild-type. aS
di
i
i SCHWOCHOW et al. 1020 1021 SCHWOCHOW et al. FI G U R E 2 A 1 Mb region on chromosome 11 is associated with autosomal barring. (a) Segregation of pigmentation phenotypes among
backcross progeny. Photograph credit: David Gourichon, INRA. (b) Genome-wide screen for genetic differentiation between three different
pools (“autosomal barred,” “unclear” and “wild-type”) of Fayoumi/Light Brown Leghorn backcross offspring using FST values in 30 kb sliding
windows. (c) SNP scoring within 20 Mb and 3.5 Mb regions on chromosomes 1 and 11, respectively, in parents (P) and offspring progeny (BC)
showing autosomal barring (AB), “unclear” patterning or wild-type plumage. The positions of MC1R and SOX10 are indicated. Please note
that a higher density of SNPs was used toward the end of chromosome 11 visually suggesting that the 1 Mb interval is taking up half of the
3.5 Mb region. The borders of the 1 Mb non-recombining interval are based on a single recombinant. Yellow color indicates homozygosity
for an allele inherited from the wild-type Light Brown Leghorn parental (WT), whereas red indicates homozygosity for a Fayoumi-derived
allele. Orange color indicates heterozygosity. The panel for chromosome 11 represents 18 SNPs with the first situated at 15.8 Mb and the
last one at 19.7 Mb. Forty-six SNPs were placed within the 20 Mb region on chromosome 1 with the first SNP located at 40 Mb and the last
at 59.8 Mb. White fields indicate missing genotypes. 3.4 | MC1R, NQO1, and CDH1 are over-expressed in
barred feathers but do not show allelic imbalance 3.4 | MC1R, NQO1, and CDH1 are over-expressed in
barred feathers but do not show allelic imbalance at the MC1R locus among the backcross progeny deviated signifi-
cantly from the expected 1:1 ratio (χ2 = 6.05, df = 1, p < .05). This is
not specific for MC1R but applies to all markers in the 1 Mb region
that did not recombine in this pedigree data. We decided to explore possible regulatory changes in gene expres-
sion for genes located in the 1 Mb interval on chromosome 11 asso-
ciated with patterning and with a putative role in melanocyte biology
(Figure 3a; Table S7). In addition to the obvious positional candidate
MC1R, we considered the NAD(P)H Quinone Dehydrogenase 1 (NQO1)
gene that codes for a cytoplasmatic 2-electron reductase, which is
reducing quinones to hydroquinones and has been shown to affect
the regulation of tyrosinase, thereby enhancing melanogenesis (Choi
et al., 2010). We also examined the expression of Cadherin 1 (CDH1),
a calcium-dependent cell-cell adhesion protein, which among others, To summarize, our genotyping data revealed a perfect associa-
tion between a patterning phenotype and the variant E*R(Fay) allele
at MC1R. It further revealed an incomplete association between the
Db allele at SOX10 and autosomal barring, which suggests that Db is
not required to form the pattern but makes it more pronounced and
easier to phenotype. There may be a third locus segregating in our
cross, which is responsible for the red belly phenotype only present
in males. Evaluating the genetic basis for this phenotype will be a
subject for future studies. g
g
p
yp
subject for future studies. ,
)
p
(
a calcium-dependent cell-cell adhesion protein, which among othe
FI G U R E 3 Expression analysis of candidate genes on chromosome 11. (a) Genes in the interval lacking recombination in the Fayoumi/
Light Brown Leghorn cross. Positional candidate genes are highlighted with red text. (b) Relative gene expression levels of candidate
genes on chromosome 11 in autosomal barred and non-barred feathers. EEF2 and βACTIN were used as housekeeping genes. Significant
differences are indicated by stars (Student's t test; *p < .05, **p < .01, ***p < .001). 3.3 | Fine mapping revealed a region of low
recombination on chromosome 11 We used our resequencing data to identify a total of 100 highly in-
formative SNPs. The SNPs were selected from the candidate regions
on chromosome 1 (n = 63) and chromosome 11 (n = 21), and in ad-
dition a few relatively high FST SNPs from chromosome 2 (n = 16)
(Table S2). The entire pedigree was genotyped for these markers
to validate the observed associations and improve the map resolu-
tion on chromosomes 1 and 11 (Table S3). A total of 46 SNPs on
chromosome 1 were informative and revealed no haplotype that
was shared among all individuals of either phenotype category
(Figure 2c). However, a large proportion of the backcross progeny
showing autosomal barring carried the Db haplotype whereas only
three that were classified as unclear carried this haplotype reveal-
ing a clear association to a patterning phenotype. For chromosome
11, 20 SNPs were highly informative and revealed a region of 1 Mb
without any recombination starting from about 18.8 Mb to the end
of chromosome 11 (Figure 2c, Table S3). All birds phenotypically
classified as autosomal barred or unclear were heterozygous for the Thus, the screen for missense mutations within the 1 Mb re-
gion on chromosome 11 revealed only one missense mutation, the
one in MC1R, that was predicted to affect protein function. Since
MC1R is an obvious candidate gene for a pigmentation phenotype,
we genotyped the entire pedigree for this missense (L133Q) muta-
tion (Table 2). All chicken displaying autosomal barring (AB) or some
kind of pattern (“unclear”) carried the variant allele at this position,
whereas all plain (WT) chicken had the wild-type allele at MC1R. Even
the 28 males that initially were phenotyped as AB but did not show
any patterning at 12 weeks of age and were grouped into the red
belly group carried the variant allele (Table S1). The segregation data 1022 SCHWOCHOW et al. 3.5 | The expression of MITF, ASIP, and KIT in
Fayoumi feather follicles Pigment bars can form due to the presence/absence of melanocytes
or by differences in melanin production (eumelanin, pheomela-
nin, or no melanin) of the melanocytes that are present (Andersson
et al., 2020; Lin et al., 2013). Autosomal barring is composed of alter-
nating black and non-black bars (Figure 1). The non-black bars may
be yellowish due to the presence of pheomelanin (Figure 1b) or more
or less non-pigmented, like in Fayoumi chicken (Figure 1a) due to the
presence of the Silver allele at SLC25A2 that inhibits expression of
pheomelanin (Gunnarsson et al., 2007). To characterize the mecha-
nism underlying autosomal barring in Fayoumi chicken, we performed
immunostaining of MITF (microphthalmia-associated transcription
factor), a marker of melanocyte progenitor cells. MITF-positive cells
are present in the proximal follicle where melanocyte stem cells are
present, as shown by positive staining for KIT (Figure 4a,c). Toward
the distal feather, barb branches start to form and pigment bars
emerge. In both pigmented and non-pigmented regions, we ob-
serve the presence of MITF-positive melanocyte progenitors. This
is particularly clear in the border region (Figure 4a,b). We explored
what may repress the activity of these melanocyte progenitors in We next reasoned that if the elevated expression levels are
the result of cis-regulatory changes, we expect to observe allelic
imbalance in gene expression at one or more of the three genes
(Figure 3c). We used SNPs in the transcripts to assess the relative
expression of alleles and genomic DNA as control in which we ex-
pect a perfect 50:50 ratio. We used the T to A polymorphism caus-
ing the MC1R/E*R(Fay) allele (L133Q; chr11: 18,841,043 bp), and
SNPs in NQO1 (T to C change; chr11: 19,037,574 bp) and CDH1 (A
to T change; chr11:18,874,575 bp) to assess allelic expression in 15
heterozygous chickens utilizing cDNA from growing feathers and
the pyrosequencing technology. The expression of the Fayoumi al-
lele associated with each investigated gene was 43.1 ± 0.75% for
MC1R, 47.7 ± 0.43% for NQO1, and 59.4 ± 1.11% for CDH1. anchors melanocytes to surrounding keratinocytes (Vasioukhin
et al., 2000) and is implicated in diseases such as vitiligo (Tarle
et al., 2015). The last gene we evaluated is coding for an E3 ubiquitin-
protein ligase (ww domain-containing protein 1, WWP1) involved in
the tanning response following UV exposure in melanocytes (Cao
et al., 2013). We measured the expression of these genes in growing
chicken feathers from 10 to 12 non-barred and 10 autosomal barred
birds. Three out of the four genes showed a statistically significant
up-regulation of expression in barred feathers compared with non-
barred feathers (Figure 3b): MC1R (fold change 2.3 ± 0.2, p = .0007,
Student's t test), NQO1 (fold change 3.2 ± 0.7, p = .004) and CDH1
(fold change 1.7 ± 0.2, p = .008). WWP1 showed no statistically sig-
nificant differential expression (p = .75, Student's t test). To summarize, MC1R, NQO1, and CDH1 located within the non-
recombining region show up-regulated expression in growing auto-
somal barred feathers but this up-regulation does not appear to be
mediated by a cis-regulatory effect since none of the three genes
shows clear allelic imbalance in favor of the Fayoumi allele. A possi-
ble explanation for this up-regulated expression is that the Fayoumi
allele underlying autosomal barring results in a higher proliferation
of melanocytes. 3.4 | MC1R, NQO1, and CDH1 are over-expressed in
barred feathers but do not show allelic imbalance (c) Relative proportion of the Fayoumi (F) allele at MC1R
NQO1, and CDH1 in either cDNA samples from F/- feathers (light gray) or genomic DNA from different genotypes (dark gray; F/-, -/- and F/F FI G U R E 3 Expression analysis of candidate genes on chromosome 11. (a) Genes in the interval lacking recombination in the Fayoumi/
Light Brown Leghorn cross. Positional candidate genes are highlighted with red text. (b) Relative gene expression levels of candidate
genes on chromosome 11 in autosomal barred and non-barred feathers. EEF2 and βACTIN were used as housekeeping genes. Significant
differences are indicated by stars (Student's t test; *p < .05, **p < .01, ***p < .001). (c) Relative proportion of the Fayoumi (F) allele at MC1R,
NQO1, and CDH1 in either cDNA samples from F/- feathers (light gray) or genomic DNA from different genotypes (dark gray; F/-, -/- and F/F) SCHWOCHOW et al. 1023 3.6 | Patterning phenotypes are strongly associated
with certain MC1R (E) alleles A number of chicken breeds are assumed to be fixed for the Pg allele
based on the interpretation of the genetic basis for various plum-
age color variants (Figure 1b; Figure S1). We therefore performed
whole-genome pooled sequencing of birds from six chicken breeds
(n = 5 each), exhibiting a variety of pigmentation patterns: Partridge
Plymouth Rock—penciling, Buttercup—autosomal barring, Silver
Sebright—single lacing, Brahma—penciling, Hamburg—spangling, and
Fayoumi—autosomal barring (Figure 1; Figure S1). This was carried out
with two purposes (i) to explore whether all breeds predicted to be
fixed for the Pg allele share any region of the genome that is identical FI G U R E 5 Region near MC1R reveals no shared haplotype associated with patterning. Predicted variants within ±5kb (top) or ±50kb
(bottom) from the MC1R locus in Fayoumi and five other breeds (Brahma, Buttercup, Hamburg, Plymouth Rock, and Sebright) showing
patterning phenotypes. The heatmaps show individual variants with vertical black lines indicating their position at the MC1R-flanking region
on chromosome 11. Each breed was sequenced as a pool of five individuals and heterozygous positions thus represent a ~50/50 split within
the pool. The MC1R coding sequence is indicated using red lines. The listed MC1R alleles use the following definitions: E*R(Fay) = 133Q,
E*B = 71T-92K-215P, and E*R = 92K. The question mark for the Hamburg pool indicates that it was predicted to be variable for E92K, V126I,
and L133P (note the P instead of Q) and thus is not fixed for E*R. Based on the data presented for Hamburg in Table S9, we deduce that the
bird sequenced here carried the two following alleles: 92K-126I-133L (E*R) and 92E-126V-133P (E*?) FI G U R E 5 Region near MC1R reveals no shared haplotype associated with patterning. Predicted variants within ±5kb (top) or ±50kb
(bottom) from the MC1R locus in Fayoumi and five other breeds (Brahma, Buttercup, Hamburg, Plymouth Rock, and Sebright) showing
patterning phenotypes. The heatmaps show individual variants with vertical black lines indicating their position at the MC1R-flanking region
on chromosome 11. Each breed was sequenced as a pool of five individuals and heterozygous positions thus represent a ~50/50 split within
the pool. The MC1R coding sequence is indicated using red lines. The listed MC1R alleles use the following definitions: E*R(Fay) = 133Q,
E*B = 71T-92K-215P, and E*R = 92K. by descent (IBD) and (ii) if these breeds share an IBD region within the
1 Mb region on chromosome 11 defined in our Fayoumi backcross
population. Thus, the reason for using only five individuals per pool
was that our aim was not to estimate allele frequencies, but to identify
sequence variants that are fixed in these breeds since all individuals
within the breeds show a patterning phenotype. The analysis of the
pooled data revealed no striking IBD region shared by all populations
neither within the chromosome 11 interval nor in the entire genome
(Figure S5). However, the most striking finding was that all populations
carried only two alleles Birchen (E*R or E*R(Fay)) or brown (E*B) at MC1R,
while not sharing any common haplotype around MC1R (Figure 5). the non-black region. In Silver Laced Wyandotte chicken, ASIP was
found to be present in the peripheral pulp facing the white region and
suppress eumelanogenesis of MITF-positive progenitor cells (Inaba &
Chuong, 2020; Lin et al., 2013). Using in situ hybridization, we de-
tected expression of ASIP in the non-black regions. This is particularly
clear in a cross section (Figure 4b). Thus, the non-black bars in autoso-
mal barring of Fayoumi chicken are not caused by the absence of mel-
anocytes, as seen in sex-linked barring (Lin et al., 2013; Schwochow
Thalmann et al., 2017). Our results suggest that eumelanogenesis is
suppressed due to the expression of ASIP in the non-black regions. A striking finding when comparing the 10 kb region harboring
MC1R is the lack of linkage disequilibrium between sequence vari-
ants in this region (Figure 5), a total contrast to the lack of recombi-
nation over a 1 Mb region reported in this study based on pedigree
analysis. This analysis also demonstrates that the Birchen (E*R) allele
previously defined based on its phenotypic effect on plumage color
is genetically heterogenous both as regards the MC1R coding se-
quence as well as the flanking sequences that may harbor regulatory
variants affecting MC1R expression. For instance, the Brahma breed
is considered fixed for the E*R allele but the Brahma birds included
in this study are apparently segregating for at least two alleles both
carrying the E92K mutation but differing as regards other missense
mutations in MC1R as well as the haplotype upstream of the coding
sequence (Figure 5). 3.5 | The expression of MITF, ASIP, and KIT in
Fayoumi feather follicles These
cDNA data and the genomic DNA control differed significantly from
each other (MC1R: Student's t test p = .0001; NQO1: Student's t test
p = .008; CDH1: Student's t test p = 1 × 10−6), but these minor differ-
ences do not support a typical allelic imbalance expected in the pres-
ence of cis-regulatory effects, and we believe that they are unlikely
to reflect actual expression differences with biological significance. FI G U R E 4 The expression of MITF, ASIP, and KIT in the Fayoumi feather follicle. (a) MITF immunostaining. Longitudinal feather sections
with enlargement shown in the right column. a–c, black and white barred region. d, proximal follicle in collar bulge region. MITF-positive
cells are seen in the basal layer of the feather filament epidermis in the stem cells region, in both black and white barb ridges. MITF-
positive cells (red) are highlighted by box a, b, c, and d. (b) Longitudinal feather sections with ASIP in situ hybridization. ASIP is expressed
in feather peripheral pulp facing non-pigmented bar regions (a’ and b’; b’ is a cross section). (c) Longitudinal feather sections with KIT in situ
hybridization showing KIT-positive melanocyte stem cells at the follicle base near collar bulge region box a’’ and b’’ FI G U R E 4 The expression of MITF, ASIP, and KIT in the Fayoumi feather follicle. (a) MITF immunostaining. Longitudinal feather sections
with enlargement shown in the right column. a–c, black and white barred region. d, proximal follicle in collar bulge region. MITF-positive
cells are seen in the basal layer of the feather filament epidermis in the stem cells region, in both black and white barb ridges. MITF-
positive cells (red) are highlighted by box a, b, c, and d. (b) Longitudinal feather sections with ASIP in situ hybridization. ASIP is expressed
in feather peripheral pulp facing non-pigmented bar regions (a’ and b’; b’ is a cross section). (c) Longitudinal feather sections with KIT in situ
hybridization showing KIT-positive melanocyte stem cells at the follicle base near collar bulge region box a’’ and b’’ SCHWOCHOW et al. 1024 3.6 | Patterning phenotypes are strongly associated
with certain MC1R (E) alleles The question mark for the Hamburg pool indicates that it was predicted to be variable for E92K, V126I,
and L133P (note the P instead of Q) and thus is not fixed for E*R. Based on the data presented for Hamburg in Table S9, we deduce that the
bird sequenced here carried the two following alleles: 92K-126I-133L (E*R) and 92E-126V-133P (E*?) SCHWOCHOW et al. 1025 Since there was no shared haplotype detected for breeds with a
patterning phenotype at MC1R or elsewhere in the genome (Figure 5
and S5), but they all carry an MC1R allele with an activating mutation,
we were considering the hypothesis that different MC1R alleles with
similar functional effects are required for patterning phenotypes. Genotyping and subsequent sequencing of 60 Fayoumi chicken from
different generations from the flock initially used for linkage mapping
revealed that they are not fixed for E*R(Fay) (L133Q) but also segre-
gate for the E*R allele (Table S9), which involves a missense mutation
(E92K) resulting in constitutive activation (Benned-Jensen et al., 2011;
Ling et al., 2003; Robbins et al., 1993) and known to be associated with
melanism in other species (Baiao & Parker, 2012; Kerje et al., 2003;
Mundy et al., 2004; Nadeau et al., 2006). Despite the segregation of
two different MC1R/E alleles, there has been no obvious heterogene-
ity in the phenotypic appearance of autosomal barring in this Fayoumi
line suggesting that the two alleles have a similar effect on patterning
in this breed. We further extended our MC1R sequencing efforts to
16 additional individuals each from three of the Pg breeds (Partridge
Plymouth Rock, Hamburg Silver Spangled and Sebright Silver) and
found that all carried the E92K mutation (Table S9). that regulatory mutations affecting MC1R expression also contribute
to the complex inheritance of within-feather patterns in chicken as
previously suggested (Ling et al., 2003). One third of the autosomal barred backcross offspring were
wild-type at the SOX10 locus, demonstrating that the Db allele is
not required for the autosomal barring phenotype. This is in con-
trast to previous reports in which Db was claimed to be required to
exhibit autosomal barring in Fayoumi chicken (Carefoot, 1984,1999;
Moore & Smyth, 1972). 4 | DISCUSSION The Patterning locus has been considered the major locus control-
ling within-feather pigmentation patterns in chickens (Figure 1b). Previous studies assigned this locus to chicken linkage group 3
(Carefoot, 1987; Moore & Smyth, 1972), now known to reside on
chromosome 1. According to this model, we expected that the seg-
regation of autosomal barring in our Fayoumi × Light Brown Leghorn
backcross should be controlled by a locus on chromosome 1 in combi-
nation with the Dark brown/SOX10 locus located on the same chromo-
some. However, the present study demonstrates that a locus located
at the distal end of chromosome 11 is underlying autosomal barring in
this pedigree. Our linkage data did not pinpoint a single gene associ-
ated with this phenotype due to the lack of recombination in a 1 Mb
region. However, other data strongly suggest that autosomal barring
in this pedigree is caused by the E*R(Fay) allele, characterized by the
missense mutation L133Q, at the MC1R locus located in this interval. Pooled genome resequencing of six different breeds all exhibiting var-
ious types of patterning (previously assumed to be controlled by the
Patterning locus) did not reveal any IBD region, which was expected
if they were sharing the same causal mutation. However, the most
striking feature was that all six breeds carried either Birchen (E*R) or
brown (E*B) alleles at the MC1R locus. We observe that MC1R exhibits
a high level of genetic diversity in its flanking sequences in addition
to diversity within the coding sequence. We propose that MC1R is
the major patterning locus in chickens; that is, different mutations
causing altered regulation of MC1R signaling promote the develop-
ment of feather patterns in interaction with other loci. One such in-
teracting locus is definitely Dark brown/SOX10, others are melanotic
(Ml) and Columbian restriction (Co) for which no underlying causal
gene has been reported yet (Figure 1b). Furthermore, it is possible The presence of an activating MC1R/E mutation may be permis-
sive but not sufficient to cause feather patterning because there are
breeds carrying the activating E92K mutation but do not show pat-
terning. In fact, none of the breeds examined in this study carried the
top dominant allele Extended Black (E), which also has the E92K mu-
tation and is associated with solid black colored feathers (Andersson
et al., 2020). 3.6 | Patterning phenotypes are strongly associated
with certain MC1R (E) alleles However, as almost all individuals carrying
the causal MC1R allele but with an “unclear” feather pattern were
wild-type at Db, we propose that the Db mutation is contributing to
the regularity and clearness of autosomal barring, although not re-
quired for its formation. It is therefore likely that breeders specif-
ically selected Db carriers for a more appealing phenotype, which
may have led to the assumption that Db is required for autosomal
barring. Thus, the genetic basis for patterning in chicken needs to be
reconsidered since it is clear that there is not a universal pattering
gene such as Pg, but that the MC1R/E locus plays a major role for
patterning. However, the present study does not completely rule out
the possibility of the existence of a Patterning locus in addition to
MC1R/E, because it is possible, but unlikely, that the Fayoumi and
Light Brown Leghorn founders used in the present study are all ho-
mozygous Pg/Pg despite the fact the latter do not show any pattern-
ing phenotype (Figure 1a). Nevertheless, our study provides strong
evidence against the previous assumption that a Patterning locus on
chromosome 1 has a predominant role for the presence/absence of
within-feather patterns in chicken (Smyth Jr, 1990). Ling et al. (2003) investigated the functional effects of differ-
ent chicken MC1R alleles using transfection studies. They found
that the E92K mutation present in the E*E, E*R, and E*B alleles is
causing constitutive activation of the receptor. In contrast, they did
not observe a similar effect for the L133Q mutation underlying the
E*R(Fay) allele, which has been phenotypically assigned to the E*R
group of MC1R alleles in chicken. However, these pharmacological
studies carried out in mammalian cells do not perfectly replicate
the endogenous conditions for chicken MC1R; for instance, im-
portant cofactors may be absent during the assay. Furthermore,
these missense mutations may occur in strong linkage disequilib-
rium with regulatory mutations that affect MC1R expression. Thus,
an important functional effect may not be manifested under these
experimental conditions. In fact, the segregation of both the E92K
and L133Q alleles in the line of Fayoumi chickens used in the pres-
ent study without an obvious heterogeneity in phenotype shows
that the two alleles must have similar effects on autosomal barring. expression of MC1R, also the closely linked NQO1 and CDH1 genes
show higher expression in autosomal barred feathers; both genes
have an established role in pigment cell biology (Choi et al., 2010;
Vasioukhin et al., 2000). Thus, the presence of the E*R(Fay) allele re-
sults in more active melanocytes and/or an expansion of the number
of melanocytes that may affect the interaction of different cell types
that together determine within-feather pigmentation patterns. This
model can be tested by gene editing and/or in vivo transfection ex-
periment manipulating MC1R and ASIP expression. The present study is also relevant for understanding the basis for
camouflage color in birds. Stippling (Figure 1b) is a wild-type camou-
flage pattern typical for female birds nesting on the ground, and some
juvenile birds present on the ground before they can fly, like gulls,
terns and certain shorebirds. Our results support a role for ASIP-
MC1R interactions in the formation of within-feather patterns, at
least in some birds, and that increased MC1R signaling not only leads
to a darker plumage but can also adjust the appearance of camouflage
patterns. The importance of ASIP-MC1R interactions for camouflage
patterns is also evident in mammals (Andersson, 2020). Dominant
MC1R mutations causing constitutive activation as well as recessive
mutations inactivating MC1R function disrupt the characteristic cam-
ouflage pattern in piglets (Kijas et al., 2001), which is also observed in
chickens since the dorsal stripes of a wild-type day-old chick are not
observed in fully black breeds, such as the Black Castellana, as well
as in the fully red breeds, such as the Rhode Island Red. In the case of
the Fayoumi, the day-old chicks show incomplete stripes on the back. Furthermore, ASIP-MC1R interactions are underlying winter white
camouflage color in snow-shoe hares (Jones et al., 2018). In birds, distinct pigment patterns can form across the body
or within a feather (Inaba & Chuong, 2020). Horizontal barring in
feathers is a striking pattern. Genetic studies of sex-linked barring
established that this phenotype is caused by the combined effect
of regulatory mutations and changes in the coding sequence of
CDKN2A (Hellström et al., 2010; Schwochow Thalmann et al., 2017). Interestingly, MITF-positive cells are absent in the white barred re-
gions, and there is no ASIP expression in the associated peripheral
pulp of sex-linked barring feathers (Lin et al., 2013). Feathers are
made from the distal to the proximal end (Chen et al., 2015). Careful
analyses revealed that the depletion of melanocytes is considered to
be based on CDKN2A-dependent precocious differentiation of me-
lanocytes. When melanocyte stem cells sense the absence of mela-
nocytes, another wave of melanocyte progenitor cells is produced to
make the next black bar (Lin et al., 2013). In contrast, in the autosomal
barring phenotype, we observed MITF-positive cells to be present
in both the black and non-black barred regions during their forma-
tion. The pattern appears to be controlled by ASIP in the peripheral
pulp region. How ASIP expression in the dermal cells is controlled
is not known. In developing Japanese quail embryos, longitudinal
pigmented stripes form on the dorsal trunk, and Japanese quail me-
lanocytes transplanted to embryonic day 6 White Leghorn chicken
embryos can induce ASIP in adjacent dermis (Inaba et al., 2019). Thus, we can speculate that melanocytes within the feather follicle
at certain functional states may also induce ASIP expression. The present finding that genetic variation at MC1R is affecting
pigmentation patterns in chicken is interesting in relation to reports
that the MC1R antagonist ASIP has a role in feather pigmentation
(Lin et al., 2013; Yoshihara et al., 2011; Zhang et al., 2013) and that
ASIP is strongly associated with differences in throat pigmentation
in golden-winged and blue-winged warblers (Toews et al., 2016) and
a plumage trait in white wagtails (Semenov et al., 2021). These data
together suggest that ASIP-MC1R interaction may play a pivotal role
for pigmentation patterns in birds in general and that this interaction
may have a greater impact in the growing feather that will develop
into a two-dimension structure, as compared to the single dimension
of the hair developing from mammalian hair follicles. 1026 | 1026 SCHWOCHOW et al. 4 | DISCUSSION Thus, further pedigree experiments are required before
we fully understand the genetic basis for the rich phenotypic diver-
sity in within-feather patterning in this species (Figure 1 and S1). We observed a surprisingly low rate of recombination in the 1 Mb
interval at the distal end of chromosome 11 given the fact that the
average recombination rate in chicken is about 4 cM/Mb. A possible
explanation for a low rate of recombination is the presence of an
inversion. However, it is unlikely that this is the case for this region
because a similarly low rate of recombination has beem reported in
previous studies (Groenen et al., 2009; Pengelly et al., 2016). The expression data in feather follicles are consistent with our
interpretation that autosomal barring is caused by an activating
coding mutation at the MC1R locus. We documented up-regulated
expression of MC1R, CDH1, and NQO1 in autosomal barred feath-
ers compared with wild-type feathers consistent with increased
pigment production or an expansion of the number of pigment cells
(Figure 4b). However, none of the three loci showed a clear allelic
imbalance as expected if the causal mutation was a cis-acting reg-
ulatory mutation affecting the expression of one or more of these
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pnas.1816107116 Carefoot, W. ACKNOWLEDGEMENTS The help of the animal caretakers of the INRA PEAT experimental
unit is gratefully acknowledged as well as Mr. Frédéric Theme and
Mr. Jacques Berge who provided blood samples from various breeds
showing feather pigmentation patterns. We are also thankful to
the wet laboratory support by Erika Manlig and thank Marie Allen
and Magdalena Bus for instructions and use of their pyrosequenc-
ing instrument. LA is supported by the Knut and Alice Wallenberg
Foundation and Vetenskapsrådet. CMC and TXJ are supported by
NIH NIAMS R37 AR060306. DS benefited from an Erasmus Mundus
fellowship within the framework of the European School of Animal
Breeding and Genetics. The National Genomics Infrastructure Why is the presence of some MC1R mutations enhancing MC1R
signaling and melanogenesis associated with within-feather pig-
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doi.org/10.1146/annurev-animal-022513-114127 (NGI)/Uppsala Genome Center and UPPMAX provided service in
massive parallel sequencing and computational infrastructure. Work
performed at NGI/Uppsala Genome Center has been funded by RFI/
VR and Science for Life Laboratory, Sweden. Choi, T.-Y., Sohn, K.-C., Kim, J.-H., Kim, S.-M., Kim, C.-H., Hwang, J.-
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tics/btv195 ORCID Doreen Schwochow
https://orcid.org/0000-0002-0090-4355
Tingxing Jiang
https://orcid.org/0000-0003-1972-8985
Jingyi Li
https://orcid.org/0000-0001-6775-5190
Bertrand Bed’Hom
https://orcid.org/0000-0002-0825-0886
Cheng-Ming Chuong
https://orcid.org/0000-0001-9673-3994
Michèle Tixier-Boichard
https://orcid.org/0000-0001-5944-7884
Leif Andersson
https://orcid.org/0000-0002-4085-6968 Garcia-Borron, J. C., Sanchez-Laorden, B. L., & Jimenez-Cervantes,
C. (2005). Melanocortin-1 receptor structure and functional
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Crooijmans, R. P., Besnier, F., Lathrop, M., Muir, W. M., Wong, G.- K.-
S., Gut, I., & Andersson, L. (2009). A high-density SNP-based linkage
map of the chicken genome reveals sequence features correlated
with recombination rate. Genome Research, 19, 510–519. https://doi. org/10.1101/gr.086538.108 DATA AVAILABILITY STATEMENT All sequencing data from this study are available through NCBI
Sequence Read Archive BioProject PRJNA694957 (SAMN17582112,
Autosomal barred pool; SAMN17582113, Dark brown (Db) pool;
SAMN17582114, Wild-type pool; SAMN17582115, unclear pool)
and PRJNA679787 (SAMN16846355, Sebright; SAMN16846356,
Brahma; SAMN16846357, Hamburg; SAMN16846358, Plymouth_
Rock; SAMN16846359, Buttercup; SAMN16846360, Fayoumi). Choi, Y., Sims, G. E., Murphy, S., Miller, J. R., & Chan, A. P. (2012). Predicting the functional effect of amino acid substitutions and
indels. PLoS One, 7, e46688. https://doi.org/10.1371/journal.pone.
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tor gene and E locus plumage color phenotype. Poultry Science, 93,
1089–1096. https://doi.org/10.3382/ps.2013-03611 CONFLICT OF INTEREST Authors declare no competing interests. melanocortin-1 receptor gene. Journal of Heredity, 103, 315–321. https://doi.org/10.1093/jhered/ess023 Besnier, F., Carlborg, Ö., Bed’hom, B., Tixier-Boichard, M., Jensen, P.,
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Y. P. (2016). The relationship between MC1R mutation and plum-
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et al. The feather pattern autosomal barring in chicken is
strongly associated with segregation at the MC1R locus. Pigment Cell Melanoma Res. 2021;34:1015–1028. https://doi. org/10.1111/pcmr.12975 How to cite this article: Schwochow D, Bornelöv S, Jiang T,
et al. The feather pattern autosomal barring in chicken is
strongly associated with segregation at the MC1R locus. Pigment Cell Melanoma Res. 2021;34:1015–1028. https://doi. org/10.1111/pcmr.12975 Robbins, L. S., Nadeau, J. H., Johnson, K. R., Kelly, M. A., Roselli-Rehfuss,
L., Baack, E., Mountjoy, K. G., & Cone, R. D. (1993). Pigmentation
phenotypes of variant extension locus alleles result from point mu-
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doi.org/10.1016/0092-8674(93)90572-8 Rubin, C.-J., Zody, M. C., Eriksson, J., Meadows, J. R. S., Sherwood, E.,
Webster, M. T., Jiang, L., Ingman, M., Sharpe, T., Ka, S., Hallböök, F.,
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Contribution of Information and Communication Technology (ICT) in Country'S H-Index
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1MARYAM FARHADI1*, HADI SALEHI2,3, MOHAMED AMIN EMBI2, MASOOD FOOLADI1,
HADI FARHADI4, AREZOO AGHAEI CHADEGANI1, and NADER ALE EBRAHIM5 *Corresponding author: Maryam Farhadi, Email: farhadim58@gmail.com *Corresponding author: Maryam Farhadi, Email: farhadim58@gmail.com Journal of Theoretical and Applied Information Technology
10th November 2013. Vol. 57 No.1
© 2005 - 2013 JATIT & LLS. All rights reserved. Journal of Theoretical and Applied Information Technology
10th November 2013. Vol. 57 No.1
© 2005 - 2013 JATIT & LLS. All rights reserved. ISSN: 1992-8645 www.jatit.org ABSTRACT The aim of this study is to examine the effect of Information and Communication Technology (ICT)
development on country’s scientific ranking as measured by H-index. Moreover, this study applies ICT
development sub-indices including ICT Use, ICT Access and ICT skill to find the distinct effect of these
sub-indices on country’s H-index. To this purpose, required data for the panel of 14 Middle East countries
over the period 1995 to 2009 is collected. Findings of the current study show that ICT development
increases the H-index of the sample countries. The results also indicate that ICT Use and ICT Skill sub-
indices positively contribute to higher H-index but the effect of ICT access on country’s H-index is not
clear Keywords: Information and Communication Technology (ICT) development, H-index, Middle East CONTRIBUTION OF INFORMATION AND
COMMUNICATION TECHNOLOGY (ICT) IN COUNTRY’S H-
INDEX 1MARYAM FARHADI1*, HADI SALEHI2,3, MOHAMED AMIN EMBI2, MASOOD FOOLADI1,
HADI FARHADI4, AREZOO AGHAEI CHADEGANI1, and NADER ALE EBRAHIM5
1Department of Accounting, Mobarakeh Branch, Islamic Azad University, Mobarakeh, Isfahan, Iran
2Faculty of Education, Universiti Kebangsaan Malaysia (UKM), Bangi, 43600, Malaysia
3Faculty of Literature and Humanities, Najafabad Branch, Islamic Azad University, Najafabad, Isfahan,
Iran
4School of Psychology and Human Development, Faculty of Social Sciences and Humanities, Universiti
Kebangsaan Malaysia (UKM), Malaysia
5Department of Engineering Design and Manufacture, Faculty of Engineering, University of Malaya, Kuala
Lumpur, Malaysia
*Corresponding author: Maryam Farhadi, Email: farhadim58@gmail.com www.jatit.org it
0
1
it
2
it
3
it
it
H
Access
Use
Skill
µ
µ
µ
µ
δ
=
+
+
+
+
(2) (2) where
's
µ
are parameters to be estimated, H
shows H-index and Access, Use and Skill stand
for ICT development sub-indices. Moreover,
subscripts i and t indicate the ith country in tth
year, respectively, and δ is the error term which
is assumed to be independent of Access, Use and
Skill variables. where
's
µ
are parameters to be estimated, H
shows H-index and Access, Use and Skill stand
for ICT development sub-indices. Moreover,
subscripts i and t indicate the ith country in tth
year, respectively, and δ is the error term which
is assumed to be independent of Access, Use and
Skill variables. H-index can measure the quantity and quality
of papers simultaneously since the total number
of published articles and the number of citations
received by that articles are considered in the
calculation of H-index. In addition, there are
many studies, which reveal the benefits of H-
index as an appropriate and fair tool for
evaluating and ranking scientists, laboratories,
scientific journals, universities, institutions and
countries [17, 18, 19, 20, 21]. 2.2.1.
ICT development The IDI, as an indicator of ICT development
is computed based on Principal Component
Analysis
(PCA),
which
combines
various
indicators in order to make single value. For the
first time, IDI is introduced in 2009 by
Measuring the Information Society [23] and
considers the degree of progress in ICTs in more
than 150 countries. Composition of this indicator
includes ICT Access showing infrastructure and
access, ICT Skill referring to ICT skill or
capability and ICT Use displaying the extent of
using ICT. Required ICT data to compute the
IDI is extracted from United Nations Educational
Scientific and Cultural Organization (UNESCO)
and ITU. Nowadays, ICT is considered as an
important factor in development of countries,
which are moving towards knowledge or Hirsch [15] proposes that H-index can be
used to appraise not only the scientists but also
countries and institutions. Hence, following
Jacso [22], current study also uses H-index as a
proxy to measure scientific ranking of countries. www.jatit.org East countries, this study applies the regression
techniques with countries’ H-index as dependent
variable and IDI as independent variable as
follows: Although, the key role of ICT is approved in
different areas such as engineering [11],
education [12], health care [13], and economy
[6], its effect on the degree of scientific ranking
of a country is yet unexplored. Therefore, the
aim of this study is to explore the impact of ICT
development as measured by ICT Development
Index (IDI) on H-index as a proxy of scientific
progress of a country. it
0
1
it
it
H
IDI
α
α
ε
=
+
+
(1) (1) where
's
α
are parameters to be estimated, H
represents
H-index,
IDI
stands
for
ICT
development index and subscripts i and t show
the ith country in tth year, respectively. ε is the
error term which is assumed to be independent of
IDI. Then, for more inspections, instead of IDI,
which captures the composite effect of ICT
development, the current study uses three ICT
sub-indices including ICT Access, ICT Use and
ICT Skill as explanatory variables. In this
approach, we can test the distinct impact of each
sub-index
on
the
country’s
H-index. Consequently, in this step of the analysis,
following equation (Equation 2) is used: In addition, in order to shed more lights on
this area, present study applies three components
of ICT development namely, ICT Skill, ICT Use
and ICT Access to examine the distinct impact of
these components on H-index as a proxy of
scientific progress of a country. To this purpose,
this study applies a multiple regression analysis
on a panel of 14 Middle East countries over the
period 1995 to 2009. The current study suggests the use of H-index
provided by SCImago database [14] to measure
the scientific ranking of country. Based on
Hirsch [15], a scientist or a group of scientists
has H-index of h when h of their papers has at
least h citations. In other words, H-index is the
number of articles with citation number higher or
equal to h [15]. The H-index, which has been
invented by Hirsch in 2005, proposes a simple
and apparently strong measure to compare the
scientific productivity and visibility of countries
and institutions [16]. 1. INTRODUCTION However, ICT revolution and use of internet
caused many structural changes [3]. As an
example, the United States productivity has been
revived during the late 1990s and the early 2000s
because of the ICT development [4]. There are
many studies introducing ICT as an important
element to facilitate economic growth in both
developing and developed countries [5, 6]. In
addition, ICT has the potential to increase health
systems through the designing new methods to
prevent and detect disease [7]. Other studies
confirm the dominant role of ICT in improving
and modernizing the learning methods and
educational systems [8, 9]. The fast progress in
technology in the last two decades has reduced
the cost of ICTs. In this situation, it is easier for
more people to use and enjoy the advantages of
ICTs [10]. However, ICT revolution and use of internet
caused many structural changes [3]. As an
example, the United States productivity has been
revived during the late 1990s and the early 2000s
because of the ICT development [4]. There are
many studies introducing ICT as an important
element to facilitate economic growth in both
developing and developed countries [5, 6]. In
addition, ICT has the potential to increase health
systems through the designing new methods to
prevent and detect disease [7]. Other studies
confirm the dominant role of ICT in improving
and modernizing the learning methods and
educational systems [8, 9]. The fast progress in
technology in the last two decades has reduced
the cost of ICTs. In this situation, it is easier for
more people to use and enjoy the advantages of
ICTs [10]. In the last decades, technology has improved
the life style and made easier the human
activities through a new civilization. A new
revolution
named
Information
and
Communication Technology (ICT) has occurred
in the present world [1]. ICT is defined as a
concept including information equipment as well
as computers and software, auxiliary equipment
that is connected to a computer (such as
calculators,
cash
registers,
photocopiers),
communications equipment, and instruments [2]. In other words, ICT is a technology with the aim
of
connecting
computers
and
other
communication equipment to gather, produce,
process, classify, manage, create, and distribute
information. After the invention of machines in
1970s, industrial revolution has been started and
machines have been used instead of handworks. 122 2.1. Empirical Model In order to examine the effect of ICT
development on scientific degree of 14 Middle 123 Journal of Theoretical and Applied Information Technology
10th November 2013. Vol. 57 No.1
© 2005 - 2013 JATIT & LLS. All rights reserved. ISSN: 1992-8645 www.jatit.org E-ISSN: 1817-3195
information-based societies. It is assumed that
these countries have experienced the process of
ICT development to turn into an information
society. Therefore, the ITU [23] suggests the
three-phase model including ICT readiness
(showing the degree of networked infrastructure
and ICT access), ICT intensity (showing the
degree of ICT use by the society) and ICT
impact (showing the result of effective and
efficient ICT use). IDI is a composite indicator
including ICT access, ICT skill and ICT use. Each sub-index is composed of a number of
indicators explained as follow:
Figure 1. Three Stages In The Evolution Towards An
Information Society (Source: ITU [25]) ed Information Technology
. Vol. 57 No.1
. All rights reserved. g E-ISSN: 1817-3195
Figure 1. Three Stages In The Evolution Towards An
Information Society (Source: ITU [25]) Journal of Theoretical and Applied Information Technology
10th November 2013. Vol. 57 No.1
© 2005 - 2013 JATIT & LLS. All rights reserved. ISSN: 1992-8645 information-based societies. It is assumed that
these countries have experienced the process of
ICT development to turn into an information
society. Therefore, the ITU [23] suggests the
three-phase model including ICT readiness
(showing the degree of networked infrastructure
and ICT access), ICT intensity (showing the
degree of ICT use by the society) and ICT
impact (showing the result of effective and
efficient ICT use). IDI is a composite indicator
including ICT access, ICT skill and ICT use. Each sub-index is composed of a number of
indicators explained as follow: information-based societies. It is assumed that
these countries have experienced the process of
ICT development to turn into an information
society. Therefore, the ITU [23] suggests the
three-phase model including ICT readiness
(showing the degree of networked infrastructure
and ICT access), ICT intensity (showing the
degree of ICT use by the society) and ICT
impact (showing the result of effective and
efficient ICT use). IDI is a composite indicator
including ICT access, ICT skill and ICT use. Each sub-index is composed of a number of
indicators explained as follow: Figure 1. Three Stages In The Evolution Towards An
Information Society (Source: ITU [25]) 1. 2.1. Empirical Model Access Sub-Index shows the availability of
ICT infrastructure and individuals’ access to
basic ICTs. This sub-index has five indicators
including fixed telephone lines per 100
inhabitants,
mobile
cellular
telephone
subscriptions
per
100
inhabitants,
international Internet bandwidth (bit/s) per
Internet user, proportion of households with a
computer and proportion of households with
Internet access at home. 2.2.2.
h-index Therefore, this subgroup has three
indicators including, adult literacy rate,
secondary gross enrolment ratio and tertiary
gross enrolment ratio. The required data for
this subgroup are extracted from the
UNESCO Institute for Statistics (UIS) [24]. The current study collects the required data
on H-index for the panel of 14 Middle East
countries over the period 1995 to 2009, from
SCImago database [14]. The SCImago Journal
and Country Rank website presents the journal
and country scientific indicators based on the
information included in the Scopus database. 2.2.2.
h-index Different sources report the H-index. Some of
these sources are subscription-based sources
such as Scopus and Web of Science (WoS) and
some of them are free access sources such as
Harzing's Publish or Perish program on the basis
of Google Scholar entries. It should be noted that
these sources report different H-index for the
same institution, country and scholar because of
their different coverage. For example, WoS
covers a high range of published journals while it
does not cover high impact conferences. Although, the coverage of publications in Scopus
is poor prior to 1996, it covers conferences
properly. Documents in Google Scholar receive
more citations in comparison with those in
Scopus and WoS. It is argued that Google
Scholar has the most coverage of journals and
conferences particularly those published after
1990 [26]. 2. Use Sub-Index shows the real degree of ICTs
strength of use based on the available
infrastructure
and
access
indicators. Considering the limitation on data for ICT
Use at the global scale and results of the
PCA, this subgroup has three indicators
including Internet users per 100 inhabitants,
fixed broadband Internet subscribers per 100
inhabitants
and
mobile
broadband
subscriptions per 100 inhabitants. 3. Skill Sub-Index captures the degree of ICT
skill in different countries. Since the required
data for many developing countries are not
collected, an appropriate indicator can be the
degree of literacy and education. Especially
in developing countries, the poor level of
education is a major obstacle to the effective
use of internet and computers. With the
inclusion of ICT in school courses, school
attendance may offer an appropriate indicator
for students’ exposure to the Internet or
computers. Therefore, this subgroup has three
indicators including, adult literacy rate,
secondary gross enrolment ratio and tertiary
gross enrolment ratio. The required data for
this subgroup are extracted from the
UNESCO Institute for Statistics (UIS) [24]. 3. Skill Sub-Index captures the degree of ICT
skill in different countries. Since the required
data for many developing countries are not
collected, an appropriate indicator can be the
degree of literacy and education. Especially
in developing countries, the poor level of
education is a major obstacle to the effective
use of internet and computers. With the
inclusion of ICT in school courses, school
attendance may offer an appropriate indicator
for students’ exposure to the Internet or
computers. 3. FINDING AND DISCUSSION In this section, we first estimate Equation 1,
which explores the impact of ICT development
on H-index as a proxy of country’s scientific
ranking. Then, applying the three ICT sub-
indices, this study presents the estimation results
for Equation 2, which is corresponding to the
effect of ICT development sub-indices on H-
index. 124 Journal of Theoretical and Applied Information Technology
10th November 2013. Vol. 57 No.1
© 2005 - 2013 JATIT & LLS. All rights reserved. ISSN: 1992-8645 www.jatit.org E-ISSN: 1817-3195
Table 1. Regression Results For The Impact Of ICT Development On H-Index
Dependent Variable: H-index
Variables
Coefficient
Standard Error
T
IDI
31.69
4.67***
6.79
Constant
24.16
13.35*
1.81
Observation = 210
F(1, 208) = 46.07***
R-squared = 0.1813
Adjusted R-squared = 0.1774
Note:
***, ** and * denote statistically significant at 1%, 5% and 10%, respectively. Standard errors are heteroskedasticity consistent. sample countries. The estimated coefficients
show a positive and significant effect of ICT Use
and ICT Skill on country’s scientific ranking. This
finding
asserts
that
increasing
the
application of ICTs and improving the ICT skills
might significantly increase the scientific degree
of a country. The results also indicate that if a
country increases the ICT Use and ICT Skill
indices by one unit, its H-index will raise by 133
and 407 units, respectively. As can be seen in
Table 2, the coefficient explaining the effect of
ICT Access on H-index is negative but not
significant. Finally, based on the adjusted R-
squared, ICT sub indices can explain 27.11% of
variations in country’s H-index. Table 1 shows the effect of ICT development
on each country’s H-index. The estimation
results indicate a positive and significant effect
of ICT development on country’s scientific
ranking as proxied by H-index. This finding
suggests that suitable policies aim at improving
the level of ICT development can significantly
increase the scientific rankings of a country. The
results also assert that if a country raises its ICT
development index by one unit, the H-index will
increase by 31 units. As can be seen in Table 1,
the adjusted R-squared is equal to 0.1774, which
means that ICT development can explain 17.74%
of variations in country’s H-index. Table 2 exhibits the impact of ICT
development sub-indices on H-index of 14 Table 2. Standard errors are heteroskedasticity consistent. ***, ** and * denote statistically significant at 1%, 5% and 10%, respectively. 4. CONCLUSION AND IMPLICATIONS [7] Haridasan, K., Rangarajan, S. and Pirio, G. “Re-Inventing Health Care Training In The
Developing World: The Case For Satellite
Applications In Rural Environments”, Online
Journal of Space Communication, 14(Winter),
2009. This paper focuses on examining the effect of
ICT development on the scientific degree of 14
Middle East countries as proxied by H-index. The
results of the regression analysis show that ICT
development has a positive and significant effect
on the H-index of these countries. Moreover,
applying the ICT development sub-indices, this
study finds that ICT Use and ICT Skill might have
positive effects on H-index but the effect of ICT
Access is not clear since its corresponding
coefficient
is
not
statistically
significant. Therefore, we can conclude that ICT induces
outcomes that leads to higher H-index values and
raises the scientific level of sample countries. In
this
situation,
policy
makers
should
aim
increasing the level of ICT development through
increasing its indicators including fixed and
mobile telephone lines, international Internet
bandwidth, proportion of households with a
computer and Internet access, Internet users, fixed
and mobile broadband Internet subscribers, adult
literacy rate, secondary and tertiary gross
enrolment ratios. [8] Sharma, A., Gandhar, K., Sharma, S. and
Seema, S. “Role of ICT in the Process of
Teaching and Learning”, Journal of Education
and Practice, Vol. 2, No. 5, 2011, pp. 1-6. [9] Sangrà, A. and González-Sanmamed, M. “The
role of information and communication
technologies in improving teaching and
learning processes in primary and secondary
schools”, Journal of Asynchronous Learning
Networks, Vol. 15, No. 4, 2011, pp. 47-59. [10]
International Telecommunication Union,
“Measuring the information society: The ICT
Development Index”, Place des Nations CH-
1211 Geneva Switzerland, 2011. [11]
C.C. Aldea, A.D. Popescu, A. Draghici
and G. Draghici, “ICT Tools Functionalities
Analysis for the Decision Making Process of
Their Implementation in Virtual Engineering
Teams”, Procedia Technology, Vol. 5, 2012,
pp. 649-658. [12]
F. Scheuermann
and
F. Pedró,
“Assessing the effects of ICT in education:
Indicators,
criteria
and
benchmarks
for
international
comparisons”,
Luxembourg:
Publications Office of the European Union,
2009. 3. FINDING AND DISCUSSION Regression Results For The Impact Of ICT Development Sub-Indices On H-Index
Dependent Variable: H-index
Variables
Coefficient
Standard Error
t
ICT Access
-25.46
49.80
-0.51
ICT Use
133.23
64.64**
2.06
ICT Skill
407.28
64.86***
6.28
Constant
-151.10
35.82***
-4.22
Observation = 210
F(3, 206) = 26.91***
R-squared = 0.2816
Adjusted R-squared = 0.2711
Note:
***, ** and * denote statistically significant at 1%, 5% and 10%, respectively. Standard errors are heteroskedasticity consistent. Table 2. Regression Results For The Impact Of ICT Development Sub-Indices On H-Index 125 © 2005 - 2013 JATIT & LLS. All rights reserved. ISSN: 1992-8645 www.jatit.org REFERENCES: [1] R.J. Gordon, “Does the ‘New Economy’
measure up to the great inventions of the
past?”, Journal of Economic Perspectives,
Vol. 14, No. 4, 2000, pp. 49-74. [13]
A.R. Bakker, “Health care and ICT,
partnership is a must”, International Journal of
Medical Informatics, Vol. 66, No. 1-3, 2002,
pp. 51-57. [2] S.D. Oliner and D.E. Sichel, “Computers and
output growth revisited: How big is the
puzzle?”, Brookings Papers on Economic
Activity, Vol. 2, 1994, pp. 273-334. [14] SCImago, SJR — SCImago Journal &
Country Rank, 2007, Retrieved March 11,
2013, from http://www.scimagojr.com [3] S.S. Cohen, J.B. Delong and J. Zysman,
“Tools for thought: What is new and important
about the E-economy?”, BRIE Working Paper,
No. 138, 2000. [15] J.E. Hirsch, “An index to quantify an
individual’s
scientific
research
output”,
Proceedings of the National Academy of
Science, Vol. 102, No. 46, 2005, pp. 16569-
16572. [4] D.W. Jorgenson and K.J. Stiroh, “Information
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methodology
for
ranking
scientific
institutions”, Scientometrics, Vol. 75, No. 1,
2008, pp. 163-174. [5] Gruber, H. and Koutroumpis, P. “Mobile
telecommunications
and
the
impact
on
economic development”, Economic Policy,
Vol. 26, No. 67, 2011, pp. 387-426. [17] P. Ball, “Index aims for fair ranking of
scientists”, Nature, Vol. 436, No. 900, 2005. doi:10.1038/436900a. [6] Farhadi, M., Ismail, R. and Fooladi, M. “Information and Communication Technology
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2012,
e48903. doi:10.1371/journal.pone.0048903 [18] G. Saad, “Exploring the h-index at the
author and journal levels using bibliometric
data of productive consumer scholars and
business-related
journals
respectively”, 126 Journal of Theoretical and Applied Information Technology
10th November 2013. Vol. 57 No.1
© 2005 - 2013 JATIT & LLS. All rights reserved. 645 www.jatit.org E-ISSN: 1817-3195
metrics, Vol. 69, No. 1, 2006, pp. 117- Journal of Theoretical and Applied Information Technology
10th November 2013. Vol. 57 No.1
© 2005 - 2013 JATIT & LLS. All rights reserved. 45 www.jatit.org E-ISSN: 1817-3195
etrics, Vol. 69, No. 1, 2006, pp. 117- ISSN: 1992-8645 Scientometrics, Vol. 69, No. 1, 2006, pp. 117-
120. [19] W. Glanzel,
“On
the
h-index
-
A
mathematical approach to a new measure of
publication activity and citation impact”,
Scientometrics, Vol. 67, No. 2, 2006, pp. 315-
321. [20] T. Braun, W. Glanzel and A. REFERENCES: Schubert, “A
Hirsch-type index for journals Scientometrics,
Vol. 69, No. 1, 2006, pp. 169-173. [21] K.M. Mclntyre, L. Hawkes, A. Waret-
Szkuta, S. Morand and M. Baylis, “The H-
Index as a Quantitative Indicator of the
Relative Impact of Human Diseases”, PLoS
ONE,
Vol. 6,
No. 5,
2011. e19558. doi:10.1371/journal.pone.0019558. [22] P. Jacso, “The h-index for countries in Web
of Science and Scopus”, Online Information
Review, Vol. 33, No. 4, 2009, pp. 831-837. [23] International
Telecommunication
Union,
“Measuring the information society: The ICT
Development Index”, Place des Nations CH-
1211 Geneva Switzerland, 2009. [24] UNESCO Institute for Statistics (UIS),
2011. http://www.uis.unesco.org. [25] International
Telecommunication
Union,
“Measuring the information society: The ICT
Development Index”, Place des Nations CH-
1211 Geneva Switzerland, 2010. [26] L.I. Meho and K. Yang, “Impact of data
sources on citation counts and rankings of LIS
faculty: Web of science versus scopus and
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and
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2125. 127
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High Prevalence of Urinary Schistosomiasis in a Desert Population: Results from an Exploratory Study Around the Ounianga Lakes in Chad
|
Research Square (Research Square)
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High Prevalence of Urinary Schistosomiasis in a
Desert Population: Results from an Exploratory
Study Around the Ounianga Lakes in Chad High Prevalence of Urinary Schistosomiasis in a
Desert Population: Results from an Exploratory
Study Around the Ounianga Lakes in Chad
Wendelin Moser
Swiss Tropical and Public Health Institute: Schweizerisches Tropen- und Public Health-Institut
Annour Aboum Batil
IRED Institut de Recherche en Elevage pour le Developpement
Rebekka Ott
Swiss Tropical and Public Health Institute: Schweizerisches Tropen- und Public Health-Institut
Moussa Abderamane
Universite de N'Djamena
Ruth Clements
Natural History Museum
Rahel Wamp§er
Swiss Tropical and Public Health Institute: Schweizerisches Tropen- und Public Health-Institut
Sven Poppert
Swiss Tropical and Public Health Institute: Schweizerisches Tropen- und Public Health-Institut
Peter Steinmann
Swiss Tropical and Public Health Institute: Schweizerisches Tropen- und Public Health-Institut
Fiona Allan
Natural History Museum
Helena Greter ( helena.greter@swisstph.ch )
Swiss Tropical and Public Health Institute: Schweizerisches Tropen- und Public Health-Institut
https://orcid.org/0000-0003-1844-3961
Research Article
Keywords: Bulinus truncatus. Chad, malacology, Ounianga, POC-CCA, prevalence, Sahara, Schistos
bovis, Schistosoma haematobium, schistosomiasis
Posted Date: October 28th 2021 Study Around the Ounianga Lakes in Chad
Wendelin Moser
Swiss Tropical and Public Health Institute: Schweizerisches Tropen- und Public Health-Institut
Annour Aboum Batil
IRED Institut de Recherche en Elevage pour le Developpement
Rebekka Ott
Swiss Tropical and Public Health Institute: Schweizerisches Tropen- und Public Health-Institut
Moussa Abderamane
Universite de N'Djamena
Ruth Clements
Natural History Museum
Rahel Wamp§er
Swiss Tropical and Public Health Institute: Schweizerisches Tropen- und Public Health-Institut
Sven Poppert
Swiss Tropical and Public Health Institute: Schweizerisches Tropen- und Public Health-Institut
Peter Steinmann
Swiss Tropical and Public Health Institute: Schweizerisches Tropen- und Public Health-Institut
Fiona Allan
Natural History Museum
Helena Greter ( helena.greter@swisstph.ch )
Swiss Tropical and Public Health Institute: Schweizerisches Tropen- und Public Health-Institut
https://orcid.org/0000-0003-1844-3961
Research Article
Keywords: Bulinus truncatus. Chad, malacology, Ounianga, POC-CCA, prevalence, Sahara, Schisto
bovis, Schistosoma haematobium, schistosomiasis ABSTRACT
23 Background: Researching a water-borne disease in the middle of the Sahara desert might
24
not seem the most relevant concern. However, nomadic Sahelian pastoralist’s health concerns
25
regarding their livestock and anecdotal reports about trematode infections of Fasciola spp and
26
Schistosoma spp in desert-raised animals justified an exploratory study focusing on the lakes
27
of Ounianga in Northern Chad. The aim was to test whether trematode parasites such as
28
Schistosoma spp occur in human populations living around the Sahara desert lakes of
29
Ounianga Kebir and Ounianga Serir in northern Chad. 30 Background: Researching a water-borne disease in the middle of the Sahara desert might
24
not seem the most relevant concern. However, nomadic Sahelian pastoralist’s health concerns
25
regarding their livestock and anecdotal reports about trematode infections of Fasciola spp and
26
Schistosoma spp in desert-raised animals justified an exploratory study focusing on the lakes
27
of Ounianga in Northern Chad. The aim was to test whether trematode parasites such as
28
Schistosoma spp occur in human populations living around the Sahara desert lakes of
29
Ounianga Kebir and Ounianga Serir in northern Chad. 30 Methods: The study comprised of three components. First, a cross sectional survey based on
31
a random sample drawn from the population to detect infections with S. haematobium and S. 32
mansoni; second, focus group discussions exploring disease priorities, access to health and
33
health seeking behaviour; and third, searching water contact sites for intermediate host snails. 34
Samples of trematode parasites and snails were confirmed on species level by molecular
35
genetics methods. 36 Methods: The study comprised of three components. First, a cross sectional survey based on
31
a random sample drawn from the population to detect infections with S. haematobium and S. 32
mansoni; second, focus group discussions exploring disease priorities, access to health and
33
health seeking behaviour; and third, searching water contact sites for intermediate host snails. 34
Samples of trematode parasites and snails were confirmed on species level by molecular
35
genetics methods. 36 Results: Among 258 participants, the overall S. haematobium prevalence using urine filtration
37
was 39.1% (95% CI 33.2% – 45.1%), with 51.5% of the infected suffering from heavy infection. 38
The intermediate host snail of S. haematobium (Bulinus truncatus) occurred at water sites near
39
both study villages, revealing the potential for local transmission. Although a positive S. Research Article Keywords: Bulinus truncatus. Chad, malacology, Ounianga, POC-CCA, prevalence, Sahara, Schistosoma
bovis, Schistosoma haematobium, schistosomiasis Posted Date: October 28th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-1016632/v1 DOI: https://doi.org/10.21203/rs.3.rs-1016632/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License: This work is licensed under a Creative Commons Attribution 4.0 International License. License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Infectious Diseases of Poverty on January
7th, 2022. See the published version at https://doi.org/10.1186/s40249-021-00930-4. High prevalence of urinary schistosomiasis in a desert population: results from an
1
exploratory study around the Ounianga lakes in Chad
2
3
Wendelin Moser1,2, Annour Adoum Batil3, Rebekka Ott1,2, Moussa Abderamane4, Ruth
4
Clements5, Rahel Wampfler1,2, Sven Poppert1,2, Peter Steinmann1,2, Fiona Allan5, Helena
5
Greter1,2*
6
7
1Swiss Tropical and Public Health Institute, Basel, Switzerland
8
2University of Basel, Basel, Switzerland
9
3Institut de Recherche en Elevage pour le Développement, Ndjamena, Chad
10
4University of Ndjamena, Geology Department, Ndjamena, Chad
11
5Department of Life Sciences, Natural History Museum, London, UK
12
13
*Corresponding author:
14
Helena Greter
15
Swiss Tropical and Public Health Institute
16
Socinstrasse 57, P.O. Box
17
CH-4002 Basel, Switzerland
18
Tel.: +41 61 284 87 30
19
E-mail: helena.greter@swisstph.ch
20
Orcid: 0000-0003-1844-3961
21
22 22 ABSTRACT
23 40
mansoni POC-CCA test result was obtained from 15.2% (10.6%-19.7%) of the samples no
41
intermediate host snails of S. mansoni were found, and the relevance of S. mansoni remains
42
uncertain. Qualitative findings underline the importance of morbidity caused by urinary
43
schistosomiasis, and the lack of access to diagnostics and treatment as a major health
44
concern. 45 Results: Among 258 participants, the overall S. haematobium prevalence using urine filtration
37
was 39.1% (95% CI 33.2% – 45.1%), with 51.5% of the infected suffering from heavy infection. 38 Results: Among 258 participants, the overall S. haematobium prevalence using urine filtration
37
was 39.1% (95% CI 33.2% – 45.1%), with 51.5% of the infected suffering from heavy infection. 38 Conclusion: This research revealed a high prevalence of urinary schistosomiasis in the
46
population living around the lakes of Ounianga in the Sahara, a UNESCO world heritage site
47
in Chad. Despite the high public health importance of the associated morbidity expressed by
48
the population there is no access to diagnostics and treatment. Further research is needed to
49
develop and test a context adapted intervention. 50 Conclusion: This research revealed a high prevalence of urinary schistosomiasis in the
46
population living around the lakes of Ounianga in the Sahara, a UNESCO world heritage site
47
in Chad. Despite the high public health importance of the associated morbidity expressed by
48
the population there is no access to diagnostics and treatment. Further research is needed to
49
develop and test a context adapted intervention. 50 2 2 51
Key words
52
Bulinus truncatus. Chad, malacology, Ounianga, POC-CCA, prevalence, Sahara,
53
Schistosoma bovis, Schistosoma haematobium, schistosomiasis
54
55 51
Key words
52
Bulinus truncatus. Chad, malacology, Ounianga, POC-CCA, prevalence, Sahara,
53
Schistosoma bovis, Schistosoma haematobium, schistosomiasis
54
55 51 Key words
52 55 3 3 BACKGROUND
56 Schistosome infections are listed among the 20 neglected tropical diseases (NTDs) targeted
57
by the World Health Organisation (WHO) for elimination by 2030 (1). In endemic regions, those
58
populations affected by schistosomiasis are often those living in poverty and / or in settings
59
with restricted access to clean water for their sanitation and hygiene needs (2). Worldwide, an
60
estimated 230 million people harbour an infection with Schistosoma spp (3). Occupational and
61
recreational activities in close contact with freshwater, e.g. fishing, doing laundry and bathing
62
present the main risk of infections. Highest prevalence is commonly observed among school-
63
aged children as they enjoy playing in stagnant water sites. Undetected and therewith
64
untreated urinary or intestinal schistosomiasis leads to chronic infections and serious
65
morbidities including a wide range of different pathologies as e.g. anaemia, stunted growth,
66
impaired cognition and organ damages, that negatively affect economic activities and therewith
67
maintain poverty (4, 5). The safe and effective drug, Praziquantel, is currently used for mass
68
drug administration programs in endemic settings as well as for treatment of individual acute
69
infections . However, its effectiveness is threatened by increasing resistance of the parasite
70
that is observed (6). 71 The majority of schistosomiasis cases occur in sub-Saharan Africa, and the disease is reported
72
from countries throughout the Sahel, including Mauretania, Mali, Niger, Chad and Sudan (7-
73
11). Infections are predominantly due to Schistosoma haematobium which has the ability to
74
maintain its life cycle in a semi-arid environment, including in the ecoregion of the Sahel (12). 75
Yet, there are old reports on schistosomiasis occurrence also more to the north, from within
76
the Sahara desert (7, 13). The occurrence of Schistosoma spp, a genus of water-transmitted
77
parasites belonging to the clades of digenean trematodes, and its occurrence in the hot and
78
hyper-arid desert may seem surprising but occurrence in at least two desert-specific
79
ecosystems have been described so far. These are (a) oases where schistosomiasis
80
transmission is linked to man-made irrigation systems (14, 15), and (b) areas with reclaimed
81
land for agriculture, made cultivable by artificial irrigation from deep wells (16). BACKGROUND
56 82 4 Anecdotal reports from nomadic Sahelian pastoralists on Fasciola spp, another digenean
83
trematode species, in livestock raised in the Chadian Sahara and recent reports about modern
84
and early Holocene finding of intermediate host snails pointed towards the occurrence and
85
potential ongoing transmission of schistosomiasis at the desert lakes of Ounianga, Chad (17). 86
Triggered by these information an exploratory study was conceptualized with the aim to
87
investigate whether trematode parasites such as Schistosoma spp occur in two settlements at
88
the lakes of Ounianga, Ennedi Ouest province, in Northern Chad. The study was covering
89
three aspects, namely epidemiology, malacology and the population’s health priorities, their
90
access to health care and treatment. 91
92
METHODS
93
Study site and study population
94
The study was carried out in January 2019 around the lakes and the two settlements of
95
Ounianga Kebir and Ounianga Serir, Ennedi Ouest province in Northern Chad (Figure 1). 96
97
<<Figure 1 near here>><<Fig 1. A map showing the lakes and the settlements of
98
Ounianga Kebir and Ounianga Serir in Northern Chad.>>
99
100
The official population estimates according to the latest national population census in Chad for
101
Ounianga Kebir counts around 9000 people and for Ounianga Serir about 1000 people
102
(RGPH2, 2009). In both communities, the primary schools were operational, yet not the
103
secondary schools. The only functional health centre of the Ounianga district is located in
104
Ounianga Kebir and its catchment population is estimated to include 30,000 people. Ounianga
105
Serir has no functional health centre; the population has set up a health post to provide basic
106
health services to the community members. 107
108
Epidemiological survey
109 Anecdotal reports from nomadic Sahelian pastoralists on Fasciola spp, another digenean
83
trematode species, in livestock raised in the Chadian Sahara and recent reports about modern
84
and early Holocene finding of intermediate host snails pointed towards the occurrence and
85
potential ongoing transmission of schistosomiasis at the desert lakes of Ounianga, Chad (17). 86
Triggered by these information an exploratory study was conceptualized with the aim to
87
investigate whether trematode parasites such as Schistosoma spp occur in two settlements at
88
the lakes of Ounianga, Ennedi Ouest province, in Northern Chad. BACKGROUND
56 The study was covering
89
three aspects, namely epidemiology, malacology and the population’s health priorities, their
90
access to health care and treatment. 91 Anecdotal reports from nomadic Sahelian pastoralists on Fasciola spp, another digenean
83
trematode species, in livestock raised in the Chadian Sahara and recent reports about modern
84
and early Holocene finding of intermediate host snails pointed towards the occurrence and
85
potential ongoing transmission of schistosomiasis at the desert lakes of Ounianga, Chad (17). 86
Triggered by these information an exploratory study was conceptualized with the aim to
87
investigate whether trematode parasites such as Schistosoma spp occur in two settlements at
88
the lakes of Ounianga, Ennedi Ouest province, in Northern Chad. The study was covering
89
three aspects, namely epidemiology, malacology and the population’s health priorities, their
90
access to health care and treatment. 91 <<Figure 1 near here>><<Fig 1. A map showing the lakes and the settlements of
98
Ounianga Kebir and Ounianga Serir in Northern Chad.>>
99 Parameters
111
used were “population survey” with two-sided confidence intervals of 95%, an expected
112
frequency of 50% and a population size of 10000, resulting in a sample size of 370. 113
Proportional to the total population estimates, the targeted sample size repartition was 330
114
people in Ounianga Kebir and 40 people in Ounianga Serir. At household level and at the
115
primary schools, individuals were randomly selected by applying the spatial sampling method
116
from the Expanded Programme of Immunization (EPI) of the World Health Organization as
117
previously published (18). After obtaining oral consent from each selected individual, or in case
118
of children from their caretakers, they were asked to produce a urine sample. A mobile field
119
laboratory was set up at the health centre, and health post, respectively. The urine samples
120
were analysed for haematuria by reagent strip testing (Hemastix; Siemens Healthcare
121
Diagnostics GmbH; Eschborn, Germany) and classified as negative, light and severe
122
haematuria as outlined by the testing handbook. Subsequently, 10 ml samples were subjected
123
to urine filtration, followed by microscopic screening of the filter content for the presence of S. 124
haematobium eggs. A point-of-care circulating cathodic antigen (POC-CCA) urine cassette test
125
(Rapid Medical Diagnostics; Pretoria, South Africa) was performed to screen for S. mansoni
126
infections. 127 <<Figure 1 near here>><<Fig 1. A map showing the lakes and the settlements of
98
Ounianga Kebir and Ounianga Serir in Northern Chad.>>
99 The official population estimates according to the latest national population census in Chad for
101
Ounianga Kebir counts around 9000 people and for Ounianga Serir about 1000 people
102
(RGPH2, 2009). In both communities, the primary schools were operational, yet not the
103
secondary schools. The only functional health centre of the Ounianga district is located in
104
Ounianga Kebir and its catchment population is estimated to include 30,000 people. Ounianga
105
Serir has no functional health centre; the population has set up a health post to provide basic
106
health services to the community members. 107 5 5 The resident population of Ounianga Kebir and Ounianga Serir, older than 5 years of age, were
110
eligible for participation. Sample size was calculated using Epi Info 7.1.3.3 (CDC). Parameters
111
used were “population survey” with two-sided confidence intervals of 95%, an expected
112
frequency of 50% and a population size of 10000, resulting in a sample size of 370. 113
Proportional to the total population estimates, the targeted sample size repartition was 330
114
people in Ounianga Kebir and 40 people in Ounianga Serir. At household level and at the
115
primary schools, individuals were randomly selected by applying the spatial sampling method
116
from the Expanded Programme of Immunization (EPI) of the World Health Organization as
117
previously published (18). After obtaining oral consent from each selected individual, or in case
118
of children from their caretakers, they were asked to produce a urine sample. A mobile field
119
laboratory was set up at the health centre, and health post, respectively. The urine samples
120
were analysed for haematuria by reagent strip testing (Hemastix; Siemens Healthcare
121
Diagnostics GmbH; Eschborn, Germany) and classified as negative, light and severe
122
haematuria as outlined by the testing handbook. Subsequently, 10 ml samples were subjected
123
to urine filtration, followed by microscopic screening of the filter content for the presence of S. 124
haematobium eggs. A point-of-care circulating cathodic antigen (POC-CCA) urine cassette test
125
(Rapid Medical Diagnostics; Pretoria, South Africa) was performed to screen for S. mansoni
126
infections. 127 The resident population of Ounianga Kebir and Ounianga Serir, older than 5 years of age, were
110
eligible for participation. Sample size was calculated using Epi Info 7.1.3.3 (CDC). Qualitative survey
129 In both communities, one focus group discussion (FGDs) with men and one with women were
130
organized. Additionally, one FGD was organized with the staff of the health centre in Ounianga
131
Kebir. In Ounianga Serir, an in-depth interview (IDI) was carried out with the person
132
responsible for the health post. The topics covered by the interview guides were disease
133
priorities and priority health issues, perceptions and health seeking behaviour. FGDs and IDI
134
were assisted by an interpreter who translated the conversation from Arabic to French, allowing
135
the study team to take notes. Digital recordings of the FGDs and IDI were transcribed and
136
translated into French, integrating the notes taken during the FGDs or IDI. 137 6 6 138 Malacological survey
139 Individual community members and school-aged children were asked to guide the team to
140
human-water contact sites. At each site, GPS coordinates and the water parameters
141
temperature (oC), pH, conductivity (µs/cm) and dissolved oxygen (mg/l) were recorded, using
142
a portable multimeter (Hach®, HQ40D, Loveland, USA)). For turbidity, a trubidimeter was used
143
(Formazin Nephelometric Units [FNU]; Hach®, 2100P Iso). The snail sampling was performed
144
adhering to standard protocols. In short, for 15 minutes, all aquatic snails were collected by
145
one person using a scoop or forceps to detach them from aquatic and subaquatic plants (19). 146
Subsequently, the snails were placed on wet cotton in petri dishes, and transferred to the field
147
laboratory. Snails were identified to the genus or, if possible to species level on site. At midday,
148
each collected snail identified as intermediate host species was placed in water for three hours
149
to induce cercarial shedding. The snail size (in mm) and weight (in mg) was measured using
150
a calibre and balance, respectively. Thereafter, all snail specimens were conserved in 70%
151
ethanol, and shipped to the National History Museum, London (NHM) for molecular analysis. 152
153 Molecular snail species and infections status confirmation
154 The snail samples selected for the molecular analyses represented individuals from each
155
collection site. All specimens were stored in 70% ethanol in the field. On arrival at the NHM,
156
the snail species identification was confirmed based on morphological characters and samples
157
re-spirited (absolute ethanol) for incorporation into the Schistosomiasis Collection at the
158
Natural History Museum (SCAN) (20). Photographic images were taken of the snail shells prior
159
to DNA extraction. Specimens were placed in TE buffer (10mM Tris, 0.1mM EDTA) pH 7.4 for
160
one hour in order to remove any remaining alcohol from within the tissue, which might interfere
161
with subsequent extraction steps. Total genomic DNA was isolated from head/foot tissue using
162
the DNeasy Blood and Tissue kit (Qiagen, UK) according to manufacturer’s instructions. DNA
163
was eluted into 200μl sterile water. 164 165 7 7 Checking of sequence data
171 The electropherograms produced were checked and Cox1 sequences edited using Geneious,
172
version 11.0.5 (http://www.geneious.com (24)). Sequences were compared to database
173
entries by performing BLAST searches via the National Center for Biotechnology Information
174
against GenBank and EMBL sequence databases; and aligned with reference material [3,4]
175
using Geneious version 11.0.5. 176 Amplification of Cox1 fragments of snail DNA
166 A polymerase chain reaction (PCR) amplification of a partial cytochrome oxidase 1 (Cox1)
167
sequence was performed using primers LCO1490 (5′GGTCAACAAATCATAAAG ATATTGG3′
168
forward) and HCO2198 (5′TAAACTTCAGGGTGACCAAAAAATCA3′ reverse) (21). PCR
169
investigations and sequencing conditions were chosen as previously outlined (22, 23). 170 sequence was performed using primers LCO1490 (5′GGTCAACAAATCATAAAG ATATTGG3′
168
forward) and HCO2198 (5′TAAACTTCAGGGTGACCAAAAAATCA3′ reverse) (21). PCR
169
investigations and sequencing conditions were chosen as previously outlined (22, 23). 170
Checking of sequence data
171
The electropherograms produced were checked and Cox1 sequences edited using Geneious,
172
version 11.0.5 (http://www.geneious.com (24)). Sequences were compared to database
173
entries by performing BLAST searches via the National Center for Biotechnology Information
174
against GenBank and EMBL sequence databases; and aligned with reference material [3,4]
175
using Geneious version 11.0.5. 176
177
Sequencing of Schistosoma spp. eggs in urine
178
Positive urine samples from Ouinanga Kebir were combined into 7 different pools of 8-12ml
179
respectively one pooled sample of 12ml for the villages of Ouinanga Serir. Samples were
180
shipped to the diagnostic center of the Swiss Tropical and Public Health Institute (Swiss TPH)
181
in Basel, Switzerland for further processing. There, each pool was centrifuged at 3000g for 10
182
minutes. Exactly 500µl of the pellet was re-suspended and transferred to a 2ml tube containing
183
garnet beads. After addition of 1 ml PBS, the sample was centrifuged 1min at 13000g and the
184
supernatant was discarded. The pellet with the garnet beads was frozen 30min at -80°C and
185
further processed as described by Barda and colleagues (25, 26). Samples were first tested
186
by simplex generic Schistosoma spp. 28S real-time PCR amplifying S. mansoni, S. 187
haematobium, S. intercalatum, S. bovis (27) and additionally S. japonicum because of
188
modifications added to the second reverse primer of the assay (table 1). The reaction mix
189
contained 1x TaqMan GenExpression MasterMix (ThermoFisher Scientific, Basel,
190
Switzerland), 800nmoles of forward primer, 400nmoles of each reverse primer and 200nmoles
191
of probe. The samples were subsequently tested by a duplex real-time PCR for the presence
192
of a specific S. mansoni TRE region and of S. haematobium dra1 sequence (27, 28). Each
193 Sequencing of Schistosoma spp. eggs in urine
178 Analytical limit of detection (LOD)
204
was tested by a plasmid dilution row ranging from 107 to 10-1 plasmids/µl containing an insert
205
with the sequence of the Schistosoma real-time PCR product, and was found to be at 10
206
plasmids/µl for all assays. On each real-time PCR plate and for each target we included
207
negative and positive low-copy plasmid controls. 208 reaction mix contained 1x TaqMan GenExpression MasterMix (ThermoFisher Scientific, Basel,
194
Switzerland), 800nmoles of each primer, 200nmoles each probe (table1). The thermoprofile
195
of all assays on the QuantStudio5 (ThermoFisher) consisted of 2min at 50°C, 10min at 95°C
196
followed by 45 cycles of 15s at 95°C and 1min at 58°C. The specificity of all assays was
197
previously tested on a variety of DNA from stool and blood samples including: Ascaris
198
lumbricoides,
Blastocystis
hominis,
Cryptosporidium
spp.,
Dientamoeba
fragilis,
199
Encephalitozoon spp., Endolimax nana, Entamoeba coli, E. dispar, E. histolytica, E. 200
moshkovskii, E. polecki, Enterocytozoon bieneusi, Giardia lamblia, Hymenolepis nana,
201
Iodamoeba bütschlii, Sarcocystis spp., Taenia spp., Strongyloides stercoralis, Trichuris
202
trichiura, Plasmodium falciparum, P. vivax, P. malariae, P. ovale, Trypanosoma cruzi, T. 203
brucei, Leishmania spp. and was found to be 100% specific. Analytical limit of detection (LOD)
204
was tested by a plasmid dilution row ranging from 107 to 10-1 plasmids/µl containing an insert
205
with the sequence of the Schistosoma real-time PCR product, and was found to be at 10
206
plasmids/µl for all assays. On each real-time PCR plate and for each target we included
207
negative and positive low-copy plasmid controls. 208 Subsequently, all samples were tested by classic PCR of the COX gene of S. haematobium
209
and S. bovis as modified from Boon and co-workers (Table 1) (29). The reaction mix contained
210
1x HotStarTaq Plus Master Mix (Qiagen, Hilden, Germany), 800nmoles of each Primer, 5µl
211
DNA in a total reaction volume of 50µl. The thermoprofile consisted of 5min at 94°C followed
212
by 40 cycles of 40s at 94°C, 40s at 58°C and 1min at 72°C and a final step of 10min at 72°C. 213
After visualization on a 2% Agarose-Gel, the positive sample of the S. bovis-COX PCR was
214
sent for Sanger sequencing with the primers of amplification at Microsynth AG (Baldach,
215
Switzerland). The Sequence was then compared to database entries by performing BLAST
216
searches via the National Center for Biotechnology Information. Sequencing of Schistosoma spp. eggs in urine
178 Positive urine samples from Ouinanga Kebir were combined into 7 different pools of 8-12ml
179
respectively one pooled sample of 12ml for the villages of Ouinanga Serir. Samples were
180
shipped to the diagnostic center of the Swiss Tropical and Public Health Institute (Swiss TPH)
181
in Basel, Switzerland for further processing. There, each pool was centrifuged at 3000g for 10
182
minutes. Exactly 500µl of the pellet was re-suspended and transferred to a 2ml tube containing
183
garnet beads. After addition of 1 ml PBS, the sample was centrifuged 1min at 13000g and the
184
supernatant was discarded. The pellet with the garnet beads was frozen 30min at -80°C and
185
further processed as described by Barda and colleagues (25, 26). Samples were first tested
186
by simplex generic Schistosoma spp. 28S real-time PCR amplifying S. mansoni, S. 187
haematobium, S. intercalatum, S. bovis (27) and additionally S. japonicum because of
188
modifications added to the second reverse primer of the assay (table 1). The reaction mix
189
contained 1x TaqMan GenExpression MasterMix (ThermoFisher Scientific, Basel,
190
Switzerland), 800nmoles of forward primer, 400nmoles of each reverse primer and 200nmoles
191
of probe. The samples were subsequently tested by a duplex real-time PCR for the presence
192
of a specific S. mansoni TRE region and of S. haematobium dra1 sequence (27, 28). Each
193 8 8 reaction mix contained 1x TaqMan GenExpression MasterMix (ThermoFisher Scientific, Basel,
194
Switzerland), 800nmoles of each primer, 200nmoles each probe (table1). The thermoprofile
195
of all assays on the QuantStudio5 (ThermoFisher) consisted of 2min at 50°C, 10min at 95°C
196
followed by 45 cycles of 15s at 95°C and 1min at 58°C. The specificity of all assays was
197
previously tested on a variety of DNA from stool and blood samples including: Ascaris
198
lumbricoides,
Blastocystis
hominis,
Cryptosporidium
spp.,
Dientamoeba
fragilis,
199
Encephalitozoon spp., Endolimax nana, Entamoeba coli, E. dispar, E. histolytica, E. 200
moshkovskii, E. polecki, Enterocytozoon bieneusi, Giardia lamblia, Hymenolepis nana,
201
Iodamoeba bütschlii, Sarcocystis spp., Taenia spp., Strongyloides stercoralis, Trichuris
202
trichiura, Plasmodium falciparum, P. vivax, P. malariae, P. ovale, Trypanosoma cruzi, T. 203
brucei, Leishmania spp. and was found to be 100% specific. Sequencing of Schistosoma spp. eggs in urine
178 haematobium egg positive in Ounianga
230
Kebir and 54.9% (95% CI 40.8% – 69.0%) in Ounianga Serir, resulting in an overall prevalence
231
of 39.2% (95% CI 33.2% – 45.1%) (Table 1). 232
233
Tab 1. Prevalence of S. haematobium and S. mansoni infection, haematuria and infection
234
intensities in the study population, Ounianga Serir and Ounianga Kebir, Chad, 2019. 235
Ounianga Kebir
Ounianga Serir
Total
Male
Female
Male
Female
Total number of participants
65
142
21
30
2
Participants age <18 years
47 (72%)
65 (46%)
14 (67%)
11 (37%)
137 (53
S. haematobium infection (egg positive)
Total no. positive
27 (41.5%)
46 (32.4%)
11 (52.4%)
17 (56.7%)
1
(39.2
Age <18 years*
24 (51.1%)
24 (36.9%)
9 (64.3%)
8 (72.7%)
65 (47.5
Heavy S. haematobium infection
Total no. positive
14 (51.9%)
21 (45.7%)
8 (72.7%)
9 (52.9%)
52 (51.5
Age <18 years*
14 (58.3%)
14 (58.3%)
8 (88.9%)
7 (87.5%)
43 (66.2
POC-CCA test results
Total no. positive
14 (21.9%)
17 (13.0%)
1 (4.8%)
5 (17.9%)
37 (15.2
Age <18 years*
14 (30.4%)
10 (16.7%)
1 (7.1%)
3 (27.3%)
28 (21.4
*(%) from all participants <18 year
236
237 Descriptive statistics of epidemiological and malacological data was performed using STATA
222
version 16.0 (STATA Corp Inc., TX, USA) and ArcGIS (Version 10.7.1.; ESRI Inc. ArcMap™
223
10.7, Redlands, CA, USA). Qualitative data analysis included full review of all transcripts,
224
followed by a descriptive and explorative thematic analysis. 225 Sequencing of Schistosoma spp. eggs in urine
178 The sequence is accessible
217
in GenBank under the number: MW937895. A table listing primers and probes is accessible in
218
the supplementary materials. 219 9 9 Descriptive statistics of epidemiological and malacological data was performed using STATA
222
version 16.0 (STATA Corp Inc., TX, USA) and ArcGIS (Version 10.7.1.; ESRI Inc. ArcMap™
223
10.7, Redlands, CA, USA). Qualitative data analysis included full review of all transcripts,
224
followed by a descriptive and explorative thematic analysis. 225
226
RESULTS
227
Epidemiological survey
228
In both study sites, urinary schistosomiasis was highly prevalent. Indeed, 35.3% (95% CI
229
28.7% – 41.8%) of the tested participants were S. haematobium egg positive in Ounianga
230
Kebir and 54.9% (95% CI 40.8% – 69.0%) in Ounianga Serir, resulting in an overall prevalence
231
of 39.2% (95% CI 33.2% – 45.1%) (Table 1). 232
233
Tab 1. Prevalence of S. haematobium and S. mansoni infection, haematuria and infection
234
intensities in the study population, Ounianga Serir and Ounianga Kebir, Chad, 2019. 235
Ounianga Kebir
Ounianga Serir
Total
Male
Female
Male
Female
Total number of participants
65
142
21
30
25
Participants age <18 years
47 (72%)
65 (46%)
14 (67%)
11 (37%)
137 (53%
S. haematobium infection (egg positive)
Total no. positive
27 (41.5%)
46 (32.4%)
11 (52.4%)
17 (56.7%)
10
(39.2%
Age <18 years*
24 (51.1%)
24 (36.9%)
9 (64.3%)
8 (72.7%)
65 (47.5%
Heavy S. haematobium infection
Total no. positive
14 (51.9%)
21 (45.7%)
8 (72.7%)
9 (52.9%)
52 (51.5%
Age <18 years*
14 (58.3%)
14 (58.3%)
8 (88.9%)
7 (87.5%)
43 (66.2%
POC-CCA test results
Total no. positive
14 (21.9%)
17 (13.0%)
1 (4.8%)
5 (17.9%)
37 (15.2%
Age <18 years*
14 (30.4%)
10 (16.7%)
1 (7.1%)
3 (27.3%)
28 (21.4%
*(%) from all participants <18 year
236
237 Descriptive statistics of epidemiological and malacological data was performed using STATA
222
version 16.0 (STATA Corp Inc., TX, USA) and ArcGIS (Version 10.7.1.; ESRI Inc. ArcMap™
223
10.7, Redlands, CA, USA). Qualitative data analysis included full review of all transcripts,
224
followed by a descriptive and explorative thematic analysis. 225
226
RESULTS
227
Epidemiological survey
228
In both study sites, urinary schistosomiasis was highly prevalent. Indeed, 35.3% (95% CI
229
28.7% – 41.8%) of the tested participants were S. Epidemiological survey
228 For each water
243
site sampled, the abundance of the intermediate host snail Bulinus truncatus is indicated.>>
244
245
Mapping of the schistosomiasis prevalence by place of living (neighbourhood) shows a slightly
246
higher prevalence for those neighbourhoods closer to a water sites where the aquatic
247
intermediate host snail Bulinus truncates was present (Fig 2, Ounianga Kebir: Yiggybeshi,
248
Ounianga Serir: Roy). Regarding the POC-CCA testing for S. mansoni, 15.2% of the urine
249
samples showed a positive test results. 250
More than half of all participants harboured a heavy S. haematobium infection (51.5%; heavy
251
infection: >50 eggs/10 ml urine) and the burden was higher in children, whereof two third were
252
heavily infected (Table 1) (30). Overall, 10.2% of all egg-negative, 69.4% of all light and 92.3%
253
of all heavily infected participants had severe haematuria (Fig 3), with no big differences
254
between age and gender. 255
256
<<Figure 3 near here>>
257
<<Fig 3. Schistosomiasis infection intensity and haematuria stratified by sex, age
258
group and place of living.>>
259
260
Qualitative survey
261
During FGDs in both study sites, abdominal issues and blood in urine were the most frequently
262
mentioned health problems among adults and also in children. Health staff mentioned that the
263
majority of patients seeking care at the centre for any cause additionally suffers from abdominal
264
issues. Among children, diarrhoeal diseases, respiratory infections and scorpion stings were
265 <<Figure 2 near here>>
241
<<Fig 2. Map showing the prevalence and snail abundance for Ounianga Kebir and
242
Serir. The prevalence among participants is displayed by neighbourhood. For each water
243
site sampled, the abundance of the intermediate host snail Bulinus truncatus is indicated.>>
244
245
Mapping of the schistosomiasis prevalence by place of living (neighbourhood) shows a slightly
246
higher prevalence for those neighbourhoods closer to a water sites where the aquatic
247
intermediate host snail Bulinus truncates was present (Fig 2, Ounianga Kebir: Yiggybeshi,
248
Ounianga Serir: Roy). Regarding the POC-CCA testing for S. mansoni, 15.2% of the urine
249
samples showed a positive test results. 250 Mapping of the schistosomiasis prevalence by place of living (neighbourhood) shows a slightly
246
higher prevalence for those neighbourhoods closer to a water sites where the aquatic
247
intermediate host snail Bulinus truncates was present (Fig 2, Ounianga Kebir: Yiggybeshi,
248
Ounianga Serir: Roy). Regarding the POC-CCA testing for S. Epidemiological survey
228 In both study sites, urinary schistosomiasis was highly prevalent. Indeed, 35.3% (95% CI
229
28.7% – 41.8%) of the tested participants were S. haematobium egg positive in Ounianga
230
Kebir and 54.9% (95% CI 40.8% – 69.0%) in Ounianga Serir, resulting in an overall prevalence
231
of 39.2% (95% CI 33.2% – 45.1%) (Table 1). 232 Tab 1. Prevalence of S. haematobium and S. mansoni infection, haematuria and infection
34 Tab 1. Prevalence of S. haematobium and S. mansoni infection, haematuria and infection
234
intensities in the study population, Ounianga Serir and Ounianga Kebir, Chad, 2019. 235
Ounianga Kebir
Ounianga Serir
Total
Male
Female
Male
Female
Total number of participants
65
142
21
30
258
Participants age <18 years
47 (72%)
65 (46%)
14 (67%)
11 (37%)
137 (53%)
S. haematobium infection (egg positive)
Total no. positive
27 (41.5%)
46 (32.4%)
11 (52.4%)
17 (56.7%)
101
(39.2%)
Age <18 years*
24 (51.1%)
24 (36.9%)
9 (64.3%)
8 (72.7%)
65 (47.5%)
Heavy S. haematobium infection
Total no. positive
14 (51.9%)
21 (45.7%)
8 (72.7%)
9 (52.9%)
52 (51.5%)
Age <18 years*
14 (58.3%)
14 (58.3%)
8 (88.9%)
7 (87.5%)
43 (66.2%)
POC-CCA test results
Total no. positive
14 (21.9%)
17 (13.0%)
1 (4.8%)
5 (17.9%)
37 (15.2%)
Age <18 years*
14 (30.4%)
10 (16.7%)
1 (7.1%)
3 (27.3%)
28 (21.4%)
*(%) from all participants <18 year
236 ab 1. Prevalence of S. haematobium and S. mansoni infection, haematuria and infection Tab 1. Prevalence of S. haematobium and S. mansoni infection, haematuria a
234 The S. haematobium prevalence was highest among children and adolescents below 18 years
238
in both villages (Table 1). In Ounianga Kebir, more boys than girls were infected (51.1% versus
239
36.9%), whereas in Ounianga Seker girls had a higher prevalence (72.7%). 240 The S. haematobium prevalence was highest among children and adolescents below 18 years
238
in both villages (Table 1). In Ounianga Kebir, more boys than girls were infected (51.1% versus
239
36.9%), whereas in Ounianga Seker girls had a higher prevalence (72.7%). 240 10 <<Figure 2 near here>>
241
<<Fig 2. Map showing the prevalence and snail abundance for Ounianga Kebir and
242
Serir. The prevalence among participants is displayed by neighbourhood. Epidemiological survey
228 mansoni, 15.2% of the urine
249
samples showed a positive test results. 250 More than half of all participants harboured a heavy S. haematobium infection (51.5%; heavy
251
infection: >50 eggs/10 ml urine) and the burden was higher in children, whereof two third were
252
heavily infected (Table 1) (30). Overall, 10.2% of all egg-negative, 69.4% of all light and 92.3%
253
of all heavily infected participants had severe haematuria (Fig 3), with no big differences
254
between age and gender. 255 11 A major constraint for people in both sites is the difficult access to health facilities. Accessible
269
facilities are usually underequipped; rarely have drugs available and the personnel had only
270
basic training. To obtain appropriate care and treatment, people needed to travel long
271
distances within Chad (to Faya, Abeche and N’Djamena) or abroad to Libya or Sudan. 272 Another common theme was the lack of safe drinking water as pumps are rare and open wells
273
are commonly used as water sources. Perceived water quality is low due to salty taste and
274
visible contamination. 275 The population was well aware of parasitic diseases, yet had limited knowledge on risk factors
276
and transmission. Blood in urine was linked to parasitic infections, low quality of drinking water,
277
water contact at the nearby lakes, or kidney issues. Kadi and Kouli are two local names for
278
parasite infections linked to abdominal pain. Kadi describes an intestinal worm infection
279
causing symptoms like intestinal spasms and flatulence, increased appetite with the tendency
280
of weight loss As a traditional treatment, infected people are given natron or an extract of the
281
roots of a plant called Boa to initiate diarrhoea, causing a worm with a red ‘mouth’ to leave the
282
body via the excrements. The symptoms described for Kouli correlate with symptoms of the
283
parasite Enterobius vermicularis such as persistent itching in the perianal area and sleep
284
disturbances. The traditional treatment administered to Kouli patients are eating butter or
285
drinking an extract of a medical plant called Chi. 286 Ouco is the local term to describe the health condition related to blood in urine combined with
287
pain while urinating and reduced male erectile function. In traditional medicine, the urine of the
288
animal called Nii (Fennec Fox, Vulpes zerda) is believed to have a curative effect. Epidemiological survey
228 289 The reported level of satisfaction with access to medical treatment for the above
290
mentioned health issues is mixed. Important challenges mentioned included stock outs of
291
medicines, lack of diagnostic means and non-effectiveness of the medical treatment received. 292
Especially the female FGD participants expressed a need for health education and
293
sensitization among the population. 294 Malacological survey
296 12 Among a total pf 17 different collection sites, 8 harboured fresh water snails (Tab 2, Fig 2). 297
Highest snail numbers were collected from the two intermediate host snail species Limnea
298
natalensis (n=42) and Bulinus truncatus (n=38), and two species of no medical importance,
299
Gyralus sp. and Polypylis sp. (n=42). Particularly high numbers of any snail species were
300
collected at two sites, namely Yoa 2 (n=35) in Ounianga Kebir and Agouta (n=34) in Ounianga
301
Kebir. Among all snails, only one B. truncatus was shedding cercariae (from Yoa 2). Upon
302
testing, they were recognized to not represent S. haematobium cercariae, and consequently
303
were not further studied. The average shell height of all L. natalensis specimen was 10.6mm
304
(95% confidence interval, 9.08mm - 12.11mm), 6.66 mm (95% CI, 5.97mm - 7.34mm) for B. 305
truncates, whereas all Gyralus sp. were juveniles with an average shell height of 1 mm or
306
below. 307 Among a total pf 17 different collection sites, 8 harboured fresh water snails (Tab 2, Fig 2). 297
Highest snail numbers were collected from the two intermediate host snail species Limnea
298
natalensis (n=42) and Bulinus truncatus (n=38), and two species of no medical importance,
299
Gyralus sp. and Polypylis sp. (n=42). Particularly high numbers of any snail species were
300
collected at two sites, namely Yoa 2 (n=35) in Ounianga Kebir and Agouta (n=34) in Ounianga
301
Kebir. Among all snails, only one B. truncatus was shedding cercariae (from Yoa 2). Upon
302
testing, they were recognized to not represent S. haematobium cercariae, and consequently
303
were not further studied. The average shell height of all L. natalensis specimen was 10.6mm
304
(95% confidence interval, 9.08mm - 12.11mm), 6.66 mm (95% CI, 5.97mm - 7.34mm) for B. 305
truncates, whereas all Gyralus sp. were juveniles with an average shell height of 1 mm or
306
below. 307 Across all sites where snails were found, the average water temperatures was 18.9oC
308
(standard deviation ±3.3), the average oxygen content was 4.8 mg/l (± 2.1) and a turbidity of
309
3.0 FNU (± 1.4). The sites without snails were characterized by a wide range of measured
310
water parameters, i.e. temperature of 22.1oC (range: 14.1-28.2), oxygen 5.9 mg/l (range: 1.1-
311
15.0) and turbidity 267.0 FNU (range: 1.0 - >1000.0). Malacological survey
296 Snails obviously preferred the pH range
312
between 7.0 and 8.8 compared the sites without snails with a pH varying between 7.0 and
313
10.5. Inconclusive results were found for the conductivity comparing sites with and without
314
snails with a range of 6.0 to 1941.0 µs/cm and 2.8 to >2500.0 µs/cm, respectively. 315 Table 3. Snail abundance and water parameters for each sampling site
17
Sampling sites with snails present
Site*
Snail species
No. of
snails
found
Temperature
[oC]
Conductivity
[µs/cm]
pH
Oxygen
[mg/l]
Turbidity
[FNU]
Ounianga Kebir
Yoa (Girki)
1*
Gyralus sp. 1
17.5
1054.0
6.8
2.7
1.3
2*
B. truncatus
28
14.5
1046.0
6.9
4.1
2.5
L. natalensis
6
Yoa (source 2)
3*
L. natalensis
19
22.7
1941.0
7.1
3.2
1.9
4*
L. natalensis
7
22.8
2.56
7.0
3.0
3.7
Ounianga Serir
Agouta
Gyralus sp. 27
14.5
8.0
8.3
4.9
3.5
L. natalensis
7
V. nilotica
√ Table 3. Snail abundance and water parameters for each sampling site 13 Djara
B. truncatus
4
19.7
6.0
8.5
6.6
2.4
G. ounaiangaensis
√
V. nilotica
√
Boku
B. truncatus
6
20.8
1524.0
8.8
8.8
5.9
L. natalensis
3
G. ounaiangaensis
√
Bedrim
G. ounaiangaensis
√
18.7
7.3
8.4
5.2
3.0
Sampling sites without snails
Ounianga Kebir
Yoa 5 (hot source)
28.4
2.8
7.0
2.9
4.9
Yoa 6 (hot source)
27.7
2107.0
7.4
3.2
16.3
Yoa 7 (lake)
14.4
290.0
10.3
1.1
158.0
Uma red
27.5
>2000.0
10.1
14.2
85.8
Uma blue
18.6
>2000.0
10.5
15.0
118.0
Uma (hot spring)
28.2
4.0
9.1
3.6
14.9
Forodone
19.1
31.1
9.8
2.0
>1000.0
Ounianga Serir
Telli
21.3
13.7
10.7
2.7
4.5
Edem
14.1
3.5
8.7
8.3
1.0
*All sampling sites are shown in Figure 2
318
319
Sequencing of Schistosoma spp. eggs in urine
320
All eight urine pools were positive in the generic Schistosoma spp. 28S real-time assay, in the
321
S. haematobium dra1 real-time assay and in the S. haematobium COX1 PCR consistent with
322
the presence of S. haematobium eggs in all pools. No pool was positive for S. mansoni TRE
323
real-time PCR. One pool from the village of Ouinanga Serir was positive for S. bovis COX1. 324
This result indicates the possibility of the presence of S. haematobium X bovis hybrids as
325
observed in previous studies in West Africa (29, 31). 326
327
DISCUSSION
328 Djara
B. truncatus
4
19.7
6.0
8.5
6.6
2.4
G. Malacological survey
296 ounaiangaensis
√
V. nilotica
√
Boku
B. truncatus
6
20.8
1524.0
8.8
8.8
5.9
L. natalensis
3
G. ounaiangaensis
√
Bedrim
G. ounaiangaensis
√
18.7
7.3
8.4
5.2
3.0
Sampling sites without snails
Ounianga Kebir
Yoa 5 (hot source)
28.4
2.8
7.0
2.9
4.9
Yoa 6 (hot source)
27.7
2107.0
7.4
3.2
16.3
Yoa 7 (lake)
14.4
290.0
10.3
1.1
158.0
Uma red
27.5
>2000.0
10.1
14.2
85.8
Uma blue
18.6
>2000.0
10.5
15.0
118.0
Uma (hot spring)
28.2
4.0
9.1
3.6
14.9
Forodone
19.1
31.1
9.8
2.0
>1000.0
Ounianga Serir
Telli
21.3
13.7
10.7
2.7
4.5
Edem
14.1
3.5
8.7
8.3
1.0
*All sampling sites are shown in Figure 2 Sequencing of Schistosoma spp. eggs in urine
320
All eight urine pools were positive in the generic Schistosoma spp. 28S real-time assay, in the
321
S. haematobium dra1 real-time assay and in the S. haematobium COX1 PCR consistent with
322
the presence of S. haematobium eggs in all pools. No pool was positive for S. mansoni TRE
323
real-time PCR. One pool from the village of Ouinanga Serir was positive for S. bovis COX1. 324
This result indicates the possibility of the presence of S. haematobium X bovis hybrids as
325
observed in previous studies in West Africa (29, 31). 326 DISCUSSION
328 In Ounianga Serir, there are no hot springs and the two freshwater lakes (Djara
347
and Boku) are used for all water-related activities. In both, B. truncatus were present and the
348
lakes’ close proximity to the quarter Roy may explain the high urinary schistosomiasis
349
prevalence. 350 The larger of the two villages, Ounianga Kebir had an overall lower schistosomiasis prevalence
335
compared to the smaller village of Ounianga Serir (35% versus 55%). In the different
336
neighbourhoods of Ounianga Kebir the prevalence varied and ranged from 21% to 42% (Fig
337
2). This may be partly explained by the proximity to the rare freshwater sites that are used for
338
washing cloth, bathing and swimming. For example, Lake Yoa with its cold temperature and
339
high salinity is fed by numerous freshwater springs. These provide habitats for the intermediate
340
host snails, and the neighbourhood with the highest prevalence was the one closest to a
341
freshwater spring. The quarters with lower prevalence were closer to freshwater sources
342
including the two hot springs (Yoa 5 and 6). Here, the high water temperature might explain
343
the absence of snails (32). The two adjacent sampling sites Yoa 3 and 4 are cold and only
344
used for irrigating the surrounding gardens or for watering livestock. Interestingly, at these two
345
sites snails of the species Limnea natalesis were found, the intermediate host of the liver fluke
346
Fasciola spp. In Ounianga Serir, there are no hot springs and the two freshwater lakes (Djara
347
and Boku) are used for all water-related activities. In both, B. truncatus were present and the
348
lakes’ close proximity to the quarter Roy may explain the high urinary schistosomiasis
349
prevalence. 350 About half of the adult participants and two thirds of the children with a positive test suffered
351
from heavy S. haematobium infections. Our data show that the infection intensity is associated
352
with the severity of haematuria, pointing towards chronic schistosomiasis caused by long-term
353
exposure and recurrent reinfection. Hence, the major health problems reported by the local
354
population, namely abdominal issues and blood in urine, may well be due to schistosomiasis,
355
and are likely the consequences of the lacking access to diagnostics and treatment options
356
and the absence of any preventive intervention, as it has also been reported from other remote
357
areas in Chad (33). DISCUSSION
328 This exploratory study was the very first time a medical research team focused on the Sahara
329
oasis of Ounianga, Ennedi Ouest province in Chad. We were able to show for the first time the
330
high prevalence of S. haematobium in the population of both villages Ounianga Kebir and Serir. 331
Living specimens of B. truncatus were found at both sites, whereas the previous findings were
332
fossils dating back to the early Holocene (17). These findings suggest the possibility of ongoing
333
local schistosomiasis transmission in this desert oasis environment. 334 This exploratory study was the very first time a medical research team focused on the Sahara
329
oasis of Ounianga, Ennedi Ouest province in Chad. We were able to show for the first time the
330
high prevalence of S. haematobium in the population of both villages Ounianga Kebir and Serir. 331
Living specimens of B. truncatus were found at both sites, whereas the previous findings were
332
fossils dating back to the early Holocene (17). These findings suggest the possibility of ongoing
333
local schistosomiasis transmission in this desert oasis environment. 334 14 The larger of the two villages, Ounianga Kebir had an overall lower schistosomiasis prevalence
335
compared to the smaller village of Ounianga Serir (35% versus 55%). In the different
336
neighbourhoods of Ounianga Kebir the prevalence varied and ranged from 21% to 42% (Fig
337
2). This may be partly explained by the proximity to the rare freshwater sites that are used for
338
washing cloth, bathing and swimming. For example, Lake Yoa with its cold temperature and
339
high salinity is fed by numerous freshwater springs. These provide habitats for the intermediate
340
host snails, and the neighbourhood with the highest prevalence was the one closest to a
341
freshwater spring. The quarters with lower prevalence were closer to freshwater sources
342
including the two hot springs (Yoa 5 and 6). Here, the high water temperature might explain
343
the absence of snails (32). The two adjacent sampling sites Yoa 3 and 4 are cold and only
344
used for irrigating the surrounding gardens or for watering livestock. Interestingly, at these two
345
sites snails of the species Limnea natalesis were found, the intermediate host of the liver fluke
346
Fasciola spp. DISCUSSION
328 358 The study was set-up as an exploratory study with the aim to reveal the presence of the
359
Schistosoma spp. lifecycle in the desert. Its scope is therefore limited and leaves several
360
factors unaddressed at this stage. For example, the men’s main activities involve working in
361
soda extraction sites, trading using traditional caravans, and raising livestock through mobile
362 15 pastoralism. Hence, during the visit of the study team, the majority of men aged 16 to 60 years
363
were absent resulting in an over-representation of women in the study population (ratio 1:2). 364
Regarding the S. mansoni diagnostics that showed a positive POC-CCA result for 15.2% of all
365
urine samples, we cannot conclude with certainty that S. mansoni is present in the study
366
population as no stool samples were collected and hence, no parasitological proof of S. 367
mansoni infection is available. Of note, according to the tests handbook, also a heavy infection
368
with S. haematobium can lead to a positive test result (34). It is also significant that no
369
intermediate host snails of the genus Biomphalaria were found. However, the exploratory study
370
was conducted in January while snail abundance is highly seasonal (35). 371 Conclusion
373 This exploratory study presents the first modern evidence of urinary schistosomiasis among
374
the population of these oasis villages. There is clearly a need for further studies to fully
375
understand the current epidemiological situation. However, apart from further studies the main
376
problems are already evident; namely the lack of health education, diagnostics and access to
377
treatment. With a combined approach, including sensitization, mass drug administration, and
378
morbidity management the control or even elimination of urinary schistosomiasis in this
379
population might be possible. 380 Ethics approval and consent to participate
383 The study received approval from the ethics committee Northwest and Central Switzerland
384
(reference no. BASEC Nr Req-2018-0120) and the ‘Comité National de Bioéthique du Tchad’
385
(CNBT) in N’Djamena, Chad (reference no. 134/PR/MESRI/SG/CNBT/2018). Research
386
authorization was granted by the Chadian Ministry of Health and its ‘Direction de la Lutte contre
387
la Maladie et de la Promotion de la Santé’ (reference no. 007/PR/MSP/DG/DLMPS/2018). 388 16 Upon arrival in the study villages, an assembly was organized with the community
389
representatives to discuss the study objectives and procedures. The traditional leaders,
390
together with the local authorities, discussed the study and decided about concrete
391
participation. Once a collective decision had been reached, written informed consent was
392
obtained from the community representatives. In line with high illiteracy rates among the
393
general population, individual participants consented orally. These consent procedures had
394
received approval by the respective ethics committees. Those participants with a positive test
395
result from either filtration or POC-CCA testing were invited to the health centre / health post
396
and were administered praziquantel in the adequate dose (40 mg/kg) by the study nurse. 397 Upon arrival in the study villages, an assembly was organized with the community
389
representatives to discuss the study objectives and procedures. The traditional leaders,
390
together with the local authorities, discussed the study and decided about concrete
391
participation. Once a collective decision had been reached, written informed consent was
392
obtained from the community representatives. In line with high illiteracy rates among the
393
general population, individual participants consented orally. These consent procedures had
394
received approval by the respective ethics committees. Those participants with a positive test
395
result from either filtration or POC-CCA testing were invited to the health centre / health post
396
and were administered praziquantel in the adequate dose (40 mg/kg) by the study nurse. 397 Consent for publication
398 All authors approved submission for publication of this manuscript. 399 Availability of data and materials
400 Data will be available on request by email to the corresponding author. 401 Authors' contributions
407 HG conceived and designed the study protocol with input from PS; AAB, HG, RO and WM
408
implemented the research in Chad; AAB, HG, MA, RO and WM carried out the field work and
409
the parasitological examinations, together with a medical team; HG and WM sampled the
410
snails, and FA and RC performed the genetic sequencing and analysis for species
411
confirmation. RW and SP performed the genetic sequencing and analysis of the Schistosoma
412 HG conceived and designed the study protocol with input from PS; AAB, HG, RO and WM
408
implemented the research in Chad; AAB, HG, MA, RO and WM carried out the field work and
409
the parasitological examinations, together with a medical team; HG and WM sampled the
410
snails, and FA and RC performed the genetic sequencing and analysis for species
411
confirmation. RW and SP performed the genetic sequencing and analysis of the Schistosoma
412 17 samples. AAB, FA, HG, RO and WM analysed and interpreted the epidemiological data; HG,
413
RO and WM drafted the manuscript; all authors critically revised the manuscript for intellectual
414
content and approved the final manuscript. HG is the guarantor of the paper. 415 samples. AAB, FA, HG, RO and WM analysed and interpreted the epidemiological data; HG,
413
RO and WM drafted the manuscript; all authors critically revised the manuscript for intellectual
414
content and approved the final manuscript. HG is the guarantor of the paper. 415 samples. AAB, FA, HG, RO and WM analysed and interpreted the epidemiological data; HG,
413
RO and WM drafted the manuscript; all authors critically revised the manuscript for intellectual
414
content and approved the final manuscript. HG is the guarantor of the paper. 415
Acknowledgments
416 We are deeply grateful to Dr. Baba Mallaye, director of the Centre d’Appui à la Recherche
417
(CNAR), N’djamena, Chad, who not only guided us through the desert to safely arrive at the
418
Lakes of Ounianga, but also introduced us and the purpose of our research visit to the local
419
authorities. We thank the population of Ounianga Kabir and Ounianga Serir for hosting us and
420
for their willingness to participate in the study. Special thanks go to the staff of the health
421
centers at the study sites for their support. Authors' contributions
407 Without the tireless engagement of our study nurse
422
Ali Abba Abakar, this work would not have succeeded. Nomadic pastoralists at Lake Chad
423
have triggered this study, and we like to express here our deepest thanks for their hospitality
424
and sharing of their wisdom. 425 We are deeply grateful to Dr. Baba Mallaye, director of the Centre d’Appui à la Recherche
417
(CNAR), N’djamena, Chad, who not only guided us through the desert to safely arrive at the
418
Lakes of Ounianga, but also introduced us and the purpose of our research visit to the local
419
authorities. We thank the population of Ounianga Kabir and Ounianga Serir for hosting us and
420
for their willingness to participate in the study. Special thanks go to the staff of the health
421
centers at the study sites for their support. Without the tireless engagement of our study nurse
422
Ali Abba Abakar, this work would not have succeeded. Nomadic pastoralists at Lake Chad
423
have triggered this study, and we like to express here our deepest thanks for their hospitality
424
and sharing of their wisdom. 425 18 References
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528
seasonal distribution of Bulinus truncatus, Bulinus forskalii and Biomphalaria pfeifferi, the
529
intermediate host snails of schistosomiasis, in N'Djamena, Chad. Geospatial health. 530
2014;9(1):109-18. 531 22
532
533
534
535
536
537 22 SUPPLEMENTARY MATERIAL
538 Table 1. Primers and probes
539
Parasite
Name
Sequence 3’-5’
Origin
Generic
Schistosoma
spp. References
428 Schisto28S_F
GTGGAGTTGAACTGCAAGC
Modified from
Cnops et al. 2012
Schisto28S_R1
CCATAGCAGACAGGCAGC
Schisto28S_R2
GCTCAACAWTAATAGTCAAACCTG
Schisto28S_P
FAM-
ACTGACAAGCAGACCCTCACACC-
BHQl
S. mansoni TRE
Sman_F
CCACGCTCTCGCAAATAATCTA
Modified from
Wichmann et al. 2013
Sman_R
AAATCGTTGTATCTCCGAAACCA
Sman_P
YYE-
ACAAACATCATAAAAATCCGTCCA-
MGB-Q5
S. haematobium
dra1
Shae_F
GATCTCACCTATCAGACGAAAC
Identical to Cnops
et al. 2013
Shae_R
TCACAACGATACGACCAAC
Shae_P
FAM-
TGTTGGTGGAAGTGCCTGTTTCGCAA
-BHQ1
S. haematobium
COX
SH_COX_F
TTTTTTGGTCATCCAGAGGTGTAT
Modified from
Boon et al. 2018
SH_COX_R
TAATAATCAATGACCCTGCAATAA
S. bovis COX
SB_COX_F
TTTTTTGGGCATCCGGAGGTGTAT
SB_COX_R
CACAGGATCAGACAAACGAGTACC
540 540 23
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English
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New vessels detected on wide-field imaging compared to two-field and seven-field imaging: implications for diabetic retinopathy screening image analysis
|
British journal of ophthalmology
| 2,015
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cc-by
| 3,884
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Copyright: This is an Open Access article distributed in accordance with the terms of the Creative Commons
Attribution (CC BY 4.0) license, which permits others to distribute, remix, adapt and build upon this work,
for commercial use, provided the original work is properly cited. See:
http://creativecommons.org/licenses/by/4.0/ Talks SJ, Manjunath V, Steel DHW, Peto T, Taylor R. New vessels detected on wide-field imaging compared to two-field and
seven-field imaging: implications for diabetic retinopathy screening image
analysis. British Journal of Ophthalmology 2015, 99(12), 1606-1609. DOI link to article: http://dx.doi.org/10.1136/bjophthalmol-2015-306719 Stephen James Talks,1 Vina Manjunath,1 David H W Steel,2 Tunde Peto,3 Roy Taylor4 Method A consecutive series of treatment naïve
patients with DR, referred from DRSS with pre-
proliferative or proliferative DR or diabetic maculopathy,
were imaged with Optomap colour images, within
3 months of DRSS referral. The incidence and
distribution of NVs were recorded in relation to two-field
and seven-field areas. ABSTRACT
Introduction Wide-field retinal imaging (Optomap),
used for detecting diabetic retinopathy (DR), has been
shown to compare well with seven-field early treatment
diabetic retinopathy study (ETDRS) photographs. An
Optomap 200° image covers 80% of the retinal surface,
compared with the standard seven-field, 30° images,
covering 30% of the retinal surface. In England, DR
screening is performed by grading two, 45° images per
eye, by the DR screening service (DRSS). eye, by t e
sc ee
g se
ce (
SS)
Purpose To assess how often retinal new vessels (NVs)
are observed on Optomap imaging, outside the DRSS
two fields and standard seven-field photography, in a
cohort of patients referred by the DRSS. Method A consecutive series of treatment naïve
patients with DR, referred from DRSS with pre-
proliferative or proliferative DR or diabetic maculopathy,
were imaged with Optomap colour images, within
3 months of DRSS referral. The incidence and
distribution of NVs were recorded in relation to two-field
and seven-field areas. Purpose To assess how often retinal new vessels (NVs)
are observed on Optomap imaging, outside the DRSS
two fields and standard seven-field photography, in a
cohort of patients referred by the DRSS. Correspondence to
Stephen James Talks,
Newcastle Eye Centre, Royal
Victoria Infirmary, Queen
Victoria Road, Newcastle upon
Tyne NE1 4LP, UK;
james.talks@nuth.nhs.uk Correspondence to
Stephen James Talks,
Newcastle Eye Centre, Royal
Victoria Infirmary, Queen
Victoria Road, Newcastle upon
Tyne NE1 4LP, UK;
james.talks@nuth.nhs.uk Method A consecutive series of treatment naïve
patients with DR, referred from DRSS with pre-
proliferative or proliferative DR or diabetic maculopathy,
were imaged with Optomap colour images, within
3 months of DRSS referral. The incidence and
distribution of NVs were recorded in relation to two-field
and seven-field areas. METHOD
A Received 1 February 2015
Revised 23 March 2015
Accepted 21 May 2015
Published Online First
13 August 2015 A consecutive series of treatment naïve patients,
referred from two DRSS in England, were imaged
with Optomap colour images, within 3 months of
referral. Referral from DRSS occurs if ‘referable’
DR is detected on analysis of two standard 45°
photographs. At DRSS images are graded for the
level of DR, and diabetic maculopathy (DMac): no
DR is denoted as R0; mild DR as R1; pre-
proliferative DR as R2; proliferative DR as R3. Potentially clinically significant DMac is repre-
sented by the M1 grade. R2, R3 and M1 are then
subsequently referred to hospital eye services. Results NVs were found in 102 of 1562 treatment
naïve eyes (6.5%) of 781 patients. Of these, 72 were
referred from DRSS as having NVs, but an additional 30
eyes (29% of NVs detected) from 25 patients were
referred with a lesser degree of DR. In 25 of the 30 eyes
without NVs reported on referral, NVs were located
outside the standard two fields taken at DRSS, and in
12, NVs were outside the area covered on seven-field
imaging (11.7% of eyes with NVs). Conclusions Wide-field imaging with Optomap
detected approximately 30% more NVs than standard
two-field imaging in patients referred from a UK DRSS. At the hospital eye clinic, certified medical photo-
graphers took three wide-field Optomap images per
eye after mydriasis, using the Optomap P2000 scan-
ning laser ophthalmoscope; straight-ahead and up
and down, with eye steering, which involves the
patient following a fixation target (figure 1). Open Access
Scan to access more
free content Date deposited: Newcastle University ePrints - eprint.ncl.ac.uk group.bmj.com
on April 5, 2016 - Published by
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Downloaded from Clinical science Received 1 February 2015
Revised 23 March 2015
Accepted 21 May 2015
Published Online First
13 August 2015 1Newcastle Eye Centre, Royal
Victoria Infirmary, Newcastle
upon Tyne, UK
2Sunderland Eye Infirmary,
Sunderland and Institute of
Genetic Medicine, Newcastle
upon Tyne, UK
3NIHR Biomedical Research
Centre at Moorfields Eye
Hospital NHS Foundation Trust
and UCL Institute of
Ophthalmology, London, UK
4Magnetic renounce centre,
Campus for ageing and vitality,
Newcastle University,
Newcastle upon Tyne, UK Stephen James Talks,1 Vina Manjunath,1 David H W Steel,2 Tunde Peto,3 Roy Taylor4 Stephen James Talks,1 Vina Manjunath,1 David H W Steel,2 Tunde Peto,3 Ro 1Newcastle Eye Centre, Royal
Victoria Infirmary, Newcastle
upon Tyne, UK
2Sunderland Eye Infirmary,
Sunderland and Institute of
Genetic Medicine, Newcastle
upon Tyne, UK
3NIHR Biomedical Research
Centre at Moorfields Eye
Hospital NHS Foundation Trust
and UCL Institute of
Ophthalmology, London, UK
4Magnetic renounce centre,
Campus for ageing and vitality,
Newcastle University,
Newcastle upon Tyne, UK 1Newcastle Eye Centre, Royal
Victoria Infirmary, Newcastle
upon Tyne, UK
2Sunderland Eye Infirmary,
Sunderland and Institute of
Genetic Medicine, Newcastle
upon Tyne, UK
3NIHR Biomedical Research
Centre at Moorfields Eye
Hospital NHS Foundation Trust
and UCL Institute of
Ophthalmology, London, UK
4Magnetic renounce centre,
Campus for ageing and vitality,
Newcastle University,
Newcastle upon Tyne, UK in a population referred from the community could
not be assessed. Grading is based on the ETDRS
studies that related retinal findings to the likelihood
of progression of the retinopathy and is based on
seven-field colour imaging. It is still unknown how
often more severe DR changes are found outside the
standard seven-field, in particular new vessel (NV)
formation. In the English DR screening service
(DRSS) two images with nominal 45° fields are
taken per eye, one centred on the fovea and the
other on the disc. This is said to have a sensitivity of
80.2% and specificity of 92.9% for detecting refer-
able DR compared to slit lamp biomicroscopy.5 In
this study, we aimed to assess how often NVs were
seen with wide-field Optomap imaging when com-
pared to the areas covered by DRSS’s two-field and
standard seven-field photography, in a cohort of
patients referred from a DRSS. ABSTRACT
Introduction Wide-field retinal imaging (Optomap),
used for detecting diabetic retinopathy (DR), has been
shown to compare well with seven-field early treatment
diabetic retinopathy study (ETDRS) photographs. An
Optomap 200° image covers 80% of the retinal surface,
compared with the standard seven-field, 30° images,
covering 30% of the retinal surface. In England, DR
screening is performed by grading two, 45° images per
eye, by the DR screening service (DRSS). Purpose To assess how often retinal new vessels (NVs)
are observed on Optomap imaging, outside the DRSS
two fields and standard seven-field photography, in a
cohort of patients referred by the DRSS. INTRODUCTION Wide-field retinal imaging (Optomap), used for
detecting
diabetic
retinopathy
(DR),
has
been
shown to compare well with seven-field early treat-
ment diabetic retinopathy study (ETDRS) photo-
graphs.1–3 As it provides a wider field of view, it
would not be surprising that more DR is seen;
however, it is thought that most potentially sight-
threatening pathology occurs between the posterior
pole and retinal mid-periphery. Silva et al4 reported
that 10% of a cohort of 206 eyes were given a
higher DR grade on Optomap images compared to
seven-field
images,
predominantly
due
to
the
finding of more haemorrhages per quadrant. In his
study, the patients were chosen from a tertiary eye
clinic to represent a range of DR severity, and so,
incidence rates of previously not recorded findings The images were then graded by an independent
reading centre and the number of eyes with NVs,
R3, recorded. The R3 images were then further
assessed to map the distribution of NVs in relation
to two-field and seven-field standard images using a
standard field map (figure 2). If there was more
than one area of NVs and any were located inside
either the two-field or seven-field areas, then they
were counted as being detected by that method. In
a few cases, where the distinction between haemor-
rhage,
intra-retinal
microvascular
abnormalities
(IRMA) and small NVs was uncertain, a fundus
fluorescein angiogram (FFA) was performed, at the
examiners discretion, on a second visit. Open Access
Scan to access more
free content Talks SJ, et al. Br J Ophthalmol 2015;99:1606–1609. doi:10.1136/bjophthalmol-2015-306719 1606 group.bmj.com
on April 5, 2016 - Published by
http://bjo.bmj.com/
Downloaded from Clinical science Figure 1
Red free Optomap images of a left eye of a diabetic referred with maculopathy in the other eye, (R1, M1), the left eye being referred as,
(R1, M0). (A) Up-steered, showing new vessels, (B) with zoom, (C) straight ahead showing new vessels outside two fields and on the edge of the
standard seven-field images, (D) down steered, showing new vessels and (E) better seen with on zoom. Clinical science Figure 1
Red free Optomap images of a left eye of a diabetic referred with maculopathy in the other eye, (R1, M1), the left eye being referred as,
(R1, M0). RESULTS
d
d Independent assessment of the wide-field imaging found NVs in
102 of 1562 treatment naïve eyes (6.5%) of 781 patients
referred from DRSS. Of these, 72 were referred from DRSS as
having NVs, but 30 eyes (29% of NVs detected) from 25
patients were referred with a lesser degree of DR: 14 were
referred as R2 and 16 as R1. In these eyes, 18 had been referred
with Dmac, (M1), the other 12 were graded as M0. The fellow
eyes of these patients were graded as R3 (proliferative DR) in
three cases, R2 in eight and R1 in nine. In nine cases both eyes
were referred with R1, having been referred because of DMac. Figure 1 shows NVs outside two fields and the benefit of
using up and down eye steering. Figure 2 shows NVs outside
seven fields confirmed on FFA. INTRODUCTION (A) Up-steered, showing new vessels, (B) with zoom, (C) straight ahead showing new vessels outside two fields and on the edge of the
standard seven-field images, (D) down steered, showing new vessels and (E) better seen with on zoom. The images were reviewed using the proprietary image review
software (Optos V2 Vantage Dx Review V.2.5.0.135; Optos,
Dunfermline, UK). Grading for each wide-field image involved
viewing
the
colour
composite,
green-wavelength
and
red-
wavelength images using all the available image enhancement tools,
including localised optimisation and magnification. However, true
NVs were recorded only if the NV was present on FFA where avail-
able and also where the NV remained clearly visible on the original
image once spotted on the manipulated image, where appropriate. FFAs were requested in 31 patients where the examiner had
some uncertainty between R2 and R3. In 14 of these cases, the
examiner had diagnosed NVs and this was confirmed in 10
cases but not found in 4 cases and in 17 cases the examiner
thought they were seeing, R2 changes, this was confirmed in 9
cases, but NVs were found in 8 cases. In 25 of the 30 eyes, in which NVs were not reported on the
two DRSS images, the NVs were found outside the standard
two-field and in 12 of these outside seven-field (11.7% of eyes
with NVs); three were just within the field of view of the two
fields but had been referred as R2 with IRMA. Two had very
small disc NVs but also had NVs beyond two fields. Images
from 23 eyes of the referred 1562 (1.4%) were deemed ungrad-
able on Optomap due to poor image quality. DISCUSSION Figure 2
Red free Optomap and fundus fluorescein angiogram of the
left eye of a diabetic referred due to maculopathy, (R1, M1 right; R1,
M1 left), showing new vessels outside standard two-field (A); and
seven-field (B) in the left eye. One factor that may have led us to detect this rate of NVs
was the use of three images per eye, using eye steering, as less
pathology is likely to be missed due to defocus or masking from
eyelashes.9 The Optos camera can take a 200° image, but the
resolution is best in a central band between the two arcades. The focus for the top and bottom areas of the retina is better by
taking the image with the patient looking up and down. Looking at three images per eye takes extra time compared to
one. Montage software is being developed to merge three pic-
tures, which will help with analysis, but is not commercially
available yet. A study comparing wide-field photographs, taken with undi-
lated and dilated pupils, found that this did not statistically
change the agreement with seven-field imaging, but reduced the
ungradable rate from 4.5% to 0%.4 We had an ungradable rate
of 1.4% using dilation and three images per eye. Figure 2
Red free Optomap and fundus fluorescein angiogram of the
left eye of a diabetic referred due to maculopathy, (R1, M1 right; R1,
M1 left), showing new vessels outside standard two-field (A); and
seven-field (B) in the left eye. Our patients with NVs not detected on DRSS images were not
‘missed’ cases, as they were correctly referred for further medical
assessment. All registered patients with diabetes are offered
annual DRSS photography in England and this has led to fewer
patients being referred from DRSS with severe NVs, and so, our
incidence figures of more peripheral pathology may be higher
than in unscreened populations. It is possible that if patients had
small NVs outside the two-field images they would have been
eventually referred as more posterior pathology developed. patient and on the photographer to use this protocol in every-
day clinical practice. In the Eurodiab paper justifying the use of
45° imaging criteria only 48 eyes were compared.6 The findings
supported the use of two-field imaging as a practical method,
with the agreement for correct DR between several examiners,
ranging between 28 and 43 of the 48 eyes, mean of 37 eyes. DISCUSSION ETDRS seven-field colour imaging is still considered the gold
standard for assessing DR; however, it is hard both on the Talks SJ, et al. Br J Ophthalmol 2015;99:1606–1609. doi:10.1136/bjophthalmol-2015-306719 1607 group.bmj.com
on April 5, 2016 - Published by
http://bjo.bmj.com/
Downloaded from group.bmj.com
on April 5, 2016 - Published by
http://bjo.bmj.com/
Downloaded from Clinical science patient and on the photographer to use this protocol in every-
day clinical practice. In the Eurodiab paper justifying the use of
45° imaging criteria only 48 eyes were compared.6 The findings
supported the use of two-field imaging as a practical method,
with the agreement for correct DR between several examiners,
ranging between 28 and 43 of the 48 eyes mean of 37 eyes
Figure 2
Red free Optomap and fundus fluorescein angiogram of the
left eye of a diabetic referred due to maculopathy, (R1, M1 right; R1,
M1 left), showing new vessels outside standard two-field (A); and
seven-field (B) in the left eye. Our study shows that on two-field DRSS imaging there is only
a small risk of missing NVs, 30/1562 (1.9%), but these repre-
sented 29% of the total number of eyes graded as having NVs. The NVs were found outside even the seven-field area in 11.7%. This is a higher rate than previously reported. In a study of 206
eyes of 103 patients, 10% were given a more severe DR grade
with wide-field imaging, using one image per eye.4 In relation to
our findings, 46 had NVs, but only two of these were found
outside the seven-field area (4% of NVs). Our study population
was much larger and represents a consecutive series referred
from DRSS, rather than a group from a highly specialised clinic. A study using wide-field FFA on 118 patients found a total of
22 eyes (10%) had pathology visible only outside a simulated
seven-field boundary. Of those eyes, 13 had peripheral retinal
non-perfusion (8%) and 9 of 54 cases (17% of NVs) had per-
ipheral NV outside seven-field. While using a different technique
for identification of cases with NVs, this study draws a similar
conclusion to ours on the relative proportion of NVs found
outside seven-field.8 In the cases where we did use FFA some
changes were made in the grading and eight additional cases of
NVs were found. Acknowledgements This article presents independent research funded by the
National Institute for Health Research (NIHR) under its Research for Patient Benefit
(RfPB) Programme (Grant Reference Number PB-PG-0609-19117). The views
expressed are those of the authors and not necessarily those of the NHS, the NIHR
or the Department of Health. We thank the photography departments at Royal
Victoria Infirmary, Newcastle upon Tyne and Sunderland Eye Infirmary for their Provenance and peer review Not commissioned; externally peer reviewed. Open Access This is an Open Access article distributed in accordance with the
terms of the Creative Commons Attribution (CC BY 4.0) license, which permits
others to distribute, remix, adapt and build upon this work, for commercial use,
provided the original work is properly cited. See: http://creativecommons.org/
licenses/by/4.0/ 6
Adlington SJ, Kohner EM, Meuer S, et al. Methodology for retinal photography and
assessment of diabetic retinopathy: the EURODIAB IDDM complications study. Diabetologia 1995;38:437–44. g
7
Diabetic eye screening. www.diabeticeye.screening.nhs.uk 8
Wessel MM, Aaker GD, Parlitsis G, et al. Ultra-wide-field angiography improves the
detection and classification of diabetic retinopathy. Retina 2012;32:785–91. 9
Rasmussen ML, Broe R, Frydkjaer U, et al. Comparison between Early Treatment
Diabetic Retinopathy Study 7-field retinal photos and non-mydriatic, mydriatic and
mydriatic steered widefield scanning laser ophthalmoscopy for assessment of diabetic
retinopathy. J Diabetes Complications 2014;29:99–104. group.bmj.com
on April 5, 2016 - Published by
http://bjo.bmj.com/
Downloaded from group.bmj.com
on April 5, 2016 - Published by
http://bjo.bmj.com/
Downloaded from Clinical science assistance during this study. Photographers Gill O’Brien and Jade Ward prepared the
figures. TP was supported by the NIHR Biomedical Research Centre at Moorfields
Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology. assistance during this study. Photographers Gill O’Brien and Jade Ward prepared the
figures. TP was supported by the NIHR Biomedical Research Centre at Moorfields
Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology. examination for evaluation of diabetic retinopathy. Am J Ophthalmol
2012;154:549–59. examination for evaluation of diabetic retinopathy. Am J Ophthalmol
2012;154:549–59. 2
Kernt M, Haritoglou C, Hadi I, et al. Assessment of diabetic retinopathy
using nonmydriatic ultra-widefield scanning laser ophthalmoscopy (Optomap)
compared with ETDRS 7-field stereo photography. Diabetes Care
2012;35:2459–63. Contributors All the authors contributed to the design of the study. TP led the
independent reading centre. SJT and VM carried out the study, recruiting and
consenting the patients. All the authors contributed to writing the paper. 3
Soliman AZ, Silva PS, Aiello LP, et al. Ultra-wide field retinal imaging in detection,
classification, and management of diabetic retinopathy. Semin Ophthalmol
2012;27:221–6. Funding National Institute for Health Research. Funding National Institute for Health Research. Competing interests None declared. Competing interests None declared. Competing interests None declared. 4
Silva PS, Cavallerano JD, Sun JK, et al. Peripheral lesions identified by mydriatic
ultrawide field imaging: distribution and potential impact on diabetic retinopathy
severity. Ophthalmology 2013;120:2587–95. Ethics approval Ethical approval was obtained from the Northern Regional Ethics
Committee and the NHS Trust Research departments at the Royal Victoria Infirmary,
Newcastle upon Tyne and the Sunderland Eye Infirmary. 5
Scanlon PH, Malhotra R, Greenwood RH, et al. Comparison of two reference
standards in validating two field mydriatic digital photography as a method of
screening for diabetic retinopathy. Br J Ophthalmol 2003;87:1258–63. Provenance and peer review Not commissioned; externally peer reviewed. DISCUSSION The kappa for interobserver and intraobserver comparisons was
good at 0.83 and 0.85, respectively. Two-field imaging, where
approximately 80% of patients are imaged yearly, using this
protocol
meets
the
appropriate
sensitivity
and
specificity
required for a screening programme and was therefore rolled
out with scale, as shown by the England DRSS.7 This study also does not clarify how much risk there is in
missing peripheral NVs, as they were not detected as a result of
a patient presenting with the complications of proliferative DR,
rather as a result of imaging a cohort of patients. However, if
NVs are missed on DRSS images, and the patient is referred
because of Dmac, appropriate management depends on the clin-
ician detecting these NVs, which may not occur in a busy
streamlined macular service. We would therefore advocate the
use of steered wide-field images in ophthalmology clinics, and
in the future hope that automated software can be developed to
allow for fast, reliable and valid identification of abnormal
vessels. In one study, seven-field ETDRS stereo images were ungrad-
able by strict grading criteria in 31.6% and in 15.3% with a
more lenient approach.5 The same paper reported good agree-
ment for detecting the difference between referable and non-
referable retinopathy between slit lamp biomicroscopy, 2×45°
field and 7×30° field photography. However, there was only
agreement on finding proliferative DR in 51/88 (58%) patients
when comparing seven-field ETDRS stereo images with slit
lamp examination. It is not clear how many had already had
laser which may have lead to confusion on definitions between
active or inactive NVs. In two cases, the clinician found NVs
outside seven-field. For the comparison of two-field to seven-
field only correlations between detecting referable from non-
referable DR were presented. Acknowledgements This article presents independent research funded by the
National Institute for Health Research (NIHR) under its Research for Patient Benefit
(RfPB) Programme (Grant Reference Number PB-PG-0609-19117). The views
expressed are those of the authors and not necessarily those of the NHS, the NIHR
or the Department of Health. We thank the photography departments at Royal
Victoria Infirmary, Newcastle upon Tyne and Sunderland Eye Infirmary for their Talks SJ, et al. Br J Ophthalmol 2015;99:1606–1609. doi:10.1136/bjophthalmol-2015-306719 1608 REFERENCES 1
Silva PS, Cavallerano JD, Sun JK, et al. Nonmydriatic ultrawide field retinal imaging
compared with dilated standard 7-field 35-mm photography and retinal specialist Talks SJ, et al. Br J Ophthalmol 2015;99:1606–1609. doi:10.1136/bjophthalmol-2015-306719 1609 group.bmj.com
on April 5, 2016 - Published by
http://bjo.bmj.com/
Downloaded from screening image analysis
imaging: implications for diabetic retinopathy
compared to two-field and seven-field
New vessels detected on wide-field imaging doi: 10.1136/bjophthalmol-2015-306719
13, 2015
2015 99: 1606-1609 originally published online August
Br J Ophthalmol http://bjo.bmj.com/content/99/12/1606
Updated information and services can be found at: #BIBL
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Endophytic Bacteria From the Roots of the Medicinal Plant Alkanna tinctoria Tausch (Boraginaceae): Exploration of Plant Growth Promoting Properties and Potential Role in the Production of Plant Secondary Metabolites
|
Frontiers in microbiology
| 2,021
|
cc-by
| 12,360
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ORIGINAL RESEARCH
published: 03 February 2021
doi: 10.3389/fmicb.2021.633488 Edited by:
Aleksa Obradovi ´c,
University of Belgrade, Serbia A selection of bacteria was then tested for the ability to induce A/S Specialty section:
This article was submitted to
Microbe and Virus Interactions with
Plants,
a section of the journal
Frontiers in Microbiology
Received: 25 November 2020
Accepted: 13 January 2021
Published: 03 February 2021 Specialty section:
This article was submitted to
Microbe and Virus Interactions with
Plants,
a section of the journal
Frontiers in Microbiology
Received: 25 November 2020
Accepted: 13 January 2021
Published: 03 February 2021 Specialty section:
This article was submitted to
Microbe and Virus Interactions with
Plants, Edited by:
Aleksa Obradovi ´c,
University of Belgrade, Serbia Reviewed by:
Carolina Chiellini,
Italian National Research Council, Italy
Debdulal Banerjee,
Vidyasagar University, India Reviewed by:
Carolina Chiellini,
Italian National Research Council, Italy
Debdulal Banerjee,
Vidyasagar University, India
*Correspondence:
Angélique Rat
angelique.rat@ugent.be 1 Laboratory of Microbiology, Department Biochemistry and Microbiology, Faculty Sciences, Ghent University, Ghent,
Belgium, 2 Laboratory of Conservation and Evaluation of Native and Floricultural Species, Institute of Plant Breeding
and Genetic Resources, Hellenic Agricultural Organization Demeter, Thessaloniki, Greece, 3 Institut für Pflanzenkultur,
Schnega, Germany, 4 Organic Chemistry Laboratory, School of Chemical Engineering, Aristotle University of Thessaloniki and
Center of Interdisciplinary Research and Innovation of AUTh (CIRI-AUTh), Natural Products Research Centre of Excellence
(NatPro-AUTH), Thessaloniki, Greece, 5 Division of Pharmacognosy and Natural Products Chemistry, Department
of Pharmacy, National and Kapodistrian University of Athens, Athens, Greece Alkannin and shikonin (A/S) are enantiomeric naphthoquinones produced in the roots of
certain plants from the Boraginaceae family such as Lithospermum spp. and Alkanna
spp. They possess antimicrobial, anti-tumoral and wound healing properties. The
production of secondary metabolites by Alkanna tinctoria might be influenced by its
endomicrobiome. To study the interaction between this medicinal plant and its bacterial
endophytes, we isolated bacteria from the roots of wild growing Alkanna tinctoria
collected near to Athens and Thessaloniki in Greece. Representative strains selected
by MALDI-TOF mass spectrometry were identified by partial 16S rRNA gene sequence
analysis. In total, 197 distinct phylotypes of endophytic bacteria were detected. The
most abundant genera recovered were Pseudomonas, Xanthomonas, Variovorax,
Bacillus, Inquilinus, Pantoea, and Stenotrophomonas. Several bacteria were then tested
in vitro for their plant growth promoting activity and the production of cell-wall degrading
enzymes. Strains of Pseudomonas, Pantoea, Bacillus and Inquilinus showed positive
plant growth properties whereas those of Bacteroidetes and Rhizobiaceae showed
pectinase and cellulase activity in vitro. In addition, bacterial responses to alkannin
and shikonin were investigated through resistance assays. Gram negative bacteria were
found to be resistant to the antimicrobial properties of A/S, whereas the Gram positives
were sensitive. Angélique Rat1*, Henry D. Naranjo1, Nikos Krigas2, Katerina Grigoriadou2,
Eleni Maloupa2, Alicia Varela Alonso3, Carolin Schneider3, Vassilios P. Papageorgiou4,
Andreana N. Assimopoulou4, Nikolaos Tsafantakis5, Nikolas Fokialakis5 and
Anne Willems1 Angélique Rat1*, Henry D. Naranjo1, Nikos Krigas2, Katerina Grigoriadou2,
Eleni Maloupa2, Alicia Varela Alonso3, Carolin Schneider3, Vassilios P. Papageorgiou4,
Andreana N. Assimopoulou4, Nikolaos Tsafantakis5, Nikolas Fokialakis5 and
Anne Willems1 Edited by:
Aleksa Obradovi ´c,
University of Belgrade, Serbia
Reviewed by:
Carolina Chiellini,
Italian National Research Council, Italy
Debdulal Banerjee,
Vidyasagar University, India
*Correspondence:
Angélique Rat
angelique.rat@ugent.be INTRODUCTION Soil microorganisms can also influence plant growth through
the secretion of growth hormones such as indole acetic acid
(IAA) or ethylene. Indeed, several studies have shown that plant
inoculation with IAA-producing bacteria promote plant growth
significantly (Patten and Glick, 2002; Mohite, 2013; Chandra
et al., 2018). Furthermore, rhizospheric bacteria can play a role
in plant defense and metabolite production (Lambers et al., 2009;
Berendsen et al., 2012; Huang et al., 2019). They can first compete
with pathogens for nutrients and space in the rhizosphere. For
instance, siderophore-producing bacteria can contend with soil
pathogens for iron uptake and consequently prevent the root
colonization by these microorganisms. As iron is essential for the
growth of many organisms and is involved in biofilm formation
that is needed for successful root colonization, its uptake by
beneficial bacteria can prevent pathogen infections (Ramey et al.,
2004; Ahmed and Holmström, 2014). Microorganisms can also
produce secondary metabolites with antimicrobial properties. For example, Burkholderia sp. showed antimicrobial activities
against the plant pathogens Phytophthora capsici, Fusarium
oxysporum, and Rhizoctonia solani through the production of
pyrrolnitrin (Jung et al., 2018). In addition, some microbes
can induce plant defenses. Asghari et al. (2020) demonstrated
that Pseudomonas sp. Sn48 and Pantoea sp. Sa14 induce plant
production of phytoalexins and polyamines resulting in a
reduction of the Agrobacterium tumefaciens gall. A
h
i
i
i
i
i h
l Studies on endophytes are indeed invaluable to the promotion
of medicinal plants’ therapeutic properties. Alkanna tinctoria
(family Boraginaceae) is a medicinal plant native to countries
of the Mediterranean region (Strid, 2016). The plant produces
quinones and phenolic compounds and has antioxidant activities. Moreover, compared to other Alkanna species, A. tinctoria
produces high amounts of alkannin/shikonin (A/S) and their
derivatives. These secondary metabolites are enantiomeric
naphthoquinones produced by root tissues. They are sequestered
to form granules in the phospholipid layer and are accumulated
in the apoplastic spaces. Thus, they can be found in the cork layer
of mature roots and their accumulation leads to a red or purple
coloration of the root (Brigham et al., 1999; Singh et al., 2010;
Tatsumi et al., 2016). The A/S derivatives are strongly involved
in the antimicrobial activity of the plant and form a chemical
barrier against soil-borne microorganisms. INTRODUCTION tissues of plants during the entire or part of their host’s lifecycle
without causing external damage (Ryan et al., 2008; Anjum
and Chandra, 2015; Orlikowska et al., 2017). Like rhizospheric
microorganisms, they can promote plant growth and influence
plant metabolites. In the case of medicinal plants, endophytes
contribute to or are responsible for their host’s pharmaceutical
properties (e.g., antioxidant and/or antimicrobial properties;
Köberl et al., 2013; Brader et al., 2014; Maggini et al., 2017). The
inoculation of Bacillus subtilis BERA 71 to chickpea increased the
plant biomass and reduced the levels of reactive oxygen species
and lipid peroxidation under salinity stress. This effect was
associated with an enhancement of the activities of enzymatic
and non-enzymatic antioxidants (Abd_Allah et al., 2017). Endophytes can also influence the antimicrobial activity as well
as the production of secondary metabolites in medicinal plants. Pandey et al. (2018) showed that endophytic fungi increase the
biomass of Withania somnifera, resulting in a higher total plant
withanolide content. Moreover, the inoculation of endophytic
bacteria stimulated the withanolide biosynthesis pathway by
upregulating the expression of key genes within this pathway
such as HMGR, DXS, and DXR (Pandey et al., 2018). Endophytes
of medicinal plants also produce a wide range of bioactive
secondary metabolites such as alkaloids, isoprenoids, flavonoids
and indoles (Tan and Zou, 2001; Barman and Bhattacharjee,
2020; Monnanda et al., 2020). Plants communicate and interact with a wide variety of
microorganisms. They release water-soluble sugars, organic
acids, ionic compounds, phenolics, hormones, and other
metabolites into the rhizosphere, thus providing nutrients to
microorganisms. Due to the richness of nutrients in this soil
region, compared to bulk soil, the rhizosphere is enriched
with microorganisms, leading to increased microbial interactions
(Sasse et al., 2018; Yu and Hochholdinger, 2018). Rhizospheric
microorganisms influence plant growth through soil nutrient
recycling and nutrient uptake. For example, bacteria such as
Acidobacterium sp., Pedobacter sp., Muciliginibacter sp., and
Cellulomonas sp. play an important role in the recycling of
plant polysaccharides such as cellulose, pectin and lignin (López-
Mondéjar et al., 2016; Poulsen et al., 2016; Belova et al., 2018). Several bacteria including those from the genera Pseudomonas,
Pantoea, and Bacillus have the ability to solubilize and therefore
make accessible insoluble phosphate to plants (Ghyselinck et al.,
2013). Similarly, rhizobia can provide plants with combined
nitrogen via biological nitrogen fixation (Mabrouk et al., 2018). Citation: Rat A, Naranjo HD, Krigas N,
Grigoriadou K, Maloupa E, Alonso AV,
Schneider C, Papageorgiou VP,
Assimopoulou AN, Tsafantakis N,
Fokialakis N and Willems A (2021)
Endophytic Bacteria From the Roots
of the Medicinal Plant Alkanna
tinctoria Tausch (Boraginaceae):
Exploration of Plant Growth
Promoting Properties and Potential
Role in the Production of Plant
Secondary Metabolites. Front. Microbiol. 12:633488. doi: 10.3389/fmicb.2021.633488 February 2021 | Volume 12 | Article 633488 1 Frontiers in Microbiology | www.frontiersin.org Rat et al. Endophytic Bacteria From Alkanna tinctoria production in hairy roots culture of A. tinctoria. Four strains belonging to Chitinophaga
sp., Allorhizobium sp., Duganella sp., and Micromonospora sp., resulted in significantly
more A/S in the hairy roots than the uninoculated control. As these bacteria can produce
cell-wall degrading enzymes, we hypothesize that the A/S induction may be related with
the plant-bacteria interaction during colonization. Keywords: endophytes, isolation, Alkanna tinctoria, alkannin, shikonin, hairy roots Frontiers in Microbiology | www.frontiersin.org Identification of the Isolates Dereplication and identification of the isolates obtained in the
different conditions were performed using MALDI-TOF MS and
16S rRNA gene sequencing. To conduct the MALDI-TOF MS,
pure 2nd generation cultures were grown. The proteins were
then extracted, spotted in duplicate onto MALDI-TOF target
plates and overlaid with matrix solution (10 mg/mL α-cyano-4-
hydroxycinnamic acid in acetonitrile-water-trifluoroacetic acid)
as previously described (Dumolin et al., 2019). The protein
profiles were read with the Bruker MicroflexTM LT/SH system. The programs SPeDE (Dumolin et al., 2019) and BioNumerics
(Applied Mats) were used to cluster the protein profiles. In SPeDe, each unique spectrum is assigned as a reference
and clusters of all similar spectra are generated (Dumolin
et al., 2019). Thus, strains from which the spectra clustered
together were considered as highly similar and only the
reference strains were selected for rRNA 16S gene sequencing
(Jain et al., 2018). After each expedition, a bacterial isolation campaign was
conducted. Three plants from each of the collections were chosen
to explore the diversity of culturable bacterial endophytes. The
roots were first cleaned with tap water to remove soil particles. Subsequently, they were immersed in a solution of 70% ethanol
for 5 min. After rinsing with sterile water, the root was further
sterilized with a solution of 1.4% of NaOCl for 20 min. The root
was rinsed again and immersed in 2% Na2S2O3 for 10 min to
neutralize the effect of bleach. A last rinse with sterile water was
performed before testing the efficiency of the sterilization process
in order to exclude non-endophytes. To do so, the external
surface of the root was imprinted onto a sterile plate containing
869 agar medium (Weyens et al., 2012; Eevers et al., 2015) before
incubation at 28◦C. Only roots without microbial growth on the
imprints were used for the isolation of endophytic bacteria. The
root material was crushed in 10 mL of sterile phosphate buffer
saline (PBS) and a 10-fold dilution series was prepared. The
dilutions 10−2–10−7 were then plated in triplicate. Genomic DNA was extracted by alkaline lysis according
to the method of Niemann et al. (1997). Amplification
of the 16S rRNA gene was performed using primers pA
(3′-AGAGTTTGATCCTGGCTCAG-5′,
forward)
and
pH
(5′-AAGGAGGTGATCCAGCCGCA-3′,
reverse). The
PCR
products were sequenced by Eurofins Genomics, Mix2seq service,
with the BKL1 primer (5′-GTATTACCGCGGCTGCTGGCA-
3′, reverse) and the sequences obtained were identified with
EzTaxon database (Yoon et al., 2017). Identification of the Isolates We considered two
sequences as belonging to the same species when they were at
least 98.7% identical and as belonging to the same genus when
they were at least 94.8% identical (Moreira and López-García,
2014; Yarza et al., 2014). INTRODUCTION They are also known
for wound healing, anticancer and anti-inflammatory properties
(Papageorgiou et al., 1999, 2008) and they comprise the active
pharmaceutical ingredients of strong wound healing medicines
approved by the National Organization for Medicines in Greece. Because of these interesting traits, A. tinctoria was chosen for the
production of A/S for medical applications (Papageorgiou et al.,
2008; Sengul et al., 2009; Tung et al., 2013). Among
the
microorganisms
interacting
with
plants,
endophytes are defined as those that colonize the internal February 2021 | Volume 12 | Article 633488 Frontiers in Microbiology | www.frontiersin.org 2 Endophytic Bacteria From Alkanna tinctoria Rat et al. Rat et al. media were compared. Nine culture media supplemented with
50 mg/L cycloheximide were tested, including the commonly
used media R2A (Difco), 1/10 TSA (Oxoid), and ISP2 (Oxoid),
as well as 1/10 869 medium (Eevers et al., 2015) which is
routinely used for the isolation of endophytes. In addition,
1/10 869 medium supplemented with either an infusion of
dried Symphytum officinale (family Boraginaceae) roots or with
allantoin was tested. To prepare the former medium supplement,
dried S. officinale roots (Bioshop, Gent, Belgium) were infused in
boiled water for 10 min (28 g/L). The infusion was then filtered
and used to resuspend the dry ingredients of medium 1/10 869. Allantoin was added to medium 1/10 869 at four concentrations
(0.32, 1.6, 9, 32 mM). Allantoin is a secondary metabolite found
in the root of several members of the Boraginaceae family (Eevers
et al., 2015; Dresler et al., 2017). In this first isolation campaign,
plates were incubated at 28◦C for 4 days after which all distinct
colony types were picked for purification. Plates of the highest
dilutions were incubated for an additional 10 days at 20◦C to
isolate slow growers. Despite the medicinal importance of wild-growing A. tinctoria
and the potential bioactive properties of its associated bacteria, no
studies have been performed so far on its endophytic bacteria and
their possible interactions with the plant. The aims of this work
were therefore (i) to explore the diversity of endophytic bacteria
associated with A. tinctoria, (ii) to screen the isolates for a number
of plant-growth promotion properties and for the production of
cell-wall degrading enzymes (iii) to evaluate the effect of the A/S
produced by the plant on the associated bacteria, and (iv) to test
their effect on plant A/S production in a bioassay. Isolation of Root Endophytic Bacteria
From Wild-Growing Alkanna tinctoria
Plants Three botanical expeditions to collect wild-growing Alkanna
tinctoria were undertaken. The first one was conducted in
December 2017, in Northern Greece (Seich Sou area close to
the Theater of the Earth, Thessaloniki, Greece), using a special
collection permit obtained by the Institute of Plant Breeding and
Genetic Resources, Hellenic Agricultural Organization Demeter
(IPBGR, HAO Demeter) which is issued annually by the
Greek Ministry of Environment and Energy. Samples from
this expedition were used to optimize the isolation conditions. The wild plants collected were taxonomically identified and
they were given the International Plant Exchange Network
(IPEN) accession number GR-1-BBGK-18,6081 for their long-
term ex situ conservation (including also asexual and in vitro
propagation trials) at the premises of IPBGR, HAO Demeter. Two further collections were made in April and May of 2018
in Southern Greece (at the University of Athens, Campus
of Zografou, Athens) and Northern Greece (Seich Sou area,
Thessaloniki), respectively. Frontiers in Microbiology | www.frontiersin.org Pectinase and Cellulase Activities Depending on the objective of the test, a minimum medium
(4 g/L KH2PO4, 6 g/L Na2HPO4, 2 g/L (NH4)2SO4, 1 g/L yeast
extract, 0.001 g/L FeSO4, 0.2 g/L MgSO4, 0.001 g/L CaCl2,
0.00001 g/L H3BO3, 0.00001 g/L MnSO4, 0.00007 g/L ZnSO4,
0.00005 g/L CuSO4, 0.00001 g/L MoO3, 15 g/L agar, pH 7)
supplemented with 5 g/L of either pectin or carboxymethyl
cellulose was used for this assay. Dense bacterial suspensions
were made in sterile PBS (OD 0.5–0.6). Then, 10 µL of each
bacterial suspension was spotted onto the center of the plate. The test was performed in triplicates and plates were incubated
at 28◦C for 5 days. Plates were then checked for the presence
of a solubilization halo. The halo was visualized by the addition
of 0.01% congo red and 1% hexadecyltrimethylammonium
bromide (CTAB) solutions for the cellulase and pectinase tests,
respectively. The strain LMG 16323 (Cellulomonas cellasea) was
used as a positive control for the cellulase assay whilst strain LMG
2404 (Pectobacterium carotovorum subsp. carotovorum) was used
as a positive control for the pectinase test. Depending on the objective of the test, a minimum medium
(4 g/L KH2PO4, 6 g/L Na2HPO4, 2 g/L (NH4)2SO4, 1 g/L yeast
extract, 0.001 g/L FeSO4, 0.2 g/L MgSO4, 0.001 g/L CaCl2,
0.00001 g/L H3BO3, 0.00001 g/L MnSO4, 0.00007 g/L ZnSO4,
0.00005 g/L CuSO4, 0.00001 g/L MoO3, 15 g/L agar, pH 7)
supplemented with 5 g/L of either pectin or carboxymethyl
cellulose was used for this assay. Dense bacterial suspensions
were made in sterile PBS (OD 0.5–0.6). Then, 10 µL of each
bacterial suspension was spotted onto the center of the plate. Plant Growth-Promoting Activity of
Endophytic Bacteria To determine the isolation conditions (medium, incubation
time and temperature) that would yield the widest bacterial
diversity, during the first isolation campaign a wide selection of To test bacteria in vitro for their plant growth promoting activity,
four
parameters
were
evaluated:
phosphate
solubilization, February 2021 | Volume 12 | Article 633488 3 Endophytic Bacteria From Alkanna tinctoria Rat et al. siderophore production, ACC deaminase activity, and indole
acetic acid production. with the culture medium free of bacteria as blanks. The test was
performed in triplicate. Incubation was at 28◦C for 72 h. The
strain R-42086 (Pseudomonas sp.) was used as a positive control
(Ghyselinck et al., 2013). 1-Aminocyclopropane-1-Carboxylate (ACC)
Deaminase Activity The test was performed in triplicates and plates were incubated
at 28◦C for 5 days. Plates were then checked for the presence
of a solubilization halo. The halo was visualized by the addition
of 0.01% congo red and 1% hexadecyltrimethylammonium
bromide (CTAB) solutions for the cellulase and pectinase tests,
respectively. The strain LMG 16323 (Cellulomonas cellasea) was
used as a positive control for the cellulase assay whilst strain LMG
2404 (Pectobacterium carotovorum subsp. carotovorum) was used
as a positive control for the pectinase test. DFS medium was prepared for the ACC deaminase test following
the protocol from Penrose and Glick (2003). The glucose and
the citric acid were filter sterilized and added to the autoclaved
medium. To conduct the experiment, the bacteria were first
grown in DFS medium + 2 g (NH4)2SO4 for 72 h at 28◦C, 100
rpm. Then, in a 96-well plate, for each strain 15 µL of the liquid
culture was added in 135 µL of DFS medium + 3 mM ACC. Each strain was tested in triplicate. The DFS medium without any
nitrogen source was used as control. As blanks, the outermost
wells of the plate were filled with the culture medium free of
bacteria. After 48 h at 28◦C, the OD was read at 590 nm. Then,
50 µL of the culture was re-transferred to fresh DFS + 0.3 M
ACC. This procedure was repeated twice to exhaust any residual
stock of nitrogen. The strain R-42058 (Pseudomonas sp.) was
used as a positive control and the ACC deaminase activity was
evaluated by comparison with the blank (Ghyselinck et al., 2013). Phosphate Solubilization NBRIP medium (2.5 g/L Ca3(PO4); 5 g/L MgCl2,6H2O; 0.25 g/L
MgSO4,7H2O; 0.2 g/L KCl, 0.1 g/L (NH4)2SO4, and 15 g/L agar)
with 10 g/L glucose was used for this test (Nautiyal, 1999). The
pH of the medium was adjusted to 7 before autoclaving and
small Petri dishes were used (diameter 5.5 cm). Dense bacterial
suspensions (OD 0.5–0.6) were made in 1 mL of PBS. Then,
10 µL of each suspension was spotted in the middle of each
plate. The test was performed in triplicate. The diameter of clear
halos around the bacterial growth was measured after 2, 4, 7, and
10 days of incubation at 28◦C. The strain R-42086 (Pseudomonas
sp.) was used as a positive control (Ghyselinck et al., 2013). After incubation, the plates were centrifuged at 20◦C,
3,700 rpm for 5 min. Then, 50 µL of the supernatant was
transferred to flat bottom 96-wells plate and 100 µL of
Salkowsky’s reagent (2% 0.5 M FeCl3 in 35% HClO4 solution)
was added and mixed by pipetting. The plate was then kept
in the dark for 30 min before measuring the optical density. A calibration curve was made with 0, 0.5, 1, 2, 5, 10, 15, 20, 25,
30 µg of IAA/mL. Enzymatic Activities Our isolates’ ability to recycle plant polysaccharides such as
cellulose, pectin and lignin was tested through phenotypic
enzymatic assays. Ligninolytic Activity This assay was conducted in two steps: a first screening on
minimum medium (4 g/L KH2PO4, 6 g/L Na2HPO4, 2 g/L
(NH4)2SO4, 1 g/L yeast extract, 0.001 g/L FeSO4, 0.2 g/L MgSO4,
0.001 g/L CaCl2, 0.00001 g/L H3BO3, 0.00001 g/L MnSO4,
0.00007 g/L ZnSO4, 0.0000 5 g/L CuSO4, 0.00001 g/L MoO3,
15 g/L agar, pH 7) supplemented with 5 g/L lignin and a second
test of selected bacteria on the same minimum medium with
25 mg/L of methylene blue. Siderophore Production To test the production of siderophores, the CAS (chrome azurol
S) blue medium was prepared according to Louden et al. (2011). Small Petri dishes were used (diameter 5.5 cm). Dense bacterial
suspensions (OD 0.5–0.6) were made in 1 mL of sterile PBS. Then, 10 µL of each suspension was spotted in the middle of each
plate. The test was performed in triplicate. The halo diameter was
observed and measured after 2, 4, 7, and 10 days of incubation
at 28◦C. The strain R-42086 (Pseudomonas sp.) was used as a
positive control (Ghyselinck et al., 2013). IAA Production Yeast malt extract medium (YM, peptone 5 g/L, yeast extract
3 g/L, malt extract 3 g/L, pH 6.2) was used to test for the
production of indole acetic acid (IAA) in liquid culture (Apine
and Jadhav, 2011; Mohite, 2013). Bacteria were grown in R2B
medium (Difco) for 72 h at 28◦C, 100 rpm. Then, 1.5 mL of
each bacterial culture was centrifuged (20◦C, 5 min, 14,000 rpm),
the supernatant was removed, and the pellet was resuspended
in sterile distilled water. Then, 100 µL of the suspension
were inoculated in 900 µL of YM medium with and without
tryptophan (1 g/L). The outermost wells of the plate were filled To screen for ligninolytic activity, dense bacterial suspensions
(OD 0.5–0.6) were made in sterile PBS. Then, 10 µL of each
bacterial suspension was spotted onto the middle of the plate. The experiment was conducted in triplicates and the plates
were incubated at 28◦C for 5 days before checking for bacterial
growth. Bacteria that were able to grow on lignin medium were
transferred two more times to the same medium to ensure that
survival was independent of the previous nutrient stock. After a total of three transfers, strains that retained the ability
to grow were selected to check for their ability to solubilize February 2021 | Volume 12 | Article 633488 Frontiers in Microbiology | www.frontiersin.org 4 Endophytic Bacteria From Alkanna tinctoria Rat et al. methylene blue. Structurally, methylene blue is very similar
to lignin and both compounds can be degraded by the same
enzymes. When methylene blue is degraded, it loses its color
and can therefore be used to identify lignin-degrading bacteria
(Bandounas et al., 2011). Dense bacterial suspensions were made
in sterile PBS. Then, 10 µL of each bacterial suspension was
spotted onto the middle of the plate. The plates were incubated
for 1 week at 28◦C and growth was checked daily. Bacteria able to
metabolize methylene blue are expected to produce bright halos
around their colonies. and transferred weekly (for a period of 3–4 weeks) to a new
MS solid medium containing 1% sucrose, 0.8% phytoagar, and
500 mg/L of cefotaxime. The roots were then propagated in liquid
MS medium supplemented with 3% sucrose (Brigham et al., 1999;
Fukui et al., 1999; Kim et al., 2009). To evaluate the ability of bacteria to induce A/S production in
the hairy roots of A. Susceptibility Assay Susceptibility assays were conducted to assess the sensitivity of
A. tinctoria endophytic bacteria isolates to the antimicrobial
effects of A/S derivatives. The diffusion assay using filter
disks described by Bauer et al. (1966) was performed. The
concentration tested was 50 µg per filter disk, following
Brigham et al. (1999). A mixture of alkannin/shikonin and
their derivatives (A/S mixture) was isolated after extraction and
further fractionation steps from A. tinctoria roots, as described
in Assimopoulou et al. (2008) and Tappeiner et al. (2013). The
mixture was first solubilized in acetone to a concentration of
46.7 mg/mL and then diluted in sterile water to a concentration
of 2.5 mg/mL. Then, 20 µL of each test solution were used to
inoculate the filter disks. In total, six filter disks were used in this
assay: three were inoculated with the A/S mixture (50 µg/filter
disk), one with acetone-water solution (5.9% acetone; used as an
A/S mixture solvent control), one with ampicillin (50 µg/filter
disk; as a control antibiotic), and one with MilliQ water (as
ampicillin solvent control). Dense bacterial suspensions were
prepared in PBS and 100 µL of each suspension was inoculated
by spreading on R2A plates. Individual filter disks containing
20 µL of each test solution were left to dry for 30 min before
being transferred onto the plates. The test was performed in
triplicate. Control strains were selected based on the results
reported by Brigham et al. (1999): LMG 8224 (Staphylococcus
aureus subsp. aureus) and LMG 7135 (Bacillus subtilis subsp. subtilis) as sensitive strains and LMG 8223 (Escherichia coli) as
a resistant strain. p
Among seven candidates showing an OD > 0.2 at 520 nm
in the first screening, four were subsequently tested in a larger
volume. Five 100 mL Erlenmeyer filled with 50 mL of MS + 1%
sucrose medium were used per treatment. Pieces of young roots
were then carefully added in each Erlenmeyer and incubated
for 1 week at 25◦C (100 rpm). The Erlenmeyers were then
randomized before being inoculated with bacteria to a final OD of
0.001. One uninoculated control (5 replicates) was also prepared. Erlenmeyers were incubated for 1 week at 25◦C (100 rpm). The
roots from each Erlenmeyer were collected and lyophilized. The
extraction was conducted overnight using 40 mg of dried roots
in 1,250 µL of methanol. Isolation and Identification of Root
Endophytic Bacteria For our study nine different populations of endogenous Alkanna
tinctoria from Northern and Southern Greece have been
explored. The first isolation campaign assessed the media that
would allow the growth of a wide diversity of culturable
bacteria and yielded 1,483 isolates. MALDI-TOF mass spectra
of these isolates were compared and grouped, resulting in
914 representative strains that were selected for genotypic
identification by 16S rRNA gene sequencing. This allowed
identification of 142 distinct phylotypes of endophytic bacteria
following the classification threshold values at genus or species
level introduced by Moreira and López-García (2014) and
Yarza et al. (2014). The number of isolates and phylotypes
recovered for each plant sample and medium is available in
Supplementary Table S1. The coverage of bacterial diversity for
each medium tested was obtained by dividing the number of
unique phylotypes from the selected medium by the total number
of phylotypes. The media that yielded the highest diversity were
medium 1/10 869 supplemented with 32 mM allantoin and IAA Production tinctoria, a first screening was performed in
6-well plates. The bacteria for this test were selected based either
on their plant growth promoting properties or their enzymatic
activities. Firstly, 7 mL of MS + 1% sucrose medium were added
to each well, followed by the aseptic addition of young root
segments. Bacteria were inoculated to a final OD of 0.001/mL. One 6-well plate was prepared per strain. In addition, an
uninoculated control plate was prepared. Plates were incubated
in the dark at 100 rpm agitation at 25◦C for 1 week. The roots
from individual wells were pooled per plate and lyophilized. The
extraction of A/S was conducted overnight with 8 mg of dried
roots in 250 µL of methanol and the OD was read at 520 nm
(Assimopoulou et al., 2009). Susceptibility Assay The OD was read at 520 nm and one-
factor Anova followed by a Neuman-Keuls test was conducted on
the results with the software R x64 3.1.0. Frontiers in Microbiology | www.frontiersin.org Plant Growth-Promoting Activity Plant Growth Promoting Activity
One hundred and twenty-seven strains which represented most
of the bacterial diversity recovered, were tested for their
plant growth promoting activity in vitro. The results are
summarized in Figure 2 (additional information is provided in
the Supplementary Table S2). The strains expressing positive
activity for all four tested parameters belonged to the genera
Pseudomonas and Bacillus. The ones expressing positive activity
for at least three of the parameters tested belonged to the genera
Pseudomonas, Pantoea, Inquilinus, Rhizobium, and Bacillus. On
the other hand, bacteria from the genus Stenotrophomonas
seem to only be able to produce siderophores. The partial 16S
sequences of these 127 tested strains are available in GenBank
database under the accession numbers MW353471–MW353597
(Supplementary Table S3). Induction of A/S Production in Hairy
Roots of A. tinctoria Members
the genera Pseudomonas were found in eight of the nin
TABLE 1 | Coverage of bacterial diversity of different media, where the number o
unique phylotypes in the selected medium was divided by the total number of
detected phylotypes is given in percentage (%). Medium
Coverage of the diversity in
1/10 869
30.3
1/10 869 + 0.32 mM allantoin
32.6
1/10 869 + 1.6 mM allantoin
28,0
1/10 869 + 9 mM allantoin
31.8
1/10 869 + 32 mM allantoin
47.0
1/10 869 + Plant extract
8.3
ISP2
22.7
1/10 TSA
40.2
R2A
35.6
Frontiers in Microbiology | www.frontiersin.org The second and third isolation campaigns using four media
only yielded 770 and 582 isolates, respectively. Together
with the isolates from the first campaign, this gave 2835
primary bacterial isolates in total. Protein profiles of all
these
isolates
were
compared
by
MALDI-TOF
MS
and
grouped. This allowed the selection of 1,428 representative
strains, 914 of which had already been identified by 16S
rRNA gene sequencing in the first isolation campaign. The
remaining 514 representative strains were also identified
by 16S rRNA gene sequencing. In total, among the 1,428
selected representative strains, 197 unique phylotypes were
recovered, belonging to 14 genera of Actinobacteria, 6 genera
of Bacteroidetes, 11 genera of Firmicutes, and 44 genera of
Proteobacteria. Also, three potentially new genera and 40
potentially new species were recovered from the roots of
wild A. tinctoria plants. The bacteria were distributed in
four phyla, i.e., Bacteroidetes, Proteobacteria, Actinobacteria,
and Firmicutes. The plants of the third isolation campaign
yielded relatively more Gram positive bacteria (Actinobacteria
and Firmicutes) compared to the other plants. Similarly, the
plants of the 1st and 2nd isolation campaigns yielded more
Alphaproteobacteria and Gammaproteobacteria, respectively
(Supplementary Figure S2). Most of the isolates belonged
to the Proteobacteria, particularly the Gammaproteobacteria,
Alphaproteobacteria,
and
Betaproteobacteria. Members
of
the genera Pseudomonas were found in eight of the nine Enzymatic Activities Enzymatic activities of the 127 strains are presented in Figure 2
(additional information is provided in Supplementary Table S2). The cellulase activity seemed to be predominant in the
Rhizobiaceae family, whereas pectinase activity was abundant
among the Bacteroidetes phylum. Among the strains tested,
some showed two degrading activities: pectinase and ligninolytic
activities were detected for R-72191 (Olivibacter sp.), R-72210
(Pseudomonas sp.), R-72249 (Pedobacter sp.), R-72394 (Pantoea
sp.), R-72464 (Xanthomonas sp.), R-7264, and R-74277 (Bacillus
sp.); cellulase and pectinase activities for the strain R-71830
(Brevibacillus sp.) and cellulase and ligninolytic activities for
R-72157 (Rhizorhapis sp.). TABLE 1 | Coverage of bacterial diversity of different media, where the number of
unique phylotypes in the selected medium was divided by the total number of
detected phylotypes is given in percentage (%). Medium
Coverage of the diversity in %
1/10 869
30.3
1/10 869 + 0.32 mM allantoin
32.6
1/10 869 + 1.6 mM allantoin
28,0
1/10 869 + 9 mM allantoin
31.8
1/10 869 + 32 mM allantoin
47.0
1/10 869 + Plant extract
8.3
ISP2
22.7
1/10 TSA
40.2
R2A
35.6 TABLE 1 | Coverage of bacterial diversity of different media, where the number of
unique phylotypes in the selected medium was divided by the total number of
detected phylotypes is given in percentage (%). Medium
Coverage of the diversity in %
1/10 869
30.3
1/10 869 + 0.32 mM allantoin
32.6
1/10 869 + 1.6 mM allantoin
28,0
1/10 869 + 9 mM allantoin
31.8
1/10 869 + 32 mM allantoin
47.0
1/10 869 + Plant extract
8.3
ISP2
22.7
1/10 TSA
40.2
R2A
35.6
Frontiers in Microbiology | www frontiersin org
6 TABLE 1 | Coverage of bacterial diversity of different media, where the number of
unique phylotypes in the selected medium was divided by the total number of
detected phylotypes is given in percentage (%). Induction of A/S Production in Hairy
Roots of A. tinctoria Hairy roots of A. tinctoria were produced with a modified
protocol (Brigham et al., 1999; Fukui et al., 1999; Kim et al.,
2009) as follows: Agrobacterium rhizogenes strain LMG 149 was
grown in 40 mL Nutrient Broth (Difco) medium at 28◦C for
48 h in the dark and at 100 rpm agitation. The leaves of
sterile A. tinctoria explants provided by IPBGR, HAO Demeter
(Thessaloniki, Greece) were cut and inoculated at different spots
with a syringe filled with a bacterial solution of A. rhizogenes
(OD = 0.5–0.6). Each inoculated leaf was transferred onto solid
Murashige and Skoog (MS, Duchefa) medium supplemented
with 3% sucrose and 0.8% phytoagar (Duchefa) and kept for 1
week at 25◦C in the dark until roots appeared. As a control,
non-inoculated leaves were cultivated under the same conditions. Developing roots (approximately 1 cm in length), were cut off February 2021 | Volume 12 | Article 633488 Frontiers in Microbiology | www.frontiersin.org 5 Endophytic Bacteria From Alkanna tinctoria Rat et al. Rat et al. medium 1/10 TSA (Table 1). Moreover, media R2A, 1/10 869
and 1/10 869 supplemented with 0.32 mM allantoin allowed
the cultivation of members of additional genera including
Acidovorax, Micromonospora, Kocuria, Diaminobutyricimonas,
and Muciliginibacter (Supplementary Figure S1). plants studied, whereas Pantoea, Xanthomonas and Bacillus
were recovered from seven A. tinctoria plants. Additionally,
representatives of Stenotrophomonas were found in six of the
nine plants (Figure 1). These genera were also isolated from
all the tested media (1/10 869, 1/10 869 + 32 mM allantoin,
1/10 TSA and R2A). The isolates that were less than 94.8%
similar to the type strain of any bacterial species represent
four potentially new genera which are related to Filimonas,
Bordetella, Cohnella and Paenibacillus. Moreover, 40 bacterial
strains that were less than 98.7% similar to the type strain
of any bacterial species probably represent potential new
species. Of these, members of the Bacteroidetes are related to
Chitinophaga, Flavobacterium, Mucilaginibacter, and Pedobacter. Potentially new species within the Phylum Firmicutes are
related to Bacillus, Cohnella, Domibacillus, Paenibacillus, and
Tumebacillus. Potentially new Proteobacteria isolates are related
to Acinetobacter, Allorhizobium, Beijerinckia, Bradyrhizobium,
Brevundimonas, Janthinobacterium, Massilia, Ochrobactrum,
Pseudomonas, Roseomonas, Rhizobium, Rhizorhapis, Shinella,
Sphingobium, Sphingomonas, and Stenotrophomonas. Finally,
potentially new isolated Actinobacteria members are related to
the genera Cellulomas, Conexibacter, Diaminobutyricimonas,
Lysinimonas, Mycobacterium, and Nocardioides. medium 1/10 TSA (Table 1). Induction of A/S Production in Hairy
Roots of A. tinctoria Moreover, media R2A, 1/10 869
and 1/10 869 supplemented with 0.32 mM allantoin allowed
the cultivation of members of additional genera including
Acidovorax, Micromonospora, Kocuria, Diaminobutyricimonas,
and Muciliginibacter (Supplementary Figure S1). For the isolation from the second and third collection
campaigns, the media R2A, 1/10 TSA, 1/10 869 and 1/10
869 + 32 mM allantoin were used to maximize the culturable
diversity. Moreover, the first isolation campaign indicated that
extended incubation at 20◦C led to a 15.2% increase in diversity at
higher dilutions. Most of these isolates were slow-growing Gram-
positive bacteria including Mycobacterium sp., Micromonospora
sp., Lysinimonas sp., and Micrococcus sp. Then in further
isolations, the plates were incubated at 20◦C for 2 weeks and
colonies were picked after 1 and 2 week(s), respectively. positive bacteria including Mycobacterium sp., Micromonospo
sp., Lysinimonas sp., and Micrococcus sp. Then in furth
isolations, the plates were incubated at 20◦C for 2 weeks an
colonies were picked after 1 and 2 week(s), respectively. The second and third isolation campaigns using four med
only yielded 770 and 582 isolates, respectively. Togeth
with the isolates from the first campaign, this gave 283
primary bacterial isolates in total. Protein profiles of a
these
isolates
were
compared
by
MALDI-TOF
MS
an
grouped. This allowed the selection of 1,428 representativ
strains, 914 of which had already been identified by 16
rRNA gene sequencing in the first isolation campaign. Th
remaining 514 representative strains were also identifie
by 16S rRNA gene sequencing. In total, among the 1,42
selected representative strains, 197 unique phylotypes we
recovered, belonging to 14 genera of Actinobacteria, 6 gene
of Bacteroidetes, 11 genera of Firmicutes, and 44 genera
Proteobacteria. Also, three potentially new genera and 4
potentially new species were recovered from the roots
wild A. tinctoria plants. The bacteria were distributed
four phyla, i.e., Bacteroidetes, Proteobacteria, Actinobacteri
and Firmicutes. The plants of the third isolation campaig
yielded relatively more Gram positive bacteria (Actinobacter
and Firmicutes) compared to the other plants. Similarly, th
plants of the 1st and 2nd isolation campaigns yielded mo
Alphaproteobacteria and Gammaproteobacteria, respective
(Supplementary Figure S2). Most of the isolates belonge
to the Proteobacteria, particularly the Gammaproteobacteri
Alphaproteobacteria,
and
Betaproteobacteria. Susceptibility Assay Susceptibility Assay Susceptibility to the antimicrobial activity of alkannin and
shikonin derivatives (in the form of a mixture of A/S pigments) February 2021 | Volume 12 | Article 633488 6 Rat et al. Endophytic Bacteria From Alkanna tinctoria was studied for the 127 strains tested for PGP and enzymatic
activities. A strain was considered as sensitive to A/S derivatives
when an inhibition zone of more than 1 mm was observed. An
example of the assay is shown in Figure 3. A pattern could be
observed in the susceptibility to the A/S mixture (50 µg): all
Gram-positive bacteria tested were sensitive whereas the Gram-
negative bacteria tested were resistant (Figure 2). No such pattern
was observed for ampicillin which was used as a control. As
shown in Figure 3, the halos observed as a result of sensitivity
to A/S derivatives were always smaller in diameter than those
produced as a result of ampicillin sensitivity. FIGURE 1 | Diversity of genera isolated from wild-growing Alkanna tinctoria
plants and the number of plant individuals in which they were found. The
number of species found per genus is specified in parentheses. Different
colors correspond to specific bacterial phyla or classes. Induction of A/S Derivatives in Hairy
Roots of A. tinctoria In the first screening, treatments with the following bacteria
resulted in hairy root extracts showing an OD higher than
the control: R-71971 (Brevibacillus sp.), R-72060 (Variovorax
sp.), R-72149 (Filimonas sp.), R-72379 (Allorhizobium sp.),
R-72395 (Duganella sp.), R-72401 (Shinella sp.), R-72406
(Stenotrophomonas sp.), R-73072 (Chitinophaga sp.), R-74161
(Acinetobacter sp.), and R-75348 (Micromonospora sp.) (Table 2). The seven bacteria resulting in an OD > 0.20 were repeated
in five replicates in a second assay. The one-factor ANOVA
conducted on the results of the second assay showed that the
p-value = 0.00179 was lower than 0.05. The bacterial treatment
had an influence on the A/S content. The comparison made with
the test of Newman Keuls are presented Table 3. Chitinophaga
sp. strain R-73072, Allorhizobium sp. strain R-72379, Massilia sp. strain R-72395, and Micromonospora sp. strain R-75348 induced
significantly more A/S derivatives in the hairy roots of A. tinctoria
when compared to the uninoculated control. DISCUSSION The cultivation of members of Boraginaceae with strong
medicinal value such as A. tinctoria and Lithospermum spp. has proven to be difficult. For example, Lithospermum spp. germinate poorly and seedlings are highly susceptible to viral
infections. Additionally, plants can only be harvested 2–3 years
after germination and the in-field maintenance of these plants
has also proved to be difficult (Yazaki, 2017). Applying suitable
endophytes to facilitate plant growth by increasing the plant
biomass and/or by inducing A/S production can be a sustainable
approach toward increasing plant yield and production of these
valuable compounds. As a first step toward the elucidation of the potential role
of root endophytic bacteria in the production for secondary
metabolites
such
as
A/S
derivatives
of
A. tinctoria,
we
isolated and characterized culturable endophytic bacteria from
A. tinctoria roots. In total, 197 unique phylotypes were recovered,
representing 4 phyla. Also, three potentially new genera and 40
potentially new species were recovered from the roots of wild
A. tinctoria plants. Most studies to date involving the isolation
of endophytic bacteria typically use one type of isolation medium
to cultivate a wide range of microorganisms (e.g., nutrient agar or
TSA), a set temperature of 28◦C and an incubation time between FIGURE 1 | Diversity of genera isolated from wild-growing Alkanna tinctoria
plants and the number of plant individuals in which they were found. The
number of species found per genus is specified in parentheses. Different
colors correspond to specific bacterial phyla or classes. February 2021 | Volume 12 | Article 633488 Frontiers in Microbiology | www.frontiersin.org 7 Rat et al. Endophytic Bacteria From Alkanna tinctoria FIGURE 2 | Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences, with Juke-Cantor model, showing the phylogenetic relationships of strains
tested in vitro for plant growth, cell-wall degrading enzymes and susceptibility to the mixture of A/S. Activities detected are shown in the outer circle. Sequences
were aligned using 361 CLC Main Workbench 7.9.1 (Qiagen). Subsequently, a phylogenetic maximum-likelihood tree (1000 bootstraps) was reconstructed and
visualized using the iTOL software 5.6.3 (Letunic and Bork, 2019). FIGURE 2 | Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences, with Juke-Cantor model, showing the phylogenetic relationships of strains
tested in vitro for plant growth, cell-wall degrading enzymes and susceptibility to the mixture of A/S. Activities detected are shown in the outer circle. Sequences
were aligned using 361 CLC Main Workbench 7.9.1 (Qiagen). DISCUSSION Subsequently, a phylogenetic maximum-likelihood tree (1000 bootstraps) was reconstructed and
visualized using the iTOL software 5.6.3 (Letunic and Bork, 2019). 48 and 72 h (Germida et al., 1998; Mbai et al., 2013; Anjum and
Chandra, 2015; Saini et al., 2015). In this study, we showed that
a higher diversity of bacteria can be obtained when more than
one medium is used. Nutrient-poor media such as R2A or diluted
TSA seem better at capturing more diversity than a rich medium
such as ISP2 (Table 1 and Supplementary Figure S1). The latter
is a medium rich in nitrogen and carbon sources and has been
designed for the routine cultivation of Streptomyces sp. (Shirling
and Gottlieb, 1966). However, as it may favor the fast-growing bacteria, it may be less suitable to isolate slow growing ones. R2A medium was designed for slow-growing bacteria (Reasoner
and Geldreich, 1985) and medium 1/10 869 for endophytes
(Eevers et al., 2015), whereas TSA medium is non-selective and
allows the growth of a wide range of microorganisms (Leavitt
et al., 1958). The dilution of TSA increased the growth of slow-
growing bacteria when compared to the non-diluted medium. Changes in the quantity and the quality of the carbon source
and the nature of electrons donors (e.g., succinate, butyrate, February 2021 | Volume 12 | Article 633488 Frontiers in Microbiology | www.frontiersin.org 8 Endophytic Bacteria From Alkanna tinctoria Rat et al. FIGURE 3 | Susceptibility of strain R-72492 (Bacillus sp.) to the mixture of
alkannin/shikonin derivatives isolated from Alkanna tinctoria roots. parameters such as the genetics of the plant (Haney et al.,
2015), local soil composition (Liu et al., 2019), or the presence
of neighboring plants (Schlatter et al., 2015; Essarioui et al.,
2016) might also influence the diversity of bacteria recovered. Microbiome analysis as well as the study of the growth conditions
of the plant in its natural environment might shed light on
bacterial variation between samples. The endophytic bacteria found in most of the root samples
were tested for a number of plant-growth promotion (PGP)
characteristics. The most interesting species for plant growth
promoting activity belong to the genera Pseudomonas, Pantoea,
Inquilinus, Rhizobium, and Bacillus (Figure 2, Supplementary
Table S2). Members of these genera were recovered on all
media used for isolation. The ability to grow easily in vitro
is promising for their application as plant growth promoting
rhizobacteria (PGPRs). DISCUSSION Some bacteria showed variable results
were obtained from the ACC deaminase test (Supplementary
Table S2). These bacteria were colored and/or were prone to form
biofilms or clumps which can lead inaccurate spectrophotometric
readings. To overcome this issue, quantitative analysis might be
applied by measuring the amount of α-ketobutyrate at 540 nm
(Penrose and Glick, 2003). FIGURE 3 | Susceptibility of strain R-72492 (Bacillus sp.) to the mixture of
alkannin/shikonin derivatives isolated from Alkanna tinctoria roots. Moreover,
several
bacteria
produced
in
vitro
cell-wall
degrading enzymes such as cellulases, pectinases and ligninases. These properties facilitate bacterial entry and spread within the
plant tissues and may contribute to the endophytic lifestyle of
these bacteria. Strains belonging to Pseudomonas sp., Pantoea sp.,
Rhizobium sp., and Bacillus sp. expressing PGP and enzymatic
activities (Figure 2 and Supplementary Table S2) might thus
show stronger impact on plant growth or A/S induction due to
their ability to actively colonize the plant. acetate) and acceptors (HEPES solution; Köpke et al., 2005), as
well as the addition of supplements like enzymes or antibiotics to
isolation media are known to generate higher microbial diversity
(Alain and Querellou, 2009). For example, the nitrogen sources
of TSA medium are casein and peptones whereas R2A and
869 media contain yeast extract. Moreover, TSA does not have
additional carbon sources such as glucose. Poor media limit the
risk that fast-growing bacteria eliminate those that grow slower
by outcompeting them for nutrients or by negatively affecting
the growth conditions (pH or the production of metabolites). Critically, slow-growers or species that grow poorly in vitro
may be naturally abundant in planta (Alain and Querellou,
2009; Kram and Finkel, 2015). Lower temperature and longer
incubation time were also more suited to recovering gram-
positive bacteria such as Actinobacteria. Indeed, studies focusing
on Actinobacteria demonstrated that an incubation period of 3–
4 weeks was necessary for their isolation (Araújo et al., 2000;
Coombs and Franco, 2003; Misk and Franco, 2011). The resistance assay demonstrated that Gram-negative
bacteria are resistant to the antimicrobial properties of A/S
derivatives (Figure 2 and Supplementary Table S2). Although
the activity of A/S derivatives at 50 µg seemed weak, the
experiment confirmed the antimicrobial properties of these
naphthoquinones. The antimicrobial activity of A/S has been
linked to their ability to form semiquinone radicals by
interacting with reactive oxygen species. Frontiers in Microbiology | www.frontiersin.org DISCUSSION 0.115
R-72097
Caulobacter sp. 0.163
R-72501
Inquilinus sp. 0.146
R-72135
Pseudomonas sp. 0.132
R-73072
Chitinophaga sp. 0.200
R-72149
Filimonas sp. 0.180
R-73074
Bradyrhizobium sp. 0.170
R-72160
Rhizobium sp. 0.162
R-73098
Xanthomonas sp. 0.129
R-72191
Olivibacter sp. 0.167
R-73110
Luteibacter sp. 0.124
R-72210
Pseudomonas sp. 0.157
R-74161
Acinetobacter sp. 0.223
R-72249
Pedobacter sp. 0.153
R-74611
Paenarthrobacter sp. 0.162
R-72251
Xanthomonas sp. 0.142
R-75348
Micromonospora sp. 0.231
The values in bold are higher than the OD value of the uninoculated control. 520 nm reflecting the total A/S content of hairy roots extracts of Alkanna tinctoria treated with selected bacteria. The values in bold are higher than the OD value of the uninoculated control. TABLE 3 | Comparisons of the OD 520 nm reflecting the total A/S content of hairy
roots extracts of Alkanna tinctoria inoculated with the best bacterial candidates
with Neuman Keuls test. Strain
Identification
OD 520 nm
Control
0.075 b
R-73072
Chitinophaga sp. 0.120 a
R-72379
Allorhizobium sp. 0.113 a
R-75348
Micromonospora sp. 0.108 a
R-72395
Massilia sp. 0.106 a
R-72060
Variovorax sp. 0.097 ab
R-74161
Acinetobacter sp. 0.092 ab
R-71971
Brevibacillus sp. 0.090 ab
Significant differences are highlighted in bold with different letters. individuals and bacteria in the presence of alkannins and
shikonins. Moreover, it has been shown that ethylene regulates
the colonization of plant tissue by bacteria: the absence of
ethylene in a plant or the addition of an ethylene inhibitor leads to
a higher degree of colonization. Bacteria that are able to affect the
ethylene level are more competent at colonizing plants (Hardoim
et al., 2008; Liu et al., 2017). The modulation of plant ethylene
levels by bacteria can occur by cleavage of 1-aminocyclopropane-
1-carboxylate (ACC), a precursor of ethylene or by inhibiting
ACC synthase and/or β-cystathionase, both being enzymes
involved in the ethylene biosynthesis pathway (Hardoim et al.,
2008; Liu et al., 2017). To confirm whether bacteria can effectively
colonize and positively affect the plant growth and the total A/S
content, in planta tests must be performed. Significant differences are highlighted in bold with different letters. p
p
Chitinophaga sp. strain R-73072, Allorhizobium sp. strain R-
72379, Massilia sp. strain R-72395, and Micromonospora sp. strain
R-75348 showed the ability to directly induce A/S production
in A. tinctoria hairy root cultures. Hairy roots are genetically
modified plant material (Gutierrez-Valdes et al., 2020). DISCUSSION They can generate
endogenous
superoxide
anion
radicals,
resulting
in
their
cytotoxicity (Papageorgiou et al., 2008, 1999; Ordoudi et al.,
2011). The peptidoglycan cell wall of the Gram-positive bacteria
is permeable to molecules with molecular weights in the range
of 30,000–57,000 Da, and hence allows for the entry of many
small antimicrobials which may partially explain their sensitivity
(Lambert, 2002). On the other hand, several mechanisms might
be involved in the resistance of the Gram-negative bacteria
to A/S, especially through redox processes. Chemical and
transcriptomic analyses may provide a better understanding of
the processes involved. Most of the bacteria having a positive
effect in the in vitro plant growth promoting activities tested
were Gram-negative and thus resistant to A/S derivatives. This
resistance may provide a competitive advantage for colonizing
the plant and living in the root tissues. Consequently, the plant
may select for plant growth promoting bacteria through the
production of A/S derivatives. Alternatively, sensitive bacteria
may colonize the plant shortly after germination when the A/S While the isolation conditions are important determining
factors for the recovery of bacterial isolates, the diversity observed
here between different roots (Figure 1 and Supplementary
Figure S2) indicates that other factors are also important. For
example, no Gammaproteobacteria such as Pseudomonas sp.,
Xanthomonas sp., or Pantoea sp. seemed to be present in
one of the root samples (Figure 1). Bacterial isolation results
can be biased by the origin and storage conditions of the
biological sample or the colony picking method, in addition
to the isolation conditions, which may have contributed to the
variation in phylotype distribution observed between roots and
isolation campaigns (Supplementary Figure S2). We studied
wild-collected plants from different locations and therefore, February 2021 | Volume 12 | Article 633488 9 Endophytic Bacteria From Alkanna tinctoria Rat et al. TABLE 2 | Optical density at 520 nm reflecting the total A/S content of hairy roots extracts of Alkanna tinctoria treated with selected bacteria. Strain
Identification
OD 520 nm
Strain
Identification
OD 520 nm
Control
0.175
R-72269
Chitinophaga sp. 0.166
R-71838
Pseudomonas sp. 0.124
R-72367
Achromobacter sp. 0.158
R-71842
Pseudomonas sp. 0.167
R-72379
Allorhizobium sp. 0.222
R-71875
Bacillus sp. 0.163
R-72395
Massilia sp. 0.201
R-71922
Bacillus sp. 0.160
R-72401
Shinella sp. 0.192
R-71971. Brevibacillus sp. 0.244
R-72406
Stenotrophomonas sp. 0.189
R-72039
Muciliginibacter sp. 0.149
R-72433
Rhizobium sp. 0.170
R-72060
Variovorax sp. 0.207
R-72495
Variovorax sp. 0.172
R-72074
Pseudomonas sp. 0.138
R-72498
Pantoea sp. Frontiers in Microbiology | www.frontiersin.org DISCUSSION Alkannin and shikonin derivatives show
antimicrobial activities and can thus be considered as plant
defense compounds. these medicinal plants by increasing the total biomass. As
demonstrated in our study, certain endophytes can also induce
A/S production in the roots, probably through to the recognition
of bacteria by the plant. These findings open-up the perspective
of using a combination of endophytes with the potential for
plant growth promotion and induction of secondary metabolite
production as a sustainable approach toward increasing the
production of secondary metabolites in selected medicinal plants. Although R-75348 did not show any cell-wall degrading
activities in vitro, several studies have demonstrated that
Micromonospora species do have a role in the breakdown of plant
cell walls through the production of hydrolytic enzymes (Hirsch
and Valdés, 2010; Trujillo et al., 2015). Our test conditions
may not have been suitable for this strain. Alternatively,
genome analysis might reveal the presence of such enzymes. Moreover, Micromonospora sp. is also known to produce
antibiotics (Boumehira et al., 2016). Inducing the production of
A/S derivatives may depend on plant-bacteria communication
through such metabolites. Indeed, some molecules are described
as antibiotics because of their effect on microorganisms under
in vitro conditions although their function in the natural habitat
can be different. Such molecules can play a role in plant-microbe
interactions but can also act as Microbe-Associated Molecular
Patterns (MAMPs). MAMPs are resistance-inducing stimuli
recognized by specific plant receptors (Köhl et al., 2019). They
can also induce pathways involved in plant defense responses. ACKNOWLEDGMENTS We would like to thank our colleague Stephanie Fordeyn for
language advise and proofreading of the manuscript. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found
online
at:
https://www.frontiersin.org/articles/10.3389/fmicb. 2021.633488/full#supplementary-material DISCUSSION The use
of MS + 1% sucrose medium, which is a poor medium for
bacterial culture, as well as the presence of agitation, might
affect the ability of the bacteria to colonize the roots or their
abilities to produce biofilm and/or metabolites. During plant
colonization, the production of bacterial enzymes or metabolites
may induce the production of A/S derivatives by the plant. Our hairy root system served as a general screening tool and
may be not optimized for specific plant-bacteria interactions. It might therefore underestimate the number of A/S inducers. Nonetheless, in vitro whole plant systems usually employ growth
medium similar to MS (Phillips and Garda, 2019) and therefore
bacteria showing inducing properties in hairy roots can likely
induce A/S in vitro plant system. The bacterial strains R-73072,
R-72379, and R-72395 inducing A/S in our system, expressed
cell-wall degrading enzymatic activities (pectinase or ligninase) derivatives content is low or they may be present in the seed and
are vertically transmitted (Truyens et al., 2014). In the future,
these hypotheses might be tested by inoculating isolates that
are sensitive to A/S at different plant growth stages and by
tracking their presence and abundance within the plant, thus
establishing whether or not these strains can colonize and survive
in the plant or not. Studies have shown that in the hairy roots of A. tinctoria,
A/S derivatives are produced by root border cells of the growing
root tips. They are then sequestered as lipid granules in the
phospholipid layer of these cells and accumulate in apoplastic
spaces. However, how the A/S derivatives are secreted into the
environment still remains unclear. Nevertheless, it is known
that this process results from plant stress and involves the actin
filaments (Brigham et al., 1999; Tatsumi et al., 2016). Depending
on where the bacteria colonize the plant, it is possible that they
do not come into contact with A/S derivatives. Colonization
assays can provide insights into the relationship between plant February 2021 | Volume 12 | Article 633488 10 Endophytic Bacteria From Alkanna tinctoria Rat et al. in vitro. Competent endophytes are able to release cell wall
degrading enzymes such as cellulases, xylanases, pectinases,
and endoglucanases, which facilitate bacterial entry and spread
within the plant tissues (Kandel et al., 2017; Pinski et al., 2019). Nonetheless, colonization remains an invasive process and thus
might induce plant defense response, resulting in the synthesis
of plant antimicrobials. DATA AVAILABILITY STATEMENT The
datasets
presented
in
this
study
can
be
found
in
online repositories. The names of the repository/repositories
and
accession
number(s)
can
be
found
in
the
article/
Supplementary Material. FUNDING We hypothesize that the endophyte’s production of cell-wall
degrading enzymes and/or secondary metabolites during plant
colonization may cause a plant stress resulting in the production
of alkannin and shikonin derivatives. This project (Micrometabolite) has received funding from the
European Union’s Horizon 2020 Research and Innovation
Programme under the Marie Skłodowska-Curie grant agreement
no. 721635. This study demonstrated the importance of the isolation
conditions for the diversity of endophytic isolates recovered from
plant roots of A. tinctoria. Nutrient-poor isolation media coupled
with an incubation temperature of 20◦C and an incubation period
of a minimum of 2 weeks allowed for a recovery of a high
diversity of culturable endophytic bacteria from A. tinctoria. Although these findings should be validated in greenhouse or
field conditions, the positive in vitro results found regarding
potential plant growth promotion features suggest that some of
these bacteria might be valuable for future in planta applications. These can be of agronomical interest to boost biomass production
or crop yield. Such endophytes also have the potential to increase
the yield of secondary metabolites such as A/S derivatives from AUTHOR CONTRIBUTIONS AW, CS, AAs, and NF designed the study and secured the
funding. AR, HN, and AAl designed and performed the
experiments. AR analyzed the data and wrote the manuscript. All
authors edited, proofread, and approved the manuscript. REFERENCES 12, 53–57. doi: 10.1007/s12892-009-
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absence of any commercial or financial relationships that could be construed as a
potential conflict of interest. Yazaki, K. (2017). Lithospermum erythrorhizon cell cultures: present and future
aspects. Plant Biotechnol. 34, 131–142. doi: 10.5511/plantbiotechnology.17. 0823a Copyright © 2021 Rat, Naranjo, Krigas, Grigoriadou, Maloupa, Alonso, Schneider,
Papageorgiou, Assimopoulou, Tsafantakis, Fokialakis and Willems. Yu, P., and Hochholdinger, F. (2018). The role of host genetic signatures on
root–microbe interactions in the rhizosphere and endosphere. Front. Plant Sci.
9:1896. doi: 10.3389/fpls.2018.01896 Frontiers in Microbiology | www.frontiersin.org REFERENCES This is an open-
access article distributed under the terms of the Creative Commons Attribution
License (CC BY). The use, distribution or reproduction in other forums is permitted,
provided the original author(s) and the copyright owner(s) are credited and that the
original publication in this journal is cited, in accordance with accepted academic
practice. No use, distribution or reproduction is permitted which does not comply
with these terms. Yoon, S.-H., Ha, S.-M., Kwon, S., Lim, J., Kim, Y., Seo, H., et al. (2017). Introducing
EzBioCloud: a taxonomically united database of 16S rRNA gene sequences
and whole-genome assemblies. Int. J. Syst. Evol. Microbiol. 67, 1613–1617. doi:
10.1099/ijsem.0.001755 Yu, P., and Hochholdinger, F. (2018). The role of host genetic signatures on
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Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah
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Volume 5 Issue 1 (2021) Pages 879-890
Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini
ISSN: 2549-8959 (Online) 2356-1327 (Print) Abstrak Penelitian ini bertujuan untuk mengetahui pengaruh pemberian bermain vegetable eating
motivation (VEM) terhadap perilaku makan sayur pada anak prasekolah di TK Asma Jaya
Kecamatan Lasolo Kabupaten Konawe Utara. Jenis penelitian ini adalah eksperimental dengan
pendekatan pre and post test without control. Populasi penelitian adalah seluruh anak
prasekolah TK Asma Jaya Kecamatan Lasolo Kabupaten Konawe Utara yang berjumlah 16
anak. Teknik pengambilan sampel menggunakan total sampling, metode analisis
menggunakan Uji Wilcoxon. Penelitian ini dilaksanakan pada bulan Juli-Agustus 2018. Hasil
penelitian menunjukkan bahwa ada pengaruh pemberian bermain VEM terhadap perilaku
makan sayur pada anak prasekolah di TK Asma Jaya Kabupaten Konawe Utara. Diharapkan
kepada orang tua agar dapat memberikan gambaran tentang cara pemberian motivasi dan
edukasi pada anak dengan pendekatan bermain VEM agar anak memiliki perilaku makan
sayur yang baik. Kata Kunci : vegetable eating motivation; perilaku makan sayur; anak prasekolah Bermain Vegetable Eating Motivation (Vem) terhadap
Perilaku Makan Sayuran pada Anak Prasekolah Islaeli Islaeli1, Ari Nofitasari2, Sri Wulandari3
Program Studi Keperawatan, Stikes Mandala Waluya Kendari
DOI: 10.31004/obsesi.v5i1.734 py g
( )
,
,
Corresponding author : Islaeli
Email Address : islaelimwkdi@gmail.com (Kendari, Sulawesi Tenggara, Indonesia)
Received 29 August 2020, Accepted 4 September 2020, Published 8 September 2020 PENDAHULUAN Anak usia dini yaitu anak yang sedang berada dalam pertumbuhan dan
perkembangan yang pesat baik itu fisik atau psikis serta anak-anak yang berusia dibawah 6
tahun. Jadi mulai dari anak lahir hingga ia mencapai umur 6 tahun ia akan dikategorikan
sebagai anak usia dini. Menurut Piaget dalam Sumantri anak usia dini berada pada masa lima
tahun pertama yang disebut The Golden Age. Masa ini merupakan masa emas perkembangan
anak (Nurdin & Anhusadar, 2020). Untuk mewadahi perkembangan anak perlu lembaga
Pendidikan yang menstimulus perkembangan anak sejak usia dini seperti Lembaga PAUD
sehingga perkembangan anak dapat dikembangkan sesuai usia anak. Menurut Musfiroh
bahwa pendidikan Anak Usia Dini merupakan pendidikan yang diselenggarakan untuk
mengembangkan keterampilan yang merupakan pendidikan dasar serta mengembangkan
diri secara utuh sesuai dengan asas pendidikan sedini mungkin dan sepanjang hayat. Aspek
yang dikembangkan dalam pendidikan anak usia dini adalah aspek pengembangan
pembiasaan meliputi sosial, emosi, kemandirian, moral, dan nilai-nilai agama, serta
pengembangan kemampuan dasar yang meliputi pengembangan bahasa, kognitif, dan fisik
motoric (Suriati, Kuraedah, Erdiyanti, & Anhusadar, 2019). Salah satu bentuk lembaga
pendidikan anak usia dini yang berada pada jalur formal adalah taman kanak-kanak (TK)
yang berusia 5-6 tahun. Tujuan pendidikan di TK untuk pencapaian perkembangan yaitu: Nilai agama dan
moral, fisik motorik kasar, motorik halus, kesehatan fisik, kognitif yaitu pengetahuan umum
dan sains, konsep bentuk warna ukuran dan pola, konsep bilangan lambang bilangan dan
huruf, bahasa yaitu menerima bahasa, mengungkapkan bahasa, keaksaraan, sosioal
emosional. Kreativitas anak usia dini dapat diupayakan melalui permainan yang dirancang
oleh Pendidik di Lembaga PAUD, karena dengan permainan anak dapat mengembangkan
serta mengintergrasikan semua potensinya, sehinga mereka lebih kreatif. Peran Pendidik
dalam kegiatan permainan anak adalah memberikan dorongan, membimbing bermain bagi
anak dan membantu anak mengembangkan potensinya, sehingga mereka menjadi anak yang
kreatif (L. O. Anhusadar & Islamiyah, 2020). Usia prasekolah (3-6 tahun) disebut juga masa keemasan (the golden years), yang hanya
datang sekali dan tidak dapat diulangi lagi, yang sangat menentukan untuk pengembangan
kualitas manusia (Suryana, 2007). Anak prasekolah merupakan masa dimana anak mulai peka
dan sensitif untuk menerima berbagai rangsangan. Pada masa inilah anak dengan mudah
menerima stimulasi dari lingkungannya sebagai dasar untuk mengembangkan kemampuan
kognitif, bahasa, motorik, nilai moral, dan sosial emosional pada anak usia dini (Masganti Sit,
2015). Untuk mengembangkan 5 aspek tersebut tentunya asupan nutrisi (gizi) sangat
mendukung dalam pertumbuhan dan perkembangan anak (Sujiono, 2015). Abstract This study aims to determine the effect of playing vegetable eating motivation (VEM) on
vegetable eating behavior in preschool children at Asma Jaya Kindergarten, Lasolo District,
Konawe Utara Regency. The research is experimental with a pre and post test approach
without control. The study population was all preschool children Asma Jaya Kindergarten,
Lasolo District, North Konawe Regency, totaling 16 children. The sampling technique used
total sampling, the method of analysis used the Wilcoxon test. This research was conducted in
July-August 2018. The results showed that there was an effect of playing VEM on vegetable
eating behavior in preschool children at Asma Jaya Kindergarten, Konawe Utara Regency. It
is hoped that parents can provide an overview of how to provide motivation and education to
children with the VEM play approach so that children have good vegetable eating behavior. Keywords: vegetable eating motivation; vegetable eating behavior; preschool children Copyright (c) 2020 Islaeli Islaeli, Ari Nofitasari, Sri Wulandari Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1) 2021 | 879 Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1) 2021 | 879 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah
DOI: 10.31004/obsesi.v5i1.734 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah
DOI: 10.31004/obsesi.v5i1.734 880 | Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1), 2021 PENDAHULUAN MDG’s (Millenium
Development Goals) merupakan salah satu tujuan dimana pada tahun 2015 setiap negara
harus berupaya terus untuk menurunkan separuh jumlah penduduk miskin dan kelaparan
yang juga terdampak pada kesehatan gizi anak-anak. Asupan makanan yang tidak sesuai
dengan kebutuhan tubuh akan memicu masalah gizi yang nantinya berdampak pada
rentannya tubuh terhadap penyakit dan hal ini sering terjadi di daerah atau negara
berkembang. Di Indonesia pencapaian MDG’s dengan indicator indikator paling menentukan untuk
memberantas kemiskinan dan kelaparan adalah prevelensi gizi kurang dan gizi buruk. Adanya penurunan untuk prevalensi gizi kurang menurun secara signifikan di tahun 1989
dari 31% menjadi 17,9% di tahun 2010. Demikian pula prevalensi gizi buruk menurun dari
12,8% (1995) menjadi 4,9% (2010). Kecenderungan ini menunjukan target penurunan
prevalensi gizi kurang dan gizi buruk menjadi 15% dan 3,5% pada tahun 2015 ini dapat
tercapai (Amirullah, Try, Putra, Daud, & Kahar, 2020). Menurut beberapa ahli keperawatan diantaranya Paplau H mengatakan bahwa
kesehatan adalah proses yang berlangsung mengarah kepda kretifitas, konstruktif, dan
produktif. Dengan demikian bahwa kesehatan adalah suatu hal yang tidak dapat dianggap 880 | Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1), 2021 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah
DOI: 10.31004/obsesi.v5i1.734 sepele keberadaannya dalam membentuk sumber daya manusia yang unggul. Bidang
kesehatan perlu mendapatkan perlakuan dan perhatian khusus guna menunjang kepentingan
pembangunan karakter bangsa (Udu, Anhusadar, Alias, & Ali, 2019). Kebutuhan nutrisi
merupakan kebutuhan yang sangat penting dalam membantu proses pertumbuhan dan
perkembangan anak serta dapat mencegah terjadinya berbagai penyakit akibat kurang nutrisi
dalam tubuh. Oleh sebab itu, makanan yang dikonsumsi anak sebaiknya beraneka ragam dan
mengandung berbagai vitamin. g
g
g
Dalam pertumbuhan anak diperlukan gizi yang seimbang, supaya seluruh anggota
badan dapat tumbuh secara wajar, pertumbuhan otot dan tulang dapat kuat, dan sehat. Beberapa sumber gizi yang sangat berguna tersebut terkandung di dalam sayuran (Sukamti,
1994) (Hermina & S, 2016). Sayuran merupakan sumber vitamin, mineral, dan kaya akan serat
yang sangat mudah ditemukan di dalam bahan makanan. Mengkonsumsi makanan berserat
sangat baik karena dapat membantu memperlancar proses pencernaan didalam tubuh,
mempengaruhi peningkatan ukuran, berat, dan melunakkan feses (Kusharto, 2007). g
g
Karena pentingnya konsumsi buah dan sayur, organisasi pangan dan pertanian dunia
Food and Agriculture Organization (FAO), merekomendasikan warga dunia untuk makan
sayur dan buah secara teratur sebanyak 75 kg/kapita/tahun begitupun dengan World Health
Organization (WHO) merekomendasikan agar konsumsi sayur dan buah sebanyak 400 gram
setiap hari (Indira, 2015). Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1) 2021 | 881 PENDAHULUAN Bermain adalah setiap Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1) 2021 | 881 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah
DOI: 10.31004/obsesi.v5i1.734 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah
DOI: 10.31004/obsesi.v5i1.734 kegiatan yang dilakukan untuk kesenangan yang ditimbulkan tanpa pertimbangan hasil
akhir. Melalui bermain anak akan memiliki berbagai manfaat bagi perkembangan, aspek ini
saling menunjang satu sama lain dan tidak dapat dipisahkan. Apabila satu aspek tidak
diberikan kesempatan untuk berkembang, maka akan terjadi ketimpangan karena bermain
adalah aktivitas yang menyenangkan dan merupakan kebutuhan yang sudah melekat dalam
diri anak (L. Anhusadar, 2016). Beberapa penelitian telah dilakukan untuk melihat pengaruh bermain untuk
meningkatkan minat makan sayuran bagi anak usia prasekolah, salah satunya dengan
bermain storytelling efektif meningkatkan pengetahuan dan sikap dalam mengkonsumsi sayur
pada anak usia prasekolah (Ilya Krisnana, Yuni Sufyanti Arief, 2015). Penelitian lain
menyimpulkan bahwa Kedua bermain peran mikro tersebut sama-sama memiliki pengaruh
dalam kecerdasan interpersonal anak, namun bermain peran mikro bebas memberikan hasil
lebih baik dalam kegiatan pembelajaran yang dilakukan dibandingkan bermain peran mikro
tepimpin. Dengan demikian, hal ini merupakan bukti empiris bahwa kecerdasan
interpersonal anak yang diberikan bermain peran mikro bebas lebih tinggi dibandingkan anak
yang diberikan bermain peran mikro terpimpin. Maka dapat direkomendasikan bahwa
bermain peran mikro bebas lebih cocok diterapkan dalam Kecerdasan interpersonal
(Damayanti, Ch, & Hapidin, 2018). Penelitian yang dilakukan oleh (Wahyuni, Syukri, & Halida, 2017) yang berjudul
“Peningkatan motivasi anak makan sayuran melalui metode bermain permainan cooking class
pada anak usia 4-6 tahun” menemukan bahwa, melalui metode bermain permainan cooking
class dapat meningkatkan motivasi anak makan sayuran pada anak usia 4-6 tahun di RA
AL- Muhajirin Rasau Jaya, Kabupaten Kubu Raya, Provinsi Kalimantan Barat. Vegetable
Eating Motivation (VEM) merupakan salah satu bentuk permainan yang dirancang oleh
peneliti dengan tujuan memberikan motivasi dan edukasi kepada anak prasekolah dengan
pendekatan bermain yang dilakukan dalam bentuk rangkaian permainan dan dilakukan
selama 6 hari, dengan tujuan untuk meningkatkan perilaku makan sayuran pada anak
prasekolah. Berdasarkan hasil observasi awal kepada 16 orang anak prasekolah di TK Asma Jaya
Kecamatan Lasolo Kabupaten Konawe Utara, 100 % Ibu selalu menghidangkan menu sayur
setiap hari. Akan tetapi, 11 Anak diantaranya menolak untuk memakan sayur, Anak lebih
memilih nasi dan lauk saja seperti telur, ikan dan ayam. PENDAHULUAN Data WHO menunjukkan bahwa kasus anak usia prasekolah
underweight di dunia sebesar 15,7% dan anak usia prasekolah overweight sebanyak 6,6%
(WHO, 2013). Secara nasional, prevalensi berat-kurang pada tahun 2013 adalah 19,6%, terdiri
dari 5,7% gizi buruk dan 13,9% gizi kurang (Balitbang Kemenkes, 2013). Berdasarkan data
Riset Kesehatan Dasar (Riskesdas) Tahun 2013 menyatakan bahwa tingkat konsumsi sayur
pada anak prasekolah di Indonesia 35 kilogram per kapita per tahun. Angka itu jauh lebih
rendah dengan angka konsumsi sayuran yang dianjurkan 75 kilogram per kapita per tahun
(Khalimatus Sa’diya, 2016). (
y
)
Menurut Soetjiningsih kebutuhan gizi, memberikan makanan yang benar pada anak
harus dilihat dari banyak aspek, seperti ekonomi, sosial, budaya, agama, disamping aspek
medik dari anak itu sendiri. Makanan pada anak usia dini harus serasi, selaras dan seimbang. Serasi artinya sesuai dengan tingkat tumbuh kembang anak. Selaras adalah sesuai dengan
kondisi ekonomi, sosial budaya serta agama dari keluarga. Sedangkan seimbang artinya nilai
gizinya harus sesuai dengan kebutuhan berdasarkan usia dan jenis bahan makanan seperti
karbohidrat, protein dan lemak (Laode Anhusadar, 2020). p
(
)
Salah satu penyebab rendahnya konsumsi sayur pada anak karena kurangnya
pengetahuan dan sikap mengabaikan pentingnya makan sayur (Indira, 2015). Tidak efektifnya
pendidikan gizi pada anak semenjak usia dini berdampak pada pengetahuan yang kurang
tentang pola konsumsi makanan yang sehat dan seimbang saat dewasa, sehingga
menyebabkan perilaku yang salah (Kemenkes, 2014). Masalah tersebut dapat berakibat buruk
bagi tumbuh kembang anak. Anak dapat mempunyai peluang besar untuk menderita kurang
gizi karena makanan yang dikonsumsi dalam jumlah sedikit sehingga tidak memenuhi
kebutuhan nutrisinya (Arifin, 2016). Selain itu, anak dapat mengalami stunting atau menjadi
balita pendek (KemenkesRI, 2016). Berdasarkan Riset Kesehatan Dasar tahun 2013, Indonesia
dengan prevalensi anak balita pendek sebesar 35,6% merupakan negara ke-5 terbesar yang
berkontribusi pada 90% stunting di dunia. Anak pendek mempunyai risiko lebih tinggi
menderita diabetes, obesitas, hipertensi dan stroke pada usia dewasa (Kemenkes, 2014). Selain
itu, Konstipasi juga menjadi salah satu penyakit yang akan dialami bila anak kurang
mengkonsumsi sayur. Bermain secara tidak langsung akan membuat anak mengembangkan kemampuan
kognitif, fisik, motorik, sosial, dan emosionalnya. Oleh karena itu memberikan stimulus
dengan pendekatan permainan kepada anak usia prasekolah merupakan hal yang sangat
penting yang harus dilakukan (Hasanah, 2016). Dalam Pendidikan Anak Usia dini ( PAUD
),bermain sambil belajar merupakan kegiatan yang baik untuk anak. PENDAHULUAN Tahap kerja
-
Memberi petunjuk pada anak cara bermain
-
Memperkenalkan jenis-jenis sayuran
-
Memberi tahu manfaat sayuran dan dampak tidak
mengkonsumsi sayuran
-
Meminta anak untuk Memilih gambar sayuran yang ingin
diwarnai
-
Kemudian anak di anjurkan untuk mewarnai gambar dengan
warna yang disukai
-
Setelah selesai mewarnai gambar, Anak dibantu untuk
melubangi bagian atas kertas gambar
-
Dipasang benang sepanjang 10 cm atau lebih pada bagian atas
yang dilubangi
-
Memberitahukan kepada orang tua untuk mengantung gambar
anak dirumah
-
Melakukan game tentang sayuran
-
Anak diminta menebak nama-nama sayuran yang mereka
gambar
-
Dan menyebutkan manfaat serta dampaknya
-
Memberi pujian pada anak bila dapat melakukan
-
Menanyakan perasaan anak setelah bermain dan menanyakan
pendapat guru dan orang tua tentang permainan. Tahap
terminasi
1. Berpamitan dengan guru, orang tua, dan anak
2. Membereskan kembali alat yang telah dipakai
3. Mencuci tangan. METODOLOGI
Jenis penelitian yang digunakan adalah penelitian kuantitatif dengan desain penelitia
eksperimental dengan pendekatan pre and post test without control. Efektifitas perlakuan dinila
dengan cara membandingkan nilai pre test dengan post test. Sampel pada penelitian in
diobservasi terlebih dahulu sebelum diberi perlakuan, kemudian setelah diberi perlakua Tabel Tahapan pelaksanaan bermain Vegetable Eating Motivation (VEM) -
Dipasang benang sepanjang 10 cm atau lebih pada bagian atas
yang dilubangi PENDAHULUAN Anak yang menolak makan sayuran
mengatakan tidak menyukai sayuran karena rasanya yang pahit, hambar, teksturnya lama
dikunyah, Peneliti juga melakukan wawancara kepada 10 orang ibu dari anak prasekolah di
TK Asma Jaya 8 orang ibu mengatakan anaknya sulit jika disuruh makan sayur, 2 orang ibu
mengatakan anaknya mau makan sayuran. Melihat perilaku anak prasekolah yang jarang
sekali suka dengan sayuran dan hari-hari mereka dipenuhi dengan bermain, maka
dibutuhkanlah sebuah terapi dengan pendekatan bermain yang dapat memotivasi dan
meningkatkan konsumsi sayuran pada anak prasekolah. Berdasarkan uraian diatas, peneliti
tertarik untuk mengadakan penelitian tentang pengaruh bermain vegetable eating motivation
(VEM) terhadap perilaku makan sayuran pada anak prasekolah di TK Asma Jaya Kecamatan
Lasolo Kabupaten Konawe Utara. p
Vegetable Eating Motivation (VEM) merupakan salah satu bentuk terapi bermain yang
dirancang oleh peneliti dengan tujuan memberikan motivasi dan edukasi kepada anak
prasekolah dengan pendekatan bermain yang dilakukan dalam bentuk rangkaian permainan
dan dilakukan selama 6 hari, dengan tujuan untuk meningkatkan perilaku makan sayuran
pada anak prasekolah. Vegetable Eating Motivation (VEM) merupakan salah satu bentuk
terapi bermain yang bertujuan memberikan motivasi dan edukasi kepada anak prasekolah
dengan pendekatan bermain untuk meningkatan perilaku makan sayuran pada anak
prasekolah. Tahapan pelaksanaan bermain Vegetable Eating Motivation (VEM) peneliti lakukan
sebagai berikut: 882 | Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1), 2021 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah
DOI: 10.31004/obsesi.v5i1.734 DOI: 10.31004/obsesi.v5i1.73
Tabel Tahapan pelaksanaan bermain Vegetable Eating Motivation (VEM)
Tujuan
Bermain
Vegetable
Eating
Motivation
1. Meningkatkan citra diri sebagai layaknya manusia
2. Dapat bersosialisasi dan berkomunikasi dengan teman sebaya
3. Untuk mengetahui lebih banyak tentang sayuran
4. Untuk mengetahui manfaat tentang sayuran
5. Dapat mewarnai gambar yang disukainya
6. Gerakan motorik halusnya lebih terarah
Persiapan
1. Guru, orang tua, dan Anak diberitahu tujuan bermain
2. Melakukan kontrak waktu
3. Anak tidak rewel
Peralatan
1. Alat mengambar (Pensil, Warna)
2. Sketsa gambar sayuran
Prosedur
pelaksanaan
1. Tahap Prainteraksi
-
Melakukan kontrak waktu
-
Mengecek kesiapan anak (tidak ngantuk, tidak rewel, keadaan
umum membaik/kondisi yang memungkinkan)
-
Menyiapkan alat
2. Tahap orientasi
-
Memberikan salam kepada guru, orang tua, dan anak
-
Menjelaskan tujuan dan prosedur pelaksanaan
-
Menanyakan persetujuan dan kesiapan
3. Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1) 2021 | 883 884 | Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1), 2021 METODOLOGI Jenis penelitian yang digunakan adalah penelitian kuantitatif dengan desain penelitian
eksperimental dengan pendekatan pre and post test without control. Efektifitas perlakuan dinilai
dengan cara membandingkan nilai pre test dengan post test. Sampel pada penelitian ini
diobservasi terlebih dahulu sebelum diberi perlakuan, kemudian setelah diberi perlakuan
sampel tersebut diobservasi kembali (Notoatmodjo, 2012). Perilaku makan sayur anak
prasekolah diukur dengan menggunakan FFQ (Food Frequncy Questionnaire) yang dilakukan Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1) 2021 | 883 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah
DOI: 10.31004/obsesi.v5i1.734 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah
DOI: 10.31004/obsesi.v5i1.734 sebelum dan sesudah intervensi bermain vegetable eating motivation dan pemberian sketsa
gambar sayuran. Kegiatan bermain dilakukan selama enam hari dan dilakukan pengukuran. Populasi dalam penelitian ini adalah seluruh anak prasekolah TK Asma Jaya Kecamatan
Lasolo Kabupaten Konawe Utara yang berjumlah 16 anak dan dengan teknik total sampling. Teknis pengumpulan data adalah dokumentasi, wawancara dan observasi. Prilaku makan sayur anak prasekolah diukur sebelum dan sesudah dilakukan metode
bermain vegetable eating motivation (vem) dengan metode ceramah dan dan pemberian sketsa
gambar sayuran. Dengan rancangan penelitian sebagai berikut. Pre Test
Perlakuan
Post Test
Keterangan :
P1a : Perilaku makan sayur sebelum pemberian terapi vegetable eating motivation (pre test)
Xa : Perlakuan (Terapi bermain vegetable eating motivation)
P2a : Perilaku makan sayur sesudah pemberian terapi vegetable eating motivation (post test)
Penelitian ini digambarkan dalam bentuk bagan berikut. Variabel Independen
Variabel Dependen
Gambar 1. Bagan Kerangka Konsep Penelitian
Keterangan :
Perilaku Makan Sayur
: Variabel dependen
Bermain vegetable eating motivation
: Variabel independent
HASIL DAN PEMBAHASAN
Bermain vegetable eating
motivation
Perilaku makan sayur
P1a
P2a
Xa Pre Test
Perlakuan
Post Test
Keterangan :
P1a : Perilaku makan sayur sebelum pemberian terapi vegetable eating motivation (pre test)
Xa : Perlakuan (Terapi bermain vegetable eating motivation)
P2a : Perilaku makan sayur sesudah pemberian terapi vegetable eating motivation (post test)
P1a
P2a
Xa Keterangan :
P1a : Perilaku makan sayur sebelum pemberian terapi vegetable eating motivation (pre test)
Xa : Perlakuan (Terapi bermain vegetable eating motivation)
P2a : Perilaku makan sayur sesudah pemberian terapi vegetable eating motivation (post test) Penelitian ini digambarkan dalam bentuk bagan berikut. Penelitian ini digambarkan dalam bentuk bagan berikut. Variabel Independen Variabel Independen Variabel Dependen Variabel Dependen Bermain vegetable eating
motivation Perilaku makan sayur Gambar 1. Bagan Kerangka Konsep Penelitian (
, %)
Distribusi responden menurut jenis kelamin, terlihat pada tabel berikut: Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1) 2021 | 885 HASIL DAN PEMBAHASAN HASIL DAN PEMBAHASAN Karakteristik responden meliputi umur dan jenis kelamin responden. Setiap
karakteristik responden, distribusi responden menurut umur terlihat pada tabel berikut : Tabel 1. Distribusi Kelompok Umur Responden Anak Pra Sekolah di TK Asma Jaya Kabupaten
Konawe Utara
No
Umur
N
%
1
4 tahun
6
37,5
2
5 tahun
7
43,8
3
6 tahun
3
18,8
Jumlah
16
100
Sumber : Data Primer, 2018 1. Distribusi Kelompok Umur Responden Anak Pra Sekolah di TK Asma Jaya Kabupaten
Konawe Utara Tabel 1. Distribusi Kelompok Umur Responden Anak Pra Sekolah di TK Asma Jaya Kabu
Konawe Utara Sumber : Data Primer, 2018 Tabel 1 menunjukkan bahwa dari 16 responden, terbanyak adalah umur 5 tahun
sebanyak 7 responden (43,8%) dan yang terkecil adalah umur 6 tahun yaitu sebanyak 3
responden (18,8%). p
(
,
)
Distribusi responden menurut jenis kelamin, terlihat pada tabel berikut: 884 | Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1), 2021 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah
DOI: 10.31004/obsesi.v5i1.734 Tabel 2. Distribusi Kelompok Jenis Kelamin Responden Anak Pra Sekolah di TK Asma Jaya
Kabupaten Konawe Utara
No
Jenis Kelamin
N
%
1
Laki-Laki
7
43,8
2
Perempuan
9
56,2
Jumlah
16
100
Sumber : Data Primer, 2018 l 2. Distribusi Kelompok Jenis Kelamin Responden Anak Pra Sekolah di TK Asma Jaya
Kabupaten Konawe Utara Tabel 2 menunjukkan bahwa dari 16 responden terdapat 7 responden (43,8%) yang
berjenis kelamin laki-laki dan terdapat 9 responden (56,2%) yang berjenis kelamin perempuan. Distribusi responden berdasarkan perilaku makan sayur pre test dapat terlihat pada Tabel 2 menunjukkan bahwa dari 16 responden terdapat 7 responden (43,8%) yang
berjenis kelamin laki-laki dan terdapat 9 responden (56,2%) yang berjenis kelamin perempuan. Tabel 2 menunjukkan bahwa dari 16 responden terdapat 7 responden (43,8%) yang
berjenis kelamin laki-laki dan terdapat 9 responden (56,2%) yang berjenis kelamin perempuan. Distribusi responden berdasarkan perilaku makan sayur pre test dapat terlihat pada
tabel berikut : Distribusi responden berdasarkan perilaku makan sayur pre test dapat terlihat pada
tabel berikut : Tabel 3. Distribusi Responden Perilaku Makan Sayur Pre Test di TK Asma Jaya Kabupaten
Konawe Utara
No
Perilaku Makan Sayur Pre Test
N
%
1
Cukup
2
12,5
2
Kurang
14
87,5
Jumlah
16
100
Sumber : Data Primer, 2018 abel 3. Distribusi Responden Perilaku Makan Sayur Pre Test di TK Asma Jaya Kabupaten
Konawe Utara Tabel 3. HASIL DAN PEMBAHASAN Distribusi Responden Perilaku Makan Sayur Pre Test di TK Asma Jaya Kabupa
Konawe Utara Sumber : Data Primer, 2018 Tabel 3 menunjukkan bahwa sebelum diberikan terapi bermain Vegetable Eating
Motivation (VEM) dari 16 responden, terdapat 2 responden (12,5%) yang memiliki perilaku
makan sayur cukup dan terdapat 14 responden (87,5%) yang memiliki perilaku makan sayur
kurang. Sedangkan distribusi responden berdasarkan perilaku makan sayur post test
dapat terlihat pada tabel berikut : Tabel 4. Distribusi Responden Perilaku Makan Sayur Post Test di TK Asma Jaya Kabupaten
Konawe Utara
No
Perilaku Makan Sayur Post Test
N
%
1
Cukup
15
93,8
2
Kurang
1
6,3
Jumlah
16
100
Sumber : Data Primer, 2018 abel 4. Distribusi Responden Perilaku Makan Sayur Post Test di TK Asma Jaya Kabupaten
Konawe Utara Sumber : Data Primer, 2018 Tabel 4 menunjukkan bahwa setelah diberikan terapi bermain Vegetable Eating
Motivation (VEM) dari 16 responden, terdapat 15 responden (93,8%) yang memiliki perilaku
makan sayur cukup dan terdapat 1 responden (6,3%) yang memiliki perilaku makan sayur
kurang. Pada uji normatitas data dalam penelitian ini adalah sesuai tebel berikut ini: Tabel 5. Uji normalitas data dengan menggunakan Shapiro-Wilk
Tests of Normality
Kolmogorov-Smirnova
Shapiro-Wilk
Statistic
df
Sig. Statistic
Df
Sig. Pre_test
.278
16
.002
.871
16
.028
Post_test
.204
16
.075
.906
16
.100 Tabel 5. Uji normalitas data dengan menggunakan Shapiro-Wilk Tabel 5 menunjukkan bahwa hasil uji normalitas data menggunakan uji Shapiro-Wilk
pada variabel perilaku makan sayur pre test menunjukkan bahwa nilai p value (0,028) < α
(0,05), dan variabel perilaku makan sayur post test menunjukkan bahwa nilai p value (0,100) Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1) 2021 | 885 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah
DOI: 10.31004/obsesi.v5i1.734 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah
DOI: 10.31004/obsesi.v5i1.734 > α (0,05) sehingga dapat disimpulkan bahwa data berdistribusi tidak normal. Karena data
berdistribusi tidak normal maka menggunakan uji Wilcoxon. > α (0,05) sehingga dapat disimpulkan bahwa data berdistribusi tidak normal. Karena data
berdistribusi tidak normal maka menggunakan uji Wilcoxon. Test Statisticsa
post_test - pre_test
Z
-3.455b
Asymp. Sig. (2-tailed)
.001 Berdasarkan hasil analisis uji Wilcoxon diperoleh nilai p value = 0,001 < 0,05 maka Ho
ditolak dan Ha diterima berati ada pengaruh pemberian bermain vegetable eating motivation
(VEM) terhadap perilaku makan sayur pada anak prasekolah di TK Asma Jaya Kabupaten
Konawe Utara. Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah
DOI: 10.31004/obsesi.v5i1.734 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah
DOI: 10.31004/obsesi.v5i1.734 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah
DOI: 10.31004/obsesi.v5i1.734 Sayuran memiliki banyak manfaat yang baik bagi kesehatan. Mengonsumsi sayuran
dan buah sangat bermanfaat untuk memenuhi kebutuhan gizi (Aswatini, Noveria, & Fitranita,
2008). Hasil penelitian menunjukkan bahwa sebelum diberikan terapi bermain Vegetable Eating
Motivation (VEM) dari 16 responden, terdapat 2 responden (12,5%) yang memiliki perilaku
makan sayur cukup dan terdapat 14 responden (87,5%) yang memiliki perilaku makan sayur
kurang. Hal ini menunjukkan bahwa banyak anak yang kurang mengkonsumsi sayur. Hal ini
sesuai dengan teori bahwa pada masa usia prasekolah anak mengalami penurunan nafsu
makan dan hanya mau makan makanan yang disukai (Siti Aizah, 2003). Siswa yang kurang
mengkonsumsi sayur dikarenakan beberapa faktor seperti anak tidak suka dengan rasa sayur
yang kurang enak, tidak suka dengan aroma sayur, serta model penyajian sayur yang kurang
disukai oleh anak. Selain itu masih banyak juga anak yang kurang mengetahui tentang
manfaat mengkonsumsi sayuran. Hal ini sesuai dengan teori bahwa pada masa usia
prasekolah anak mengalami penurunan nafsu makan dan hanya mau makan makanan yang
disukai. Orang tua harus berusaha keras memaksa anak untuk mau makan sayuran sehingga
membuat suasana makan tidak nyaman dan anak menjadi rewel. Sikap ini merupakan suatu
kasus global dan merupakan suatu kesulitan bagi orang tua untuk memberi anak mereka
makanan yang mengandung serat dan banyak vitamin yaitu sayur mayur (Asy’ariyah, Arief,
& Krisnana, 2014). Selanjutnya setelah diberikan terapi bermain Vegetable Eating Motivation (VEM) dari 16
responden, terdapat 15 responden (93,8%) yang memiliki perilaku makan sayur cukup dan
terdapat 1 responden (6,3%) yang memiliki perilaku makan sayur kurang. Hal ini
menunjukkan bahwa terjadi peningkatan jumlah anak yang signifikan dalam hal perilaku
mengkonsumsi sayur setelah diberikan terapi bermain VEM oleh peneliti. Hal ini dapat
disebabkan oleh pemberian terapi VEM yang disukai dan diminati oleh anak prasekolah
sehingga banyak siswa yang antusias dalam mengikuti kegiatan yang diadakan oleh peneliti. Anak yang antusias dikarenakan belum pernah mendapatkan kegiatan mewarnai gambar
sayuran sebelumnya, serta model gambar yang menarik sehingga anak suka dengan kegiatan
menggambar sayuran. Peneliti juga menjelaskan kepada siswa tentang manfaat
mengkonsumsi setiap sayuran dan akibat tidak mengkonsumsi jenis sayuran yang digambar
oleh siswa. Sayur dan buah merupakan pangan yang mudah diperoleh di Indonesia, dan
sangat bermanfaat bagi tubuh, karena kaya akan zat gizi, seperti vitamin, mineral, dan serat
untuk mengatur proses pencernaan dalam tubuh. Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1) 2021 | 887 HASIL DAN PEMBAHASAN Uji normalitas data dilakukan untuk mengetahui distribusi data, apakah data tersebut
berdistribusi normal atau tidak yang akan menentukan jenis uji yang akan digunakan dalam
melakukan analisis. Untuk lebih jelasnya dapat disajikan pada tabel uji normalitas di bawah
ini. Tabel 6 : Hasil Uji Normalitas Perilaku Makan Sayur Sebelum dan Sesudah Diberikan Terapi
Bermain Vegetable Eating Motivation (VEM)
Variabel Penelitian
P value
α
Perilaku makan sayur pre test
0,168
0,05
Perilaku makan sayur post test
0,520
0,05
Data primer diolah 2018 bel 6 : Hasil Uji Normalitas Perilaku Makan Sayur Sebelum dan Sesudah Diberikan Terapi
Bermain Vegetable Eating Motivation (VEM) Tabel 6 menunjukkan bahwa hasil uji normalitas data menggunakan uji Kolmogorov-
Smirnov pada variabel perilaku makan sayur pre test menunjukkan bahwa nilai p value
(0,168) > α (0,05), dan variabel perilaku makan sayur post test menunjukkan bahwa nilai p
value (0,520) > α (0,05) sehingga dapat disimpulkan bahwa data berdistribusi normal. Karena
data berdistribusi normal maka memenuhi syarat untuk dilakukan uji Paired T Test. Pengaruh pemberian bermain vegetable eating motivation (VEM) terhadap perilaku
makan sayur pada anak prasekolah di TK Asma Jaya Kecamatan Lasolo Kabupaten Konawe
Utara. Distribusi pengaruh pemberian bermain vegetable eating motivation (VEM) terhadap
perilaku makan sayur pada anak prasekolah di TK Asma Jaya Kecamatan Lasolo Kabupaten
Konawe Utara, yaitu: Tabel 7. Analisis pengaruh pemberian bermain vegetable eating motivation (VEM) terhadap
perilaku makan sayur pada anak prasekolah di TK Asma Jaya Kabupaten Konawe Utara
Perilaku makan
sayur
n
Mean
SD
t value
P value
Pre Test
16
1,69
0,793
8,777
0,000
Post Tes
16
4,56
1,209
Sumber : Data Primer, 2018 Tabel 7. Analisis pengaruh pemberian bermain vegetable eating motivation (VEM) terhada
perilaku makan sayur pada anak prasekolah di TK Asma Jaya Kabupaten Konawe Utara Tabel 8 menunjukkan bahwa perilaku makan sayur pada pre tes memiliki nilai mean
1,69 dengan nilai SD 0,793. Sedangkan pada post tes memiliki nilai mean 4,56 dengan nilai SD
1,209. Berdasarkan hasil analisis uji paired t-test diperoleh nilai t hit = 8,777 dengan taraf
kepercayaan 95% (0,05) df = 15 diperoleh t tabel (1,753). Jadi t hit > t tabel dan p value = 0,000
< 0,05 maka Ho ditolak dan Ha diterima berati ada pengaruh pemberian bermain vegetable
eating motivation (VEM) terhadap perilaku makan sayur pada anak prasekolah di TK Asma
Jaya Kabupaten Konawe Utara. 886 | Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1), 2021 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah
DOI: 10.31004/obsesi.v5i1.734 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah
DOI: 10.31004/obsesi.v5i1.734 Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah
DOI: 10.31004/obsesi.v5i1.734 Hasil penelitian juga menunjukkan masih terdapat 1 responden (6,3%) yang tetap
kurang mengkonsumsi sayuran setelah diberikan terapi VEM. Hal ini bisa disebabkan oleh
beberapa hal seperti anak yang kurang serius dan antusias mengikuti kegiatan terapi bermain
VEM. Anak juga kurang mendengar penjelasan yang disampaikan oleh peneliti sehingga anak
tetap kurang suka mengkonsumsi sayuran. Berdasarkan hasil analisis uji paired t-test
diperoleh nilai t hit = 8,777 dengan taraf kepercayaan 95% (0,05) df = 15 diperoleh t tabel
(1,753). Jadi t hit > t tabel dan p value = 0,000 < 0,05 maka Ho ditolak dan Ha diterima berati
ada pengaruh pemberian bermain vegetable eating motivation (VEM) terhadap perilaku
makan sayur pada anak prasekolah di TK Asma Jaya Kabupaten Konawe Utara. y
p
p
y
p
Hasil penelitian ini sejalan dengan penelitian menyimpulkan bahwa Hasil uji statistik
didapatkan nilai rata-rata konsumsi sayur sebelum diberikan intervensi yaitu 38,22 dengan
standar deviasi (SD) 18,236 dan setelah diberikan intervensi yaitu 68,11 dengan standar
deviasi (SD) 18.304. Hasil analisa diperoleh p value sebesar 0,000 < α (0,05), maka dapat
disimpulkan terjadi peningkatan konsumsi sayuran pada anak prasekolah setelah diberikan
terapi bermain Vegetable Eating Motivation (VEM) (Noviyanti emulyani, 2017). Penelitian ini
juga sejalan dengan penelitian (Burhannudin Ichsan , Bayu Hendro Wibowo, 2015) bahwa
hasil penelitian dengan mengggunakan uji t (deoendent t test) dengan pendekatan pre test dan
post tes diperoleh nilai p-value = 0,000 (< 0,05). Simpulannya yaitu terdapat peningkatan
pengetahuan tentang pentingnya sayuran bagi anak-anak yang bermakna setelah mengikuti
sesi penyuluhan. SIMPULAN Perilaku makan sayuran anak pra sekolah sebelum bermain Vegetable Eating
Motivation (VEM) Di TK Asma Jaya Kecamatan Lasolo Kabupaten Konawe Utara sebagian
besar yang memiliki nafsu makan kurang. Perilaku makan sayuran anak pra sekolah sesudah
bermain Vegetable Eating Motivation (VEM) Di TK Asma Jaya Kecamatan Lasolo Kabupaten
Konawe Utara sebagian besar nafsu makan cukup meningkat dibandingkan sebelum bermain
Vegetable Eating Motivation. Ada pengaruh bermain Vegetable Eating Motivation (VEM)
Terhadap Perilaku Makan Sayuran Pada Anak Prasekolah Di TK Asma Jaya Kecamatan
Lasolo Kabupaten Konawe Utara p<α (ρ=0,000<α=0,05). 888 | Jurnal Obsesi : Jurnal Pendidikan Anak Usia Dini, 5(1), 2021 UCAPAN TERIMA KASIH Terima kasih penulis ucapkan kepada para pendidik TK Asma Jaya Kecamatan Lasolo
Kabupaten Konawe Utara yang telah meluangkan waktunya untuk mengisi instrument
penelitian ini dan semua pihak yang telah membantu pelaksanaan penelitian dan penulisan
artikel ini. Tidak lupa ucapan terima kasih kepada editor dan reviewer Jurnal Obsesi yang
sudah memberikan kesempatan sehingga jurnal bisa untuk diterbitkan. Bermain Vegetable Eating Motivation (Vem) terhadap Perilaku Makan Sayuran pada Anak Prasekolah
DOI: 10.31004/obsesi.v5i1.734 Di Indonesia menyarankan untuk
mengkonsumsi sayur sebanyak 3-5 porsi dan buah-buahan 2-3 porsi dalam sehari. WHO
menyarankan konsumsi sayur dan buah-buahan sebanyak 400 gram setiap hari (Rahmad &
Almunadia, 2017). )
Menurut peneliti bahwa adanya peningkatan jumlah anak prasekolah setelah
diberikan terapi bermain VEM disebabkan oleh beberapa faktor yaitu terapi yang diberikan
secara berkelompok sehingga penerimaan informasi lebih jelas dan mudah dipahami oleh
anak. Pemberian terapi VEM juga dipraktekkan langsung oleh siswa sehingga meningkatkan
minat anak dalam mengkonsumsi sayuran. Kegiatan bermain VEM ini memiliki manfaat yang
besar dalam menunjang perkembangan kemandirian konsumsi anak. Karena dengan bermain
Vegetable eating motivation (VEM) ini menyediakan waktu dan ruang bagi anak untuk belajar
memahami mamfaat mengkonsumsi sayuran. Hasil penelitian lain menyimpulkan bahwa
Identifikasi hasil dari konsumsi sayur sebelum diberikan pendidikan Kesehatan dengan
metode storytelling di SDN Mulyoagung 04 Dau Malang memiliki nilai rata-rata (mean) 90,21
gram perhari dengan persentase 33,3% cukup (>150 gram perhari) dan 66,7% kurang (<150
gram perhari). Identifikasi hasil dari konsumsi sayur sesudah diberikan pendidikan
Kesehatan dengan metode storytelling di SDN Mulyoagung 04 Dau Malang memiliki nilai
rata-rata (mean) 128,71 gram perhari dengan persentase 70,8% cukup (>150 gram perhari) dan
29,2% kurang (<150 gram perhari). Ada perbedaan konsumsi sayur sebelum dan sesudah
pendidikan Kesehatan dengan metode storytelling pada anak sekolah dasar di SDN
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Knockout of a PLD gene in Schizochytrium limacinum SR21 enhances docosahexaenoic acid accumulation by modulation of the phospholipid profile
|
Research Square (Research Square)
| 2,023
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cc-by
| 8,145
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Knockout of a PLD gene in Schizochytrium
limacinum SR21 enhances docosahexaenoic acid
accumulation by modulation of the phospholipid
profile
Yiting Zhang
Xiamen University
Xiaowen Cui
Xiamen University
Shuizhi Lin
Xiamen University
Tao Lu
Xiamen University
Hao Li
Xiamen University
Yinghua Lu
Xiamen University
Mingfeng Cao
Xiamen University
Xihuang Lin
Third Institute of Oceanography, Ministry of Natural Resources
Xueping Ling ( xpling@xmu.edu.cn )
Xiamen University
Research Article
Keywords: Schizochytrium, phospholipase D, docosahexaenoic acid, phospholipomics, acyl CoAindependent pathway
Posted Date: September 28th, 2023
DOI: https://doi.org/10.21203/rs.3.rs-3376770/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
Read Full License
Page 1/28
Additional Declarations: No competing interests reported.
Version of Record: A version of this preprint was published at Biotechnology for Biofuels and Bioproducts
on January 30th, 2024. See the published version at https://doi.org/10.1186/s13068-024-02465-w.
Page 2/28
Abstract
Background:
The hydrolysis and transphosphatidylation of phospholipase D (PLD) play important roles in the
interconversion of phospholipids (PLs), which has been shown to profoundly impact lipid metabolism in
plants. In this study, the effect of the PLD1gene of Schizochytrium limacinum SR21 on lipid
metabolismwas investigated.
Results:
PLD1knockout had little impact on cell growth and lipid production, but it significantly improved the
percentage of polyunsaturated fatty acids in lipids, of which docosahexaenoic acid (DHA) content
increased by 13.3% compared to the wild-type strain. Phospholipomics and qRT-PCR analysis revealed
the knockout of PLD1reduced the interexchange and increasedde novo synthesis of PLs, which altered
the composition of PLs, accompanied by a decrease in phosphatidylcholine (PC) and an increase in
phosphatidylinositol, lysophosphatidylcholine, and phosphatidic acid levels. PLD1 knockout also
increased DHA content in triglycerides (TAGs) and decreased it in PLs.
Conclusions:
These results indicate that PLD1 mainly performs the transphosphatidylation activity in Schizochytrium,
and its knockout promotes the migration of DHA from PLs to TAGs, which is conducive to DHA
accumulation and storage in TAGsvia an acyl CoA-independent pathway. This study provides a novel
approach for identifying the mechanism of DHA accumulation and metabolic regulation strategies for
DHA production in Schizochytrium.
1. Background
Schizochytrium is a natural representative strain for the industrial production of docosahexaenoic
acid (DHA) owing to its fast growth rate and high lipid content, containing more than 50% DHA [1-4]. The
mechanism of DHA synthesis in Schizochytrium has been extensively studied and is known to involve
two distinct biochemical pathways: the aerobic fatty acid synthesis (FAS) pathway and the anaerobic
polyketide synthase (PKS) pathway [5-10], which are the main targets for regulating the synthesis of
polyunsaturated fatty acids (PUFAs) in microorganisms [11-14]. Recent studies have revealed that DHA
production depends not only on the pathway of DHA synthesis but also on the migration, accumulation,
and assembly of DHA stored after synthesis [15-16]. Although DHA is mainly stored as DHA-triglyceride
(DHA-TAG) in Schizochytrium and Thraustochytrium [17], it has been reported that the synthesized DHA
might first be incorporated into phospholipids (PLs) to form DHA-PLs, before being channeled and
assembled into DHA-TAG for storage [18-19]. This suggests that the final DHA yield is closely related to
PL metabolism. PLs are the main components of cell membranes and play important roles in biological
Page 3/28
reproduction, cell division, and membrane transport [20]. The regulation of PL metabolism in
Schizochytrium may be effective for DHA production.
Phospholipase D (PLD) is capable of converting different types of PLs through transphosphatidylation or
hydrolyzing the polar head of PLs to produce phosphatidic acid (PA) [21], which has a significant role in
cell growth and lipid metabolism [22, 23]. Currently, studies on PLD expression in the regulation of lipid
metabolism mainly focus on plants [24, 25]. Overexpression of the soybean PLDγ gene (GmPLDγ)
in Arabidopsis resulted in increased seed weight, a greater number of smaller lipid droplets, and
significant upregulation of glycerolipid metabolism-related genes, suggesting a regulatory role for
GmPLDγ in TAG synthesis and fatty acid remodelling [26]. Knockout of PLDα1 in soybean resulted in the
higher unsaturation of TAG and major membrane lipids and lower unsaturation of PA and
lysophospholipids during seed development. The study also found that phospholipid:diacylglycerol
acyltransferase (PDAT) facilitated the conversion of acyl-CoA between phosphatidylcholine (PC) and
diacylglycerol (DAG), promoting the unsaturation of TAG [27]. These results suggest that the regulation of
PLD expression exerts an important influence on lipid metabolism in plants, particularly on the allocation
of fatty acids between TAG and PLs. There are few publications on the function of PLD in lipid synthesis
of microorganisms; therefore, it is of interest to explore the effect of PLD regulation on the patterns of PLs
and DHA production in Schizochytrium, a premium engineered strain used for lipid production.
In this study, we first mined two PLD genes, PLD1 and PLD2, from Schizochytrium and constructed the
corresponding knockout strains, D PLD1 and D PLD2. The mutant strain with the higher DHA yield, D PLD1,
was further analyzed using phospholipomics and qRT-PCR to reveal the potential regulatory mechanism
of PLD on DHA production in Schizochytrium.
2. Results and Discussion
2.1 Mining of PLD genes
Seventeen species of PLD corresponding to the EC3.1.4.4 enzyme classification were found in the JGI
database. The sequence similarity of the PLD genes and the corresponding proteins among species was
not high, indicating that the PLDs found in the JGI database were independent and non-repetitive (Fig.
S1). Notably, PLD superfamily domains were found in PLD-46 and PLD-100463 proteins. Phylogenetic
tree analysis of PLD-46 and PLD-100463 genes showed that they were closely related to the PLD and
PLDα1 genes of Aurantiochytrium sp. FCC1311, respectively, and had 59.20% and 67.02% sequence
homology, respectively (Fig. 1). Therefore, in this study, we chose to regulate the expression of PLD-46
and PLD-100463, which were named PLD1 and PLD2, respectively. Gene sequences are listed in Fig. S2
and S3.
2.2 Construction of transgenic S. limacinum SR21 with knockout of PLD1 or PLD2
Both positive transformants were screened for zeocin resistance. The PCR validations of the zeocin gene
are shown in Fig. S4. The target band of the zeocin resistance gene (375 bp) appeared in the knockout
Page 4/28
strains and positive control groups but not in the negative control group, indicating that the
PLD1 or PLD2 gene had been successfully knocked out in S. limacinum SR21.
2.3 Effects of PLD1 or PLD2 knockout on cell growth and lipid synthesis
As shown in Fig. 2a, compared with the wild type, there was no significant change in the biomass of
the D PLD1 strain before 96 h, but a decrease of 11.0% (P < 0.01) and 7.4% (P<0.05) occurred at 120 h and
144 h, respectively. During the whole fermentation, the total lipid yield of D PLD1 strain showed little
change when compared with the wild type. Fig. 2b shows that the D PLD1 and wild-type strains had the
same glucose consumption rate throughout the fermentation process. Glucose was almost completely
used up at 96 h, which was consistent with the observations of the same cell growth patterns in both
strains before 96 h. The small decrease in biomass in the D PLD1 strain at the later stage was probably
the result of knocking out PLD1, which slightly affected cell membrane function, resulting in minor cell
damage. The lipid content in the two strains also showed no significant change during the full growth
stage (Fig. 2c); however, DHA production was improved in the D PLD1 strain (Fig. 2d). The highest yield of
DHA from shake flask fermentation of D PLD1 strain achieved 9.61 g/L, which was 12.3% higher than that
of the wild type (P<0.01). These results suggest that knockout of PLD1 does not affect cell growth or total
lipid production but enhances DHA accumulation.
As shown in Fig. 3a, compared with the wild type, the biomass of the D PLD2 strain declined drastically
from 72 h to 144 h, and the highest decrease was observed at 120 h (32.8%; P < 0.01). The total lipids of
the D PLD2 strain showed a similar reduction as that observed for biomass, decreasing by 17.6% (P <
0.01) at 120 h. Fig. 3b shows that the glucose consumption rate in the D PLD2 strain decreased at the
early stage but increased at the middle stage compared to the wild-type strain. The D PLD2 strain
consumed the same amount of glucose as the wild-type strain. The lipid content of the D PLD2 strain
showed no apparent change compared to that of the wild type before 96 h (Fig. 3c) but was higher at 120
and 144 h. This indicated that the two strains had the same ability to produce lipids before 96 h, and that
the D PLD2 strain could improve the ability of the unit cell to produce lipids after 120 h. We postulated
that the knockout of PLD2 severely impaired cell membrane function, leading to decreased cell growth
and lipid yield. To maintain a certain amount of growth, cells of D PLD2 strain used glucose to synthesize
other metabolites that resist cell damage and after 120 h, mature cells of the D PLD2 strain showed
enhanced lipid synthesis. DHA production in the PLD2 knockout strain decreased significantly during all
stages compared to the wild-type strain (Fig. 3d). These results indicated that PLD2 knockout is not
conducive to cell growth, total lipid production, or DHA yield.
2.4 Effect of PLD1 or PLD2 knockout on fatty acid composition
Table 1 shows that the PLD1 knockout decreased the percentage of saturated fatty acids (SFAs) and
increased the proportion of PUFAs in the lipids of Schizochytrium. In the D PLD1 strain, the proportion of
DHA in lipids increased by 13.3% and 12.5% at 120 h and 144 h, respectively. These results suggest that
PLD1 knockout facilitates the synthesis of PUFAs, particularly DHA, in Schizochytrium. Knockdown of the
Page 5/28
PLDα1 gene in soybeans resulted in higher unsaturation of TAG [27], which is similar to the results of the
present study.
Table 1 Fatty acid composition of wild-type strain and D PLD1 strain at late stages of fermentation
Fatty acid
120 h
144 h
WT
D PLD1
significance
WT
D PLD1
significance
C14:0
3.38±0.03
3.78±0.02
**
3.20±0.07
3.78±0.08
**
C15:0
1.27±0.03
0.96±0.01
**
1.24±0.04
0.94±0.01
**
C16:0
49.76±0.20
43.20±0.20
**
49.05±0.34
42.96±0.99
**
C17:0
0.48±0.01
0.30±0.00
**
0.49±0.02
0.30±0.00
**
C18:0
1.74±0.01
1.67±0.02
**
1.85±0.03
1.63±0.01
**
EPA
0.39±0.03
0.36±0.00
0.48±0.01
0.39±0.01
**
DPA
6.92±0.03
8.32±0.01
**
7.10±0.09
8.38±0.13
*
DHA
33.11±0.16
37.52±0.18
**
33.52±0.31
37.69±0.80
**
Others
2.94±0.12
3.89±0.11
*
3.09±0.09
3.94±0.18
**
SFAs
56.63±0.20
49.91±0.23
**
55.81±0.48
49.60±1.10
**
PUFAs
40.43±0.16
46.19±0.19
**
41.10±0.41
46.46±0.92
**
Table 2 shows that the PLD2 knockout had little effect on the proportion of PUFAs and SFAs in
Schizochytrium. However, a decrease in the proportion of even-numbered carbon fatty acids, such as
C14:0, C16:0, and C18:0, was observed, and the proportion of odd-numbered carbon fatty acids (OCFAs)
increased significantly, of which C15:0 and C17:0 increased by 246.2% (P < 0.01) and 259.6% (P < 0.01),
respectively. OCFA concentration in human plasma has been reported to be negatively correlated with the
risk of type 2 diabetes and cardiovascular disease [14, 28, 29]; therefore, increasing the production of
OCFAs helps increase the commercial potential of Schizochytrium. The above results suggest that PLD2
knockout mainly inhibited cell growth to reduce lipid yield but did not affect the synthesis pathway of
PUFAs and SFAs in Schizochytrium.
Table 2 Fatty acid composition of wild-type strain and △PLD2 strain at late stages of fermentation
Page 6/28
Fatty acid
120 h
144 h
WT
△PLD2
significance
WT
△PLD2
significance
C14:0
2.96±0.17
2.08±0.14
**
2.83±0.46
1.92±0.07
**
C15:0
1.22±0.30
4.00±0.32
**
1.00±0.05
3.47±0.52
**
C16:0
50.77±0.57
48.81±1.13
**
50.51±0.55
46.54±0.75
**
C17:0
0.56±0.16
2.29±0.25
**
0.46±0.06
2.09±0.27
**
C18:0
1.65±0.03
1.88±0.06
**
1.74±0.12
1.89±0.03
**
EPA
0.42±0.05
0.35±0.02
0.57±0.14
0.55±0.12
**
DPA
6.90±0.35
6.64±0.21
**
6.95±0.17
7.06±0.38
*
DHA
32.48±0.69
30.63±0.26
**
32.34±0.35
32.80±0.68
**
Others
3.04±0.13
3.32±0.39
*
3.60±0.86
3.68±0.37
**
SFAs
57.15±1.06
59.06±0.68
**
56.54±0.79
55.91±1.53
**
PUFAs
39.80±1.09
37.62±0.48
**
39.86±0.28
40.41±1.18
**
OCFAs
1.78±0.46
6.29±0.57
**
1.46±0.10
5.56±0.78
**
Note: When P > 0.05, the difference is insignificant; when 0.01 < P < 0.05, the difference is significant and
represented by *; when P < 0.01, the difference is extremely significant and represented by **.
The regulation of PLD can influence phospholipid metabolism by affecting hydrolysis and
transphosphatidylation and subsequently the function of the cell membrane and the glycerophospholipid
metabolic pathway, thereby influencing the cell growth and lipid metabolism of Schizochytrium [30, 31].
The different results for the PLD1 and PLD2 knockouts may be related to their preference for hydrolysis or
transphosphatidylation, allowing them to have different roles in phospholipid metabolism. PLD1 is
thought to play a role in transphosphatidylation, which affects phospholipid composition and the
regulation of fatty acid synthesis, whereas PLD2 mainly hydrolyzes phospholipid, which damages cell
function and inhibits cell growth and lipid production. Given that the PLD1 knockout promoted DHA
synthesis without a significant effect on cell growth and lipid production, whereas PLD2 knockout
showed a severe reduction in cell growth and lipid synthesis compared to the wild strain,
the D PLD1 strain was chosen for the subsequent experiments and analysis.
2.5 Effect of PLD1 knockout on transcriptional levels of related genes
The transcriptional levels of genes involved in lipid metabolism in the wild-type and knockout strains
were measured during the logarithmic growth period (60 h) and lipid transformation period (108 h) (Fig.
4). The fatty acid synthase (FAS) gene encodes a series of proteins related to the de novo synthesis of
fatty acids in the form of gene clusters that can synthesize saturated 14-carbon fatty acids from acetyl
Page 7/28
CoA via the traditional FAS pathway [32]. The chain length factor (CLF) gene is located in the PKS gene
cluster and is responsible for chain lengthening during PUFA synthesis [33]. At 60 h, expression levels of
both FAS and CLF genes were significantly upregulated in the D PLD1 strain; at 108 h, in the lipid
transformation stage, the expression of CLF in the D PLD1 strain increased by 96.6%, whereas the
expression of FAS did not change significantly (Fig. 4a and b). This supports the observation that PLD1
knockout enhanced DHA synthesis (Fig. 2d). Phosphatidic acid phosphatase (PAP) catalyzes the
synthesis of DAG from PA, a hydrolytic product of PLD. PLD1 knockout improved the expression level of
PAP, which increased by 1.71 times in the lipid transformation stage compared to the wild-type strain
(Fig. 4c). This results in more PA being transformed into DAG in the D PLD1 strain, suggesting that PLD1
may play a greater role in transphosphatidylation than hydrolysis. PLD1 knockout regulates phospholipid
metabolism by weakening the transphosphatidylation of phospholipids, thereby promoting the Kennedy
pathway and improving TAG synthesis. CDP-diacylglycerol synthase (CDS) and phosphocholine cytidyl
transferase (CCT) are the key enzymes involved in phospholipid synthesis, including that of
phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), and PC. At the 106 h
timepoint, the expression level of CDS and CCT in the D PLD1 increased by 1.76 and 7.18 times (Fig. 4d
and e), respectively, compared to the control. This indicates that PLD1 knockout increases the synthesis
of phospholipids and further demonstrates that PLD1 is not responsible for hydrolysis of phospholipids
in Schizochytrium. If PLD1 played a role in hydrolytic activity, the D PLD1 strain would accumulate the
hydrolytic substrates of phospholipids and the hydrolytic product of PA would be reduced, thereby
inhibiting the synthetic pathway of phospholipids and TAG synthesis for PA. Therefore, it is assumed that
PLD1 regulates phospholipid composition by the transphosphatidylation of phospholipids, which
influences fatty acid synthesis and DHA accumulation in Schizochytrium.
For TAG synthesis, PDAT uses PC as an acyl donor and DAG as an acyl acceptor to transfer the acyl
group of a phospholipid to DAG [27]. PLD1 knockout promoted PDAT transcription at 60 h and 108 h (Fig.
4f). Correspondingly, the transcriptional level of the diacylglycerol acyltransferase gene (DGAT), which
catalyzes DAG and acyl-CoA to form TAG via the Kennedy pathway, was downregulated at both stages
(Fig. 4g). These results indicate that the knockout of PLD1 increases TAG synthesis via the acyl-CoAindependent pathway of PDAT and reduces TAG synthesis via the Kennedy pathway of DGAT, which
favors the production of unsaturated TAG [27]; this promotes DHA accumulation in TAG.
2.6 Effects of PLD1 knockout on phospholipid metabolism
2.6.1 Phospholipid content and fatty acid composition
As shown in Fig. 5, PLD1 knockout led to an apparent increase in total PLs at 120 h, probably because
PLD1 knockout reduced the transesterification activity of PLD, resulting in weakened interconversion and
enhanced de novo synthesis of PLs (Fig. 4 d and e). In Thraustochytrium sp. 26185, DHA preferentially
integrates into PLs rather than directly into TAG and then migrates to TAG to form DHA-TAGs at a later
stage [8]. Yue [18] et al. found that DHA accumulates in both TAG and PC during the growth of
Schizochytrium sp. A-2 and that DHA migrated from PC to TAG at a later stage of fermentation. The
Page 8/28
increase in total phospholipids may facilitate the assembly and accumulation of DHA, which could also
explain the increased DHA production caused by PLD1 knockout (Fig. 2d).
Table 3 shows the main bound fatty acids in the TAGs and PLs of S. limacinum SR21. Both TAG and PLs
mainly bound SFAs, which accounted for approximately 40–55%, of which C16:0 accounted for the
largest proportion (>90%). However, the proportions of PUFAs in the TAG and PLs were significantly
different. The proportion of PUFAs in TAGs was approximately 35%, including DHA, DPA, and EPA,
whereas PUFAs in PLs only accounted for 10–15%, mainly consisting of DHA and DPA. These results
indicate that PUFAs or (DHA) are mainly bound to TAG. Notably, EPA was not detected in the PLs and was
thought to be at too low a level to be detected. This suggests that EPA was mostly bound to TAG, which
provides a new theory for the study of EPA synthesis in S. limacinum SR21. Comparison of the fatty acid
composition at different time points showed that the content of PUFAs or DHA in the TAG of both strains
was higher at 120 h than at 72 h, and the content of SFAs decreased accordingly. However, the content of
PUFAs or DHA in PLs at 120 h was lower than that at 72 h. It was assumed that DHA migrated from PLs
to TAG in the later stages, which is consistent with previously reported results [18, 19]. Comparison of the
fatty acid composition of the different strains at 72 and 120 h showed that the PLD1 knockout increased
the content of DHA in TAG and decreased the content of DHA in PLs, indicating that PLD1 knockout
promoted the migration of PUFAs or DHA from PLs to TAG, which is conducive to the accumulation and
storage of DHA (Fig. 2d).
Table 3 Main fatty acid composition in TAG and PLs of wild-type strains and △PLD1 strains
Culture condition
Lipid class
Percent of fatty acids (%)
C14:0
C16:0
C18:0
SFAs
EPA
DPA
DHA
PUFAs
WT
TAG
0.12
52.0
2.43
54.6
0.14
5.64
28.8
34.6
72 h
PL
2.03
34.8
13.7
50.5
0.00
3.18
11.1
14.3
△PLD1
TAG
0.17
44.0
2.57
46.7
0.18
7.71
32.6
40.5
72 h
PL
2.55
28.9
17.0
48.5
0.00
1.05
3.81
4.86
WT
TAG
0.30
46.9
2.86
50.1
0.22
6.02
30.7
36.9
120 h
PL
1.26
25.7
13.2
40.2
0.00
3.09
6.69
9.78
△PLD1
TAG
0.26
42.6
1.67
44.5
0.20
7.75
34.0
42.0
120 h
PL
2.30
20.9
11.2
34.4
0.00
0.57
3.22
3.79
2.6.2 Phospholipid types and composition in S. limacinum SR21
The polar head structure of phospholipids determines the phospholipid species. As shown in Table 4, 43
phospholipid molecules were identified; including 15 PC, nine lysophosphatidylcholine (LPC), 10 PG, four
phosphatidylethanolamine (PE), two PI, two PS, and one PA. The three main phospholipids in S.
Page 9/28
limacinum SR21 were PC, PI, and PG; and PC accounted for half of the total PLs. PC is also the main
phospholipid in other Schizochytrium strains [18, 34, 35] and is related to DHA accumulation and
migration to TAG [18, 19]. PI has an inositol head group that plays an important role in cell signal
transduction and metabolic regulation [36]. PG is closely related to cell growth and affects the lipid
dependence of cellular stress responses and adaptation mechanisms [37]. The levels of PE, PS, and PA in
S. limacinum SR21 were low (<5%); however, they also play important roles in cell membrane structure
and function [30].
Table 4 Phospholipid species in S. limacinum SR21 (120 h)
Page 10/28
Type
Unsaturation
Acyl composition
Molecular formula
m/z
Content
(μg/g)
PC
C30:0
C14:0/C16:0
C38H76O8NP
750.5285
6.45
C32:0
C16:0/C16:0
C40H80O8NP
778.5598
16.10
C36:5
C14:0/C22:5
C44H78O8NP
824.5442
15.53
C36:6
C14:0/C22:6
C44H76O8NP
822.5285
18.19
C37:5
C15:0/C22:5
C45H80O8NP
838.5598
6.45
C37:6
C15:0/C22:6
C45H78O8NP
836.5442
19.19
C38:5
C16:0/C22:5
C46H82O8NP
852.5755
661.44
C38:6
C16:0/C22:6
C46H80O8NP
850.5598
2076.51
C40:6
C18:0/C22:6
C48H84O8NP
878.5911
14.32
C40:7
C18:1/C22:6
C48H82O8NP
876.5755
28.22
C42:10
C20:4/C22:6
C50H80O8NP
898.5598
124.96
C42:11
C20:5/C22:6
C50H78O8NP
896.5442
270.73
C44:10
C22:5/C22:5
C52H84O8NP
926.5911
58.27
C44:11
C22:5/C22:6
C52H82O8NP
924.5755
525.61
C44:12
C22:6/C22:6
C52H80O8NP
922.5598
1518.21
C14:0
C14:0
C22H46NO7P
512.2988
4.19
C15:0
C15:0
C23H48NO7P
526.3145
1.30
C16:0
C16:0
C24H50NO7P
540.3301
59.88
C18:0
C18:0
C26H54NO7P
568.3614
1.10
C18:1
C18:1
C26H52NO7P
566.3458
2.79
C18:2
C18:2
C26H50NO7P
564.3301
2.28
C20:5
C20:5
C28H48NO7P
586.3145
2.44
C22:5
C22:5
C30H52NO7P
614.3458
109.23
C22:6
C22:6
LPC
C30H50NO7P
Page 11/28
612.3301
489.56
PG
PI
PE
PS
PA
C30:0
C14:0/C16:0
C36H71O10P
693.4707
0.35
C31:0
C15:0/C16:0
C37H73O10P
707.4863
1.20
C32:0
C16:0/C16:0
C38H75O10P
721.502
147.72
C38:5
C16:0/C22:5
C44H77O10P
795.5176
11.97
C38:6
C16:0/C22:6
C44H75O10P
793.502
98.12
C40:6
C18:0/C22:6
C46H79O10P
821.5333
0.80
C40:7
C18:1/C22:6
C46H77O10P
819.5176
1.34
C42:10
C20:4/C22:6
C48H75O10P
841.502
3.60
C44:11
C22:5/C22:6
C50H77O10P
867.5176
2.57
C44:12
C22:6/C22:6
C50H75O10P
865.502
5.03
C38:5
C16:0/C22:5
C47H81O13P
883.5337
1069.19
C38:6
C16:0/C22:6
C47H79O13P
881.518
2005.56
C38:5
C16:0/C22:5
C43H76O8NP
764.523
74.82
C38:6
C16:0/C22:6
C43H74O8NP
762.5074
211.59
C44:11
C22:5/C22:6
C49H76O8NP
836.523
20.26
C44:12
C22:6/C22:6
C49H74O8NP
834.5074
56.60
C28:0
C14:0/C14:0
C34H66NO10P
678.4346
63.49
C44:12
C22:6/C22:6
C50H74NO10P
878.4972
12.93
C44:12
C22:6/C22:6
C47H69O8P
791.4652
180.21
The two acyl chains of PLs determine the phospholipid diversity. Fatty acids with different structures and
characteristics play different roles in the structure and function of PLs. C16:0 and C22:6 were the two
major fatty acids of lipids in S. limacinum SR21 (Table 4) and also the main fatty acids of acyl chains
binding to PLs (Table 3). PC tended to bind to unsaturated fatty acids (UFAs), and the two most important
types of PC were PC (16:0/22:6) and PC (22:6/22:6). The proportion of UFAs in PC was 73.3%, of which
DHA accounted for 77.7%. Yue [18] et al. reported that in Schizochytrium sp. A-2, PC (22:6/22:6) and PC
(22:5/22:6) accounted for half of the total PC. The LPC and PE in this study also showed a preference for
binding UFAs. The proportions of UFAs in LPC and PE reached 90.1% and 60.6%, respectively, of which
DHA accounted for 80.7% and 78.4%, respectively. PI bound to both SFAs and UFAs, of which DHA
accounted for 31.6%. PG and PS tend to bind SFAs, and in this study, they accounted for 75.1% and
Page 12/28
83.1% of SFAs respectively. Eriko [34] found that in Schizochytrium sp. F26-b, DHA was the main fatty
acid at the acyl ends of PC, LPC, PE, and PI, whereas there was almost no PUFA at the acyl end of PS.
Guang [35] also reported that in S. limacinum, PUFAs accounted for 86% and 71.6% of PC and PE,
respectively, and SFAs only accounted for 11.8% and 23.5 % of PC and PE, respectively. Meanwhile, SFAs
in PG reached 64.2%. These results indicate that DHA synthesized by Schizochytrium may first be
incorporated into PLs, in particular PC, for storage.
2.6.3 Phospholipidomic analysis in wild-type and D PLD1 strains
The proportions of various PLs in the wild-type and D PLD1 strains are shown in Fig. 6. Changes in PL
content with fermentation time were compared. The percentage of PG in both strains decreased
remarkably as fermentation proceeded, which may be closely related to the cell growth period. The
conversion of glucose to glycerol 3-phosphate is much easier compared to that for other phospholipid
polar head groups; therefore, it may be easier to synthesize PG with a sufficient carbon source to meet the
membrane phospholipid supply during exponential cell production. When glucose is depleted, cells stop
proliferating, and membrane PLs metabolism shifts from PL synthesis to interconversion among PLs,
resulting in phospholipid composition changes. Furthermore, a decrease in PG levels increases the
cellular stress response and lipid dependence of the adaptation mechanism [37]. We observed that the
decrease in the proportion of PG was accompanied by a drastic increase in PI. The proportion of PI in the
PLs was lower than that of PC. PI has an inositol head group, and its phosphorylation at different
positions in inositol is a decisive factor in distinguishing biofilms [38, 39]. PI is also an important
signaling molecule that regulates cell signal transduction and the metabolic process [36]. The increase in
PI at 120 h in both strains might be the result of a more active signaling pathway after entering the lipid
transformation period, especially lipid metabolism, which requires more PI for signal transduction and
metabolic regulation. LPC also increased at 120 h in both strains. LPC has one less acyl chain than PC
and exhibits a strong surface activity. A high proportion of LPC may cause cell membrane rupture and
necrosis [40]. The conversion between PC and LPC is not only related to the acyl remodelling of
phospholipids but also to the transfer of fatty acids from PC to TAG, catalyzed by PDAT [41, 42]. The
increase in LPC may be due to the rupture and necrosis of cells entering the late phase of growth or might
be related to the deacylation of PC to LPC. PC and PA levels showed different changes over time in both
strains. In the wild-type strain at 120 h, PC increased by 9.2%, whereas PA decreased by 26.2% compared
to the levels at 72 h. In the PLD1 knockout strain at 120 h, PC decreased by 20.9%, whereas PA increased
by 178.9% compared to the levels at 72 h. This further proves that PLD1 mainly plays the role of
transesterification rather than hydrolysis of PLs. The knockout of PLD1 reduced the interconversion
among PLs, which affected their composition and strengthened the migration of DHA from PC to TAG at
the lipid transformation stage (Fig. 4f). This resulted in decreased PC, increased LPC, and enhanced DHA
accumulation.
The changes in the PL content of the different strains were compared. For the three major PLs at 72 h,
knockout of PLD1 resulted in no change in PC, a 32.9% decrease in PG, and a 20.3% increase in PL
content compared to the wild-type strain. This suggests that the knockout of PLD1 mainly reduced PG
Page 13/28
synthesis at the cell growth stage, which resulted in a decrease in biomass (Fig. 2a). PI not only affects
the transport of substances between membranes by regulating ion channels, ion pumps, transporters,
endocytosis, and exocytosis but also regulates lipid metabolism and distribution and is closely related to
lipid transporters [38]. The increase in PI in the D PLD1 strain demonstrates that the knockout of PLD1
could upregulate the PI signaling pathway to increase cell metabolism. For other minor PLs at 72 h,
knockout of PLD1 showed no clear change in PA and PS content compared with the wild-type strain;
however, LPC and PE content showed a small increase. This increase in PE may be related to the mutual
transformation of lipid types in polar lipids. In addition, the phospholipid composition significantly
affects the fluidity of the cell membrane, and PE can increase the mobility of the membrane owing to its
head group and anionic properties [43]. The increase in LPC was not accompanied by a decrease in PC,
indicating that the GPC acylation pathway was activated to synthesize LPC; this is currently thought to be
the main pathway for DHA-PC synthesis [44-46]. For PG and PI at 120 h, knockout of PLD1 resulted in
similar changes to those seen at 72 h compared to the wild-type strain. However, PC showed a different
change and decreased by 27.2% at 120 h compared to the wild-type strain, indicating that PC had been
converted. Accordingly, LPC and PA showed a 45.0% and 275% increase, respectively, at 120 h compared
with the wild-type strain, indicating that the acyl migration from PC to DAG to form TAG via the acyl CoAindependent pathway [47, 48] is increased in the D PLD1 strain (Fig. 4f). This requires that more DAG
forms TAG via PDAT catalysis, thereby increasing the synthesis of PA to provide more DAG by the
Kennedy pathway (Fig. 4c) and resulting in an increase in total lipids (Fig. 2a).
Synthesized DHA may first be incorporated into PC to form DHA-PC, then channeled to DAG and
assembled into DHA-TAG for storage [18, 19]. The results of the PL content and fatty acid composition
also showed that PC tended to bind UFAs (73.3%) and that DHA accounted for 57.0% of total fatty acids
in PC (Table 4). The increase of DHA production in the D PLD1 strain (Fig. 2d) combined with the
upregulation of genes related to the fatty acid synthesis and acyl CoA-independent pathways (Fig. 4) and
the observed changes in different PLs (Fig. 6) allows us to infer the method by which DHA is synthesized
and accumulated in Schizochytrium. DHA is initially incorporated mainly into PLs, particularly PC, and
then migrated to DAG to produce TAG via the acyl CoA-independent pathway depending on PDAT
catalysis, which can be promoted by regulating phospholipid metabolism through PLD1.
3. Conclusion
This study is the first to explore the effect of PLD expression on lipid synthesis in Schizochytrium. The
genes PLD1 and PLD2 identified in S. limacinum SR21 have different effects on lipid synthesis. In S.
limacinum SR21, the knockout of PLD1 demonstrated a promoting effect on PUFA synthesis and DHA
accumulation without affecting biomass and total lipid production. PLD1 is presumed to play a role in
transphosphatidyl activity in S. limacinum SR21, and the knockout of PLD1 reduces PL interconversion
and enhances de novo synthesis of PLs, thereby improving the total PL yield and affecting their
metabolism. This results in an increase in the binding of DHA to PLs and allows DAG to be assembled
into DHA-TAG for storage by an acyl CoA-independent pathway. These results suggest that PLD exerts an
Page 14/28
important influence on the allocation of fatty acids between TAG and PLs and is closely related to the
enzyme activity of PDAT in the acyl CoA-independent pathway. Future studies should investigate its exact
mechanisms and functions.
4. Materials and methods
4.1 Strains, media, and culture conditions
The S. limacinum SR21 (ATCC MYA-1381) strain was purchased from the American Type Culture
Collection (Manassas, VA, USA) and was used as the original strain. The seed media and fermentation
broths used in this study were the same as those in our previous study [30]. The modified inorganic salt
stock solution contained Na2SO4, 240 g/L; MgSO4, 40 g/L; kH2PO4, 20 g/L; (NH4)2SO4, 20 g/L; K2SO4, 13
g/L; and KCl, 10 g/L. The medium was sterilized at 115 °C for 20 min before use. The trace element and
vitamin solutions were filter-sterilized using a 0.2 μm filter. The activated single colonies were cultured in
seed medium at 28 °C, 200 rpm (SKY-2102C double-layer thermostatic shaker, Suzhou, China) for 24 h.
After the second generation culture, the seed culture (4% v/v) was transferred to the fermentation broth
and incubated at 28 °C and 200 rpm for 144 h or longer.
4.2 Mining of PLD genes
PLD-related information for S. limacinum SR21 was obtained from the Joint Genome Institute (JGI)
database. The similarity between the PLD gene sequence and the corresponding protein sequence was
analyzed using DNAstar Megalign (Madison, WI, USA), and phylogenetic tree analysis of the selected PLD
genes was performed using the NCBI database.
4.3 Construction of gene knockout strains
PLD knockout strains were constructed using homologous recombination technology, as shown in Fig.
S5. The up- and downstream sequences of PLD were amplified by PCR using primers from S.
limacinum SR21 genomic DNA (Table S1). The PCR reagent PrimeSTAR HS (Premix) was purchased
from TaKaRa Bio (TaKaRa Biotechnology Co.,Ltd, Dalian, China), and PCR primers were synthesized by
Xiamen Boshang Biotechnology Company. The pBlue-zeo plasmid was previously constructed in our
laboratory [30] and included an integration region with multiple cloning sites and zeocin expression
cassettes for resistance screening. The plasmid pBlue-zeo-PLD was constructed by inserting the
homologous arm of the PLD gene into the multiple cloning site of the vector.
The disrupted fragment was PCR-amplified with primers from the constructed vectors and transformed
into S. limacinum SR21 by electroporation, according to the method by Ling et al. [30]. The disrupted
fragment combined the targeted gene with the homologous arms to replace it [31]. After electroporation,
the cells were cultivated for 3 h at 28 °C in a seed medium containing 1 M sorbitol (Macklin Biochemical,
Shanghai, China) and then recovered for 3–5 days in a solid medium containing 50 μg/mL zeocin
(Sangon Biotech, Shanghai, China). The positive transformants were screened by zeocin resistance
Page 15/28
plates and cultured at 28 °C and 200 rpm in a shaker (SKY-2102C double-layer thermostatic shaker) for
PCR validation and fermentation experiments.
4.4 Measurement of biomass and glucose content
One milliliter of fermentation broth was added to a 1.5 mL centrifuge tube and centrifuged at 10,000 rpm
for 2 min at 28 ℃. The supernatant was collected to measure glucose concentration using the 3, 5dinitrosalicylic acid (DNS) method. The cell pellets were washed twice with normal saline and stored in a
refrigerator at −20 °C. The biomass was calculated after 24 h of vacuum freeze-drying.
4.5 Lipid extraction and fatty acid composition analysis
Three milliliters of fermentation broth was mixed with 4 mL 12 M HCl and incubated in a water bath at 65
°C for 45 min. Total lipids (TLs) from the mixture were extracted four times with 3 mL of n-hexane, and
the lipid extract was then purified and dried by evaporation. Total fatty acid (TFA) production was
calculated by subtracting unsaponifiable matter (UM) from TLs; UM was isolated from lipids by
saponification [49]. The preparation of fatty acid methyl esters and analysis of fatty acid composition
were performed as previously described [30].
4.6 Real-time quantitative PCR (qRT-PCR)
Total RNA was extracted from 1 mL of broth using the RNA Plant Plus reagent (TaKaRa, Japan)
according to the manufacturer’s instructions, and 1 μg from each sample was reverse-transcribed using
the QuantScript RT Kit reagent (TaKaRa, Japan). qRT-PCR amplification was performed using the One
Step SYBR PrimeScript PLUS RT-PCR Kit (TaKaRa, Japan). Primers used are listed in Table S2. The mRNA
expression level was normalized using the actin gene as the internal control, and the relative gene
expression level was calculated by using 2−ΔΔCT method [50].
4.7 Quantification of phospholipids
Lipids were first extracted from S. limacinum SR21 using the Bligh–Dyer method [51] with some
modifications. Five milliliters of fermentation broth was added to 2 mL of deionized water, 3 mL of
chloroform, and 6 mL of methanol before ultrasonication for 30 min; 3 mL of chloroform was added for
ultrasonication for 30 min. Then, 3 mL of deionized water was added, followed by incubation for 30 min.
One milliliter of saturated NaCl solution was then added the solution allowed to stand for 2 h. The
solutions were centrifuged at 3,000 ×g for 10 min at 28 ℃. After centrifugation, the lower layer of the
lipids was transferred to a glass bottle and dried under a nitrogen stream. The obtained lipid samples
were weighed and stored at −20 °C until required. Phospholipids were separated from the lipid samples
using solid-phase extraction [52].
Page 16/28
Phospholipids were determined using ultra performance liquid chromatography-mass
spectrometry (Waters UPLC Acquity H-Class-Xevo-G2 Q-ToF, Milford, MA, USA). The chromatographic
experimental conditions were as follows: chromatographic column: ACQUITY UPLC BEH HILIC column
(150 mm × 2.1 mm × 1.7 μm); mobile phase: A is acetonitrile, and B is 20 mM ammonium formate
aqueous solution (0.1% formic acid added to obtain pH 3.5); the flow rate was 0.2 mL/min; injection
volume: 2 μL; elution procedure: 0–4 min, 95% A and 5% B; 4–22 min, 95–60% A and 5–40% B; 22–25
min, 60 % A and 40% B; 25–25.1 min, 60–95 %A and 40–5% B; 25.1–30 min, 95% A and 5% B. The mass
spectrometry experimental conditions were as follows: electrospray negative ion mode (ESI-); analyzer
mode: sensitivity mode; data acquisition time: 2.5–20 min; scanning range: 250–1000 Da; scanning time:
0.5 s; capillary voltage: 2 kV; sample cone voltage: 30 V; extraction cone voltage: 4 V; ion source
temperature: 100 °C; desolvation gas temperature: 350 °C; cone gas flow rate: 50 L/h; the desolvation gas
flow rate was 400 L/h.
4.8 Statistical analysis
Statistical significance between different strains or groups was evaluated using a t-test; 0.01 < P < 0.05
was considered statistically significant, and P < 0.01 was considered extremely significant. Three parallel
experiments were conducted. Values are expressed as the means ± SD (standard deviation).
Declarations
Authors' contributions
XPL and YHL conceived and designed the research. YTZ and XWC conducted the experiments and wrote
the manuscript. SZL contributed analytical tools. HL, TL and YTZ analyzed the data. XPL, MFC and YHL
modified the manuscript. All authors read and approved the manuscript.
Data availability
The authors can confirm that all relevant data are included in the article and supplementary materials
files.
Acknowledgments
We thank Editage (https://www.editage.cn) for English language editing during the preparation of this
manuscript. We gratefully acknowledge the Analysis and Test Center of the Third Institute of
Oceanography for its continuous support.
Funding
This work was supported by the Natural Science Foundation of Xiamen, China (No.
3502Z20227183), National Key Research and Development Program of China (No. 2022YFC2104600),
and National Natural Science Foundation of China (No. 31871779).
Page 17/28
Declaration of competing interests
The authors declare no competing financial interests or personal relationships that influence the work
reported in this study.
Ethical approval
This study does not contain any studies with human participants or animals performed by any of the
authors.
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Figures
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Figure 1
Phylogenetic tree analysis of the (a) PLD-46 and (b) PLD-100463 gene sequences of S. limacinum SR21.
Bootstrap values (>50%, repeated 1,000 times) are displayed on each internal branch
Page 22/28
Figure 2
(a) Biomass and total lipids, (b) glucose concentration, (c) lipid content, and (d) DHA yield of the wild-type
and the ΔPLD1 strains. * and ** represent 0.01 < P < 0.05 and P < 0.01, respectively. All data are
expressed as mean ± SD of three independent experiments
Page 23/28
Figure 3
(a) Biomass and total lipids, (b) glucose concentration, (c) lipid content, and (d) DHA yield of the wild-type
and the ΔPLD2 strains. * and ** represent 0.01 < P < 0.05 and P < 0.01, respectively. All data are
expressed as mean ± SD of three independent experiments
Page 24/28
Figure 4
Transcription levels of related genes in wild-type and ΔPLD1 strains: (a) fatty acid synthase (FAS), (b)
chain length factor (CLF), (c) phospholipid acid phosphatase (PAP), (e) CDP-diacylglycerol synthase
(CDS), (e) choline phosphate transferase (CCT), (f) phosphatidylglycerol acyltransferase (PDAT), (g)
diacylglycerol acyltransferase (DGAT). There was a significant difference at the 0.01 < P < 0.05 (*) or P <
Page 25/28
0.01 (**) level between the wild-type strain and the ΔPLD1 strain. All data were expressed as mean ± SD
of three independent experiments
Figure 5
Phospholipid content in 72 and 120 h samples of the wild-type and ΔPLD1 strains. All data were
expressed as mean ± standard deviation (mean ± SD) of three independent experiments
Page 26/28
Figure 6
Fractionation of total phospholipids extracted from wild strain and ΔPLD1 strains at 72 h and 120 h,
respectively. PC, phosphatidylcholine; LPC, lysophosphatidylcholine; PG, phosphatidylglycerol; PI,
phosphatidylinositol; PE, phosphatidylethanolamine; PS, phosphatidylserine; PA, phospholipid acid. All
data were expressed as mean ± standard deviation (mean ± S.D.) of three independent experiments
Page 27/28
Supplementary Files
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Supplementarymaterialsforsubmission.pdf
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https://openalex.org/W4283786673
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https://nottingham-repository.worktribe.com/preview/12892630/s42234-022-00099-7.pdf
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English
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Electric Field Responsive Nanotransducers for Glioblastoma
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bioRxiv (Cold Spring Harbor Laboratory)
| 2,022
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cc-by
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Abstract Background: Electric field therapies such as Tumor Treating Fields (TTFields) have emerged as a bioelectronic treat-
ment for isocitrate dehydrogenase wild-type and IDH mutant grade 4 astrocytoma Glioblastoma (GBM). TTFields
rely on alternating current (AC) electric fields (EF) leading to the disruption of dipole alignment and induced dielec-
trophoresis (DEP) during cytokinesis. Although TTFields have a favourable side effect profile, particularly compared
to cytotoxic chemotherapy, survival benefits remain limited (~ 4.9 months) after an extensive treatment regime (20
hours/day for 18 months). The cost of the technology also limits its clinical adoption worldwide. Therefore, the discov-
ery of new technology that can enhance both the therapeutic efficiency and efficacy of these TTFields will be of great
benefit to cancer treatment and decrease healthcare costs worldwide. Methods: In this work, we report the role of electrically conductive gold (GNPs), dielectric silica oxide (SiO2), and
semiconductor zinc oxide (ZnO) nanoparticles (NPs) as transducers for enhancing EF mediated anticancer effects
on patient derived GBM cells. Physicochemical properties of these NPs were analyzed using spectroscopic, electron
microscopy, and light-scattering techniques. Results: In vitro TTFields studies indicated an enhanced reduction in the metabolic activity of patient-derived Glioma
INvasive marginal (GIN 28) and Glioma contrast enhanced core (GCE 28) GBM As per our journal style, article titles
should not include capitalisedletters unless these are proper nouns/acronyms. We have therefore usedthe article title
“Electric field responsive nanotransducers forglioblastoma” as opposed to “Electric Field Responsive Nanotransduc-
ersfor Glioblastoma” as given in the submission system. Please check ifthis is correct.cells in groups treated with NPs
vs. control groups, irrespective of NPs dielectric properties. Our results indicate the inorganic NPs used in this work
enhance the intracellular EF effects that could be due to the virtue of bipolar dielectrophoretic and electrophoretic
effects. Conclusions: This work presents preliminary evidence which could help to improve future EF applications for bio-
electronic medicine. Furthermore, the merits of spherical morphology, excellent colloidal stability, and low toxicity,
make these NPs ideal for future studies for elucidating the detailed mechanism and efficacy upon their delivery in
GBM preclinical models. Keywords: Tumor Treating Fields, Inorganic nanoparticles, Electric fields, Glioblastoma Bioelectronic Medicine Bioelectronic Medicine Bioelectronic Medicine Jain et al. Bioelectronic Medicine (2022) 8:17
https://doi.org/10.1186/s42234-022-00099-7 Open Access © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. Background While the
full extent of how TTFields work is currently unclear,
two proposed mechanisms explain the mode of action:
dipole alignment and DEP. In the first instance, spindle
formation during mitosis is affected. Microtubules are
influenced by the dipole moment of the building blocks,
therefore cell division is limited (Kirson et al., 2004). In
the second instance, an inhomogeneous distribution of
the EF within dividing cells causes molecules to become
polarised. These polar molecules then move to regions
with higher EF intensities, notably the cleavage furrow in
mitotic cells, and thereby interfere with cytokinesis (Kir-
son et al., 2007).f Despite TTFields affecting rapidly dividing cancer cells,
there appears to be no effect on healthy cells with rela-
tively slower cell division. Even cells that exhibit rapid cell
division such as those found in bone marrow or intestine
are not affected as they are protected by high impedance
of the bone, and slower replication times compared to
cancerous cells respectively (Blatt et al., 2021). Tight junctions between epithelial cells at the blood-
brain barrier (BBB) are known to inhibit the influx
and efflux of many molecules to the brain (Brightman
& Reese, 1969; Reese & Karnovsky, 1967). This barrier
can be overcome by taking advantage of the leaky vas-
culature surrounding brain tumours which leads to the
accumulation of NPs in the tumour, via the Enhanced
Permeation and Retention (EPR) effect. NPs are fre-
quently utilised to take advantage of this EPR effect,
with studies showing that NPs of a size range of 20-100
nm are ideal to allow for maximum accumulation in
the cells, tumour and longer clearance times (Perrault
et al., 2009). Gold NPs (GNPs) are of particular interest
for biomedical and bioelectronics applications due to During last decade, there has been much experi-
ment work into the mechanism of action of TTFields,
but there has been limited efforts to enhance TTFields
using NPs. One example is biocompatible barium titan-
ate NPs (BTNPs) with a high dielectric constant, which
were investigated as breast cancer sensitisers in cells that
were resistant to TTFields (Yoon et al., 2020). This study
found that BTNPs accumulate within the cytoplasm
when exposed to TTFields where they are then polar-
ised by the inhomogeneous EF as discussed earlier, caus-
ing the BTNPs to migrate to the cleavage furrow and the
cells to undergo apoptosis. Background This significant improvement to OS is not
accompanied by any major side effects, with the only
reported effect being contact dermatitis at the site of the
electrodes. their biocompatibility and tuneable properties (Shukla
et al., 2005; Perrault & Chan, 2009; Sanjuan-Alberte
et al., 2019). In recent years there have been numerous
clinical trials utilising GNPs to treat a range of can-
cers, including glioblastoma (Libutti et al., 2010; Gad
et al., 2012; Kumthekar et al., 2021). ZnO NPs (semi-
conductors) and SiO2 NPs (insulators), have also been
well researched as a potential treatment for cancers,
with the former showing the preferential killing of can-
cer cells over normal cells, while the latter has shown
advantages of being highly tuneable, allowing for tar-
geted drug delivery (Hanley et al., 2008; Wang et al.,
2009; Murugan et al., 2017). g
Electric fields have been used as external stimuli for the
delivery of drugs from NPs for cancer and tissue regener-
ation (Kolosnjaj-Tabi et al., 2019). However, the behavior
of nanoparticles under EFs in cellular environment needs
further studies. Apart from the classic endocytosis medi-
ated uptake of NPs in cancer cells, electric fields have
shown to enhance the uptake of NPs by permeabilizing
cancer cell membranes via electroporation and by modu-
lating bioelectricity through voltage-gated ion channels
(Aguilar et al., 2021; Chang et al., 2018). This has opened
new area of research to develop new tools to study intra-
cellular interaction of NPs with EFs. Apart from the well-
known electrophoretic and dielectrophoretic movement
of NPs, EFs have been shown to induce electrostatic
induction and charge separation of nanomaterials. This
electric polarization leads to generation of numerous
bipolar nanoelectrodes which acts as transducer of EFs
(Guo et al., 2021). Using modelling approaches Tiwari
et al. demonstrated that spherically capped gold nanow-
ire enhanced EFs inside the cells. They inferred that due
to the uniform and homogenous distribution of EFs over
nanomaterials (due to charge separation is over a short
distance ≈ nm) addition source and sinks are gener-
ated. This causes a local enhancement in electric field
strengths around nanomaterials in contact with cytoplas-
mic entities leading to the rupture of plasma membrane
and eventually apoptosis (Tiwari et al., 2009). TTFields are directional, mainly influencing cell behav-
iour when the electric field and axis of cell division are
parallel to one another (Kirson et al., 2007). Background Isocitrate dehydrogenase wild-type GBM is a form of
highly aggressive brain tumour accounting for 49.1% of
primary malignant brain tumours with less than 7% of
patients surviving after 5 years post-diagnosis (Ostrom
et al., 2021). The current standard of care is known as
the ‘Stupp regimen’ and consists of surgical resection *Correspondence: Akhil.Jain@nottingham.ac.uk; Frankie. Rawson@nottingham.ac.uk 1 Bioelectronics Laboratory, Division of Regenerative Medicine and Cellular
Therapies, School of Pharmacy, Biodiscovery Institute, University
of Nottingham, Nottingham NG7 2RD, UK
Full list of author information is available at the end of the article 1 Bioelectronics Laboratory, Division of Regenerative Medicine and Cellular
Therapies, School of Pharmacy, Biodiscovery Institute, University
of Nottingham, Nottingham NG7 2RD, UK
Full list of author information is available at the end of the article © 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/. Jain et al. Bioelectronic Medicine (2022) 8:17 Jain et al. Bioelectronic Medicine (2022) 8:17 Page 2 of 9 Jain et al. Bioelectronic Medicine followed by treatment with radiotherapy and the alkylat-
ing chemotherapeutic agent, temozolomide, increasing
overall survival (OS) to a median of 14.6 months (Stupp
et al., 2005). Since this finding in 2005, there has been
little progression in the identification of new treatments
for GBM that are United States Food and Drug Adminis-
tration (FDA) approved, except for TTFields. Indeed, no
molecular targeted therapeutics predicated on genome
biology has shown efficacy in phase III trials to date. These low intensity (<4 V/cm), intermediate frequency
(100-500 kHz) and AC EFs, have been shown to further
enhance cell death when in conjunction with the Stupp
regimen, increasing OS by a median of 4.9 months (Stupp
et al., 2017). TTFields GIN 28 and GCE 28 cells were seeded on a 22 mm cell
culture treated coverslip at a density of 3.5 × 104 and
incubated for 24 hours at 37°C and 5% CO2. Next, the
media was replaced with media containing GNPs/SiO2/
ZnO NPs at a concentration of 5 μg/mL or 25 μg/mL and
incubated at 37°C for 4 hours. Next, the coverslips were
transferred to ceramic Petri dishes of the inovitro™ sys-
tem (Novocure, Haifa, Israel). Finally, the TTFields were
applied for a duration of 48 or 72 hours using the ino-
vitro system which consists of two pairs of perpendicular
transducer arrays on the outer walls of the ceramic plate
containing the Petri dishes. A sinusoidal waveform gen-
erator was attached to the transducer arrays producing
alternating EFs set at a frequency of 300 kHz and 1V/cm
intensity. TTFields of 300 KHz was chosen for this work,
this is based on previous studies that used range of fre-
quencies from 200-300 KHz for GBM cells (Branter et al.,
1982; Linder et al., 2021). TTFields were applied bi-direc-
tionally (perpendicular to each other), which switches
between the two direction every second. The temperature
was measured to be 37°C inside the dishes by thermistors
attached to the ceramic walls. Finally, the change in met-
abolic activity/ viability of GIN 28 and GCE 28 cells in
response to TTFields or NP + TTFields, was determined
using the PrestoBlueTM assay as described above. Methods
Materials All the reagents were of analytical grade and were used
as supplied without further purification unless speci-
fied. Citrate-capped spherical GNPs, ZnO, and SiO2 NPs
of size 50 nm were purchased from Sigma Aldrich, UK. PrestoBlue cell viability reagent and cell culture treated
22 mm coverslips (Nunc™ Thermanox™ Coverslips) were
purchased from ThermoFisher Scientific, UK. In vitro toxicity In vitro toxicity
HA-COR, GIN 28 and GCE 28 cells were seeded in a
96-well plate at a density of 4.5 × 103 cells per well and
incubated for 24 hours at 37°C and 5% CO2. Culture
media was replaced with medium containing GNPs/
SiO2/ZnO NPs (concentration = 0.1, 0.5, 1, 2, 5, 10, 20, 50
or 100, 200 μg/ mL) and incubated for 4 hours. Next, the
media was replaced with fresh media and cells were incu-
bated for 48 or 72 hours. Finally, metabolic activity was
determined using PrestoBlueTM. For each well, the media
was replaced with media containing 10% PrestoBlue cell
viability reagent and incubated at 37°C for 2 hours in an
incubator before transferring the coloured metabolic
product to a black-bottom 96-well plate. Finally, the
fluorescence of the plate was read using a plate reader
(TECAN Infinite 2000) with an excitation wavelength of
570 nm and an emission wavelength of 600 nm. Mean ±
S. D values are presented relative to negative controls. Background While this study is the first
example of using NPs to enhance TTFields, BTNPs are Jain et al. Bioelectronic Medicine (2022) 8:17 Jain et al. Bioelectronic Medicine Page 3 of 9 not FDA-approved, creating a barrier to translating these
findings to a clinical setting. were maintained at 37°C in an incubator with humidi-
fied atmosphere, containing 5% CO2. Cells were routinely
tested for mycoplasma where they were grown in an
antibiotic-free medium for one week before mycoplasma
testing. All cells used were mycoplasma-free. i
g
g
Here, we investigate the underlying mechanism with
the hypothesis that conductive particles would enhance
the effects of TTFields. NP-enhanced TTFields mecha-
nism of action was investigated, which is of paramount
importance for the discovery of new approaches that
can enhance these TTFields-induced anticancer effects. The NPs chosen were gold, zinc oxide (ZnO), and silica
(SiO2), which are FDA-approved conductive, semi-
conductive, and insulating NPs respectively (Rasmus-
sen et al., 2010; Zhou et al., 2018). As the two suggested
modes of TTFields action are due to dipole alignment
and DEP, the effects of using GNPs and ZnO NPs with
different electrical conductivities were chosen to address
the first mechanism, while dielectric SiO2 NPs have
potential to enhance TTFields by the second mechanism. From a clinical application perspective, the overall toxic-
ity, pharmacokinetics, and therapeutic efficacy of the NPs
must be evaluated in an accurate in vitro model that can
reflect the cancer heterogeneity observed in clinics. Fur-
thermore, as observed in clinics, the efficacy of TTFields
varies across patients which is attributed to heterogeneity
in GBM. Therefore, we utilised patient-derived GIN 28
(isolated from the invasive margin) and GCE 28 (isolated
from the contrast-enhanced core), which reflect GBM
tumour characteristics that are observed clinically. Cell culture GIN 28 cells were isolated from the 5-aminolevulinic acid
(5ALA) fluorescing infiltrative tumour margin and GCE
28 were isolated from the core central region of a GBM
patient who underwent surgery at the Queen’s Medi-
cal Centre, University of Nottingham (Nottingham, UK)
using the method described earlier (Smith et al., 2017;
Smith et al., 2020). Low-passage patient-derived GIN 28
and GCE 28 cells were cultured in DMEM (Gibco) sup-
plemented with 10% FBS, 1% Penicillin/Streptomycin,
and 1% L-Glutamine. Human derived cortical astrocytes
(HA-COR) were obtained from ScienCell (Cat. No. 1800,
Batch No. 24490) and were cultured in astrocyte medium
(AM) containing 2% FBS, 1% astrocyte growth supple-
ment, 1% Penicillin/Streptomycin from ScienCell. Cells Page 4 of 9 Jain et al. Bioelectronic Medicine (2022) 8:17 Jain et al. Bioelectronic Medicine (2022) 8:17 Jain et al. Bioelectronic Medicine resonance of 45 nm GNPs (Haiss et al., 2007), while the
365 nm peak in ZnO arises from the intrinsic band-gap
absorption due to electron transitions from the valence
band to the conduction band (O → Zn ) (Khokhra et al.,
2017). Furthermore, a sharp and narrow absorption peak
is a characteristic of uniform dispersion of monodisperse
GNPs and ZnO NPs. FTIR of SiO2 NPs (Fig. 1E) showed
two broad absorption peaks cantered at 795 cm-1 and
1055 cm-1 corresponding to bending vibrational modes
of the Si-O-Si groups (Brassard et al., 2011). To under-
stand the colloidal behaviour of these NPs, we carried out
ζ (Fig. 1F) and dynamic light scattering (DLS) (Fig. 1G)
measurements to determine their hd and surface charge. Previous studies have reported that ζ and hd of NPs can
influence their interaction with cells. For instance, NPs
with smaller hd have higher diffusion constant but weak
interaction with cells and vice versa with ζ (Villanueva-
Flores et al., 2020). Furthermore, surface charge and hd
plays a key role in determining the polarization and
movement of conducting and insulating NPs under the
influence of EF via electrophoresis and DEP, respectively
(Zhao & Bau, 2009). Therefore, it is important to balance
hd and ζ of the selected NPs for optimal cellular and EF
interaction. In general, NPs with ζ values of ≥ - 30 mV
or ≥ + 30 mV are considered to have optimal colloidal
stability for biological application due to the presence of
sufficient repulsive forces (Skoglund et al., 2017). Cell culture Citrate
capped GNPs and ZnO NPs showed a mean ζ value of
– 39.2 ± 2.2 mV and -36.5 ± 1 mV which is attributed
to the charge of citrate and oxide ions, respectively, sug-
gesting good physical colloidal stability. In contrast, SiO2
NPs showed a positive zeta potential value of +12.2 ± 0.9
mV due to the presence of -NH2 groups on the surface
of these NPs indicating presence of weak repulsive forces
and moderate colloidal stability. DLS analysis indicated
a monodisperse sample of GNPs with a hd of 43.8 ± 3.2
nm. A slight increase in the size of ZnO (hd = 69.4 ± 6.7
nm ) and SiO2 (hd = 50.7 ± 5.5 nm) NPs compared to
TEM measurements, further confirmed the agglomera-
tion of these NPs in colloidal solution. Collectively, the
data indicate that these inorganic NPs have optimal phys-
icochemical properties for biological applications. Physicochemical Properties of NPs Physicochemical Properties of NPs Inorganic NPs such as GNPs, SiO2, and ZnO present
several advantages for biological application such as
excellent biocompatibility, wide surface conjugation
chemistry, and colloidal stability (Sperling et al., 2008;
Hosseinpour et al., 2020; Jiang et al., 2018). Importantly
due to a large difference in the dielectric constant (SiO2
> ZnO > GNPs) (Abdelhalim et al., 2011; Ahmad et al.,
2019; Dutta & De, 2007), these NPs are best suited to gain
further insight into the role of EF in GBM therapy. Previ-
ous literature indicates that nano-bio interaction depends
on size; therefore, we chose spherical ~50 nm NPs to
demonstrate EF effects as this NP diameter is shown to
be optimum for achieving high cell uptake (Shang et al.,
2014; Zhu et al., 2013; Chithrani et al., 2006; Mittag et al.,
2021). Characterizationh Collectively, the
data indicate that these inorganic NPs have optimal phys-
icochemical properties for biological applications. The size and morphology of the NPs were analyzed
using a transmission electron microscope (JEOL 2000
FX TEM) operating at 200 kV accelerating voltage. TEM
samples were prepared by dropping 15 mL of NP solu-
tion on a carbon-coated copper grid (400 Mesh, Agar
Scientific), where the samples were allowed to sit on
the grid for at least 15 minutes before analyses. Fourier-
transform infrared spectroscopy (FTIR) was carried out
by drying silica NPs at 37°C for 48 hours and finally plac-
ing the dry sample onto a Cary 630 FTIR spectrometer
(Agilent Technologies Ltd) for the measurement of trans-
mittance spectra. UV-Vis absorption spectrum of ZnO
and GNPs was recorded on a Cary 3500 UV-Vis (Agilent
Technologies Ltd). The hydrodynamic diameter (hd) and
Zeta potential (ζ) of the NPs were monitored on a Mal-
vern Zetasizer Nano-ZS (Malvern Instruments, UK). Statistical analysis All the statistical analyses were performed using Graph-
Pad Prism v9.2.0 software (GraphPad Software, Inc). All the data are expressed as mean ± S.D., unless speci-
fied. For responses that were affected by more than one
variable, a two-way ANOVA with a Tukey multiple com-
parison post-test was used, and a p-value of ≥ 0.05 was
considered significant. Characterizationh resonance of 45 nm GNPs (Haiss et al., 2007), while the
365 nm peak in ZnO arises from the intrinsic band-gap
absorption due to electron transitions from the valence
band to the conduction band (O → Zn ) (Khokhra et al.,
2017). Furthermore, a sharp and narrow absorption peak
is a characteristic of uniform dispersion of monodisperse
GNPs and ZnO NPs. FTIR of SiO2 NPs (Fig. 1E) showed
two broad absorption peaks cantered at 795 cm-1 and
1055 cm-1 corresponding to bending vibrational modes
of the Si-O-Si groups (Brassard et al., 2011). To under-
stand the colloidal behaviour of these NPs, we carried out
ζ (Fig. 1F) and dynamic light scattering (DLS) (Fig. 1G)
measurements to determine their hd and surface charge. Previous studies have reported that ζ and hd of NPs can
influence their interaction with cells. For instance, NPs
with smaller hd have higher diffusion constant but weak
interaction with cells and vice versa with ζ (Villanueva-
Flores et al., 2020). Furthermore, surface charge and hd
plays a key role in determining the polarization and
movement of conducting and insulating NPs under the
influence of EF via electrophoresis and DEP, respectively
(Zhao & Bau, 2009). Therefore, it is important to balance
hd and ζ of the selected NPs for optimal cellular and EF
interaction. In general, NPs with ζ values of ≥ - 30 mV
or ≥ + 30 mV are considered to have optimal colloidal
stability for biological application due to the presence of
sufficient repulsive forces (Skoglund et al., 2017). Citrate
capped GNPs and ZnO NPs showed a mean ζ value of
– 39.2 ± 2.2 mV and -36.5 ± 1 mV which is attributed
to the charge of citrate and oxide ions, respectively, sug-
gesting good physical colloidal stability. In contrast, SiO2
NPs showed a positive zeta potential value of +12.2 ± 0.9
mV due to the presence of -NH2 groups on the surface
of these NPs indicating presence of weak repulsive forces
and moderate colloidal stability. DLS analysis indicated
a monodisperse sample of GNPs with a hd of 43.8 ± 3.2
nm. A slight increase in the size of ZnO (hd = 69.4 ± 6.7
nm ) and SiO2 (hd = 50.7 ± 5.5 nm) NPs compared to
TEM measurements, further confirmed the agglomera-
tion of these NPs in colloidal solution. In vitro Toxicity of NPs TEM analysis revealed that the mean diameter of GNPs
is ~ 42 ± 4 nm (Fig. 1A) with homogenous spherical
morphology, while SiO2 (Fig. 1B) and ZnO (Fig. 1C) were
observed to agglomerate with a mean diameter of ~ 46 ±
7 nm and ~ 40 ± 11 nm, respectively. UV-Vis spectros-
copy of GNPs and ZnO NPs (Fig. 1D) dispersed in phos-
phate buffer saline (PBS) showed distinctive absorption
peaks at 529 nm and 365 nm, respectively. The absorp-
tion peak at 529 nm is attributed to surface plasmon Before investigating the effect of using NPs in conjunc-
tion with TTFields, it was important to ascertain the
toxicity of the different NPs on HA-COR (healthy cells),
GIN 28 and GCE 28 cells. An experiment was therefore
carried out to investigate the effect of increasing the NP
concentration from 0 to 50 μg/ mL, by using PrestoBlue
assay, which reports on the metabolic activity of cells as
an indicator of cell viability (Peng et al., 2020; Xu et al.,
2015). However, the limitation of this assay is that it does Jain et al. Bioelectronic Medicine (2022) 8:17 Page 5 of 9 Jain et al. Bioelectronic Medicine (2022) 8:17 Fig. 1 Physico-chemical characterization of inorganic NPs (Gold – GNPs; Zinc Oxide – ZnO; Silica oxide – SiO2). Transmission electron microscopy
images of (A) GNPs, (B) SiO2 and (C) ZnO NPs. D UV-Vis absorption spectrum of ZnO and GNPs in PBS; E FTIR spectrum of SiO2 NPs; F Zeta potential;
and G Hydrodynamic diameter obtained using DLS. Error bars represent the standard deviation of the mean n=3; N= 3 Fig. 1 Physico-chemical characterization of inorganic NPs (Gold – GNPs; Zinc Oxide – ZnO; Silica oxide – SiO2). Transmissio
images of (A) GNPs, (B) SiO2 and (C) ZnO NPs. D UV-Vis absorption spectrum of ZnO and GNPs in PBS; E FTIR spectrum of S
and G Hydrodynamic diameter obtained using DLS. Error bars represent the standard deviation of the mean n=3; N= 3 Fig. 1 Physico-chemical characterization of inorganic NPs (Gold – GNPs; Zinc Oxide – ZnO; Silica oxide – SiO2). Transmission electron microscopy
images of (A) GNPs, (B) SiO2 and (C) ZnO NPs. D UV-Vis absorption spectrum of ZnO and GNPs in PBS; E FTIR spectrum of SiO2 NPs; F Zeta potential;
and G Hydrodynamic diameter obtained using DLS. TTFields and NPs Mediated Enhanced EF Effects in GBM
Cells enhancement with conductive GNPs and semiconducting
ZnO NPs, can be further explained by the ability of these
conducting and semiconducting NPs to polarize and align
themselves with the applied EF to act as bipolar nanoelec-
trodes or transducers (Guo et al., 2021; Cao et al., 2018; Li
& Anand, 2017). Furthermore, the observed enhanced elec-
tric effects could also be due to the bipolar electrophoretic
effect, as both the GNPs and ZnO are negatively charged. Moreover, TTFields are known to induce biophysical forces
on charged entities, this could have triggered the intracellu-
lar movement of GNPs and ZnO, thus enhancing the forces
on polar intracellular structures affecting dipole alignment
and disruption of mitotic spindle. In contrast, SiO2 is a
well-known dielectric material which upon cellular uptake
increases the impedance of cells. Furthermore, under
the influence of TTFields (non-uniform EFs) these NPs
experience dielectrophoretic forces which induces their
polarization. This polarization could lead to the dielectro-
phoretic movement of these particles towards the pole with
higher EF intensity thus enhancing the classic intracellular
TTField effects (Rominiyi et al., 2021). Cells
Encouraged by the optimal biocompatibility of ZnO NPs
(≤ 5 μg/mL), GNPs (≤ 50 μg/mL) and SiO2 (≤ 50 μg/mL),
we then investigated the role of these inorganic NPs in
enhancing EF effects in patient derived GBM cells. Dielec-
tric properties of tissues, as well as intracellular machin-
ery, play an important role in determining the efficacy of
TTFields as they are known to inhibit the proliferation of
cancer cells by inducing DEP of proteins involved in the
cell division process (Hershkovich et al., 2019; Wenger
et al., 2015). Therefore, we hypothesised that by intro-
ducing NPs of different electrical conductivity (dielectric
properties), the impedimetric properties of the cells can
be modified, allowing further insights into the EF medi-
ated cellular response. TTFields were delivered in GIN
28 and GCE 28 cells using the Inovitro laboratory system
(Novocure, Haifa, Israel, schematic shown in Fig. 3A) that
replicates the effect of the clinically used technique (the
OptuneTM device). Before the application of TTFields,
cells were incubated for 4 hours with either GNPs/ZnO/
SiO2 NPs to allow uptake. We observed that the applica-
tion of TTFields alone (300 kHz and1V/cm) for 48 hours
decreased the metabolic activity of both GIN 28 (Fig. 3B)
and GCE 28 cells (Fig. 3C) by ~ 25% vs. untreated con-
trol (p < 0.0003). In vitro Toxicity of NPs Error bars represent the standard deviation of the mean n=3; N= 3 concentration of 50 μg/ mL, and at 10 μg/ mL for GIN
28 and GCE 28 cells. This decrease in metabolic activ-
ity by ZnO NPs (concentration = > 5 μg/mL) has been
attributed to the generation of reactive oxygen species
at higher concentrations (Liu et al., 2017). Nevertheless,
the FDA has classified ZnO NPs as a “GRAS” (generally
regarded as safe) at lower concentrations (Rasmussen
et al., 2010). Based on the obtained data, a concentra-
tion of 5 μg/ mL of each type of NPs was chosen for the
in vitro TTFields experiment, as at this concertation no
significant change in the metabolic activity of GBM cells
was observed for all three types of the NPs. not identify the mechanism of change in metabolic activ-
ity. In the cases of GNPs and SiO2, there was no effect on
the metabolic activity of the HA-COR (Fig. S1), GIN 28
(Fig. 2A), and GCE 28 (Fig. 2B) cells across the concentra-
tion range tested. From this, we can infer GNPs and SiO2
are not toxic to the healthy HA-COR and patient derived
GBM cells used in this study at concentrations up to 50
μg/ mL and are therefore biocompatible. In contrast, as
the concentration of ZnO NPs increased, a clear effect
on cellular metabolism was observed for all cell types at
higher concentrations. ZnO nanoparticles significantly
reduced the metabolic activity of healthy HA-COR at a Jain et al. Bioelectronic Medicine (2022) 8:17 Jain et al. Bioelectronic Medicine Page 6 of 9 Fig. 2 In vitro toxicity of inorganic NPs on patient derived GBM cells. A GIN 28, and B GCE 28 were incubated with increasing concentration of GNPs,
SiO2 and ZnO NPs for 4 hours, before changing the media containing the NPs with fresh media. Metabolic activity was determined 48 hours after
changing the media, the experiment was run in triplicate, and fluorescence at 590 nm is expressed as % of control (no NPs). Results are expressed as
the mean ± S.D. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001 obtained using 2-way ANOVA with a Tukey post-test Fig. 2 In vitro toxicity of inorganic NPs on patient derived GBM cells. In vitro Toxicity of NPs A GIN 28, and B GCE 28 were incubated with increasing concentration of GNPs,
SiO2 and ZnO NPs for 4 hours, before changing the media containing the NPs with fresh media. Metabolic activity was determined 48 hours after
changing the media, the experiment was run in triplicate, and fluorescence at 590 nm is expressed as % of control (no NPs). Results are expressed as
the mean ± S.D. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001 obtained using 2-way ANOVA with a Tukey post-test TTFields and NPs Mediated Enhanced EF Effects in GBM
Cells TTFields and NPs Mediated Enhanced EF Effects in GBM
Cells from triplicate or quadruplicate repeats and two independent experiments (
ld
d
l
d
)
d
d
h
d
ff Fig. 3 TTFields and inorganic NPs (Gold – GNPs; Zinc oxide – ZnO; Silica oxide – SiO2) mediated enhanced EF effects on patient-derived GBM cells. A Schematic representation of TTFields setup consisting of – a base plate containing 8 ceramic dishes is connected to TTFields generator via a flat
cable. Cells were seeded on a coverslip placed within a ceramic dish. The base plate is placed inside an incubator where the cells were maintained
at 37°C and 5% CO2. B GIN 28 cells and C GCE 28 cells were treated with TTFields at 300 kHz, 1V/cm, and 48 hours at NPs concentration of 5 μg/
mL. D GNPs mediated enhanced TTFields effect on GIN 28 cells at 300 kHz, 1V/cm after 48 and 72 hours at a concentration of 25 μg/mL. Error bar
represents mean ± S.E.M. from triplicate or quadruplicate repeats and two independent experiments Fig. 3 TTFields and inorganic NPs (Gold – GNPs; Zinc oxide – ZnO; Silica oxide – SiO2) mediated enhanced EF effects on patient-derived GBM cells. A Schematic representation of TTFields setup consisting of – a base plate containing 8 ceramic dishes is connected to TTFields generator via a flat
cable. Cells were seeded on a coverslip placed within a ceramic dish. The base plate is placed inside an incubator where the cells were maintained
at 37°C and 5% CO2. B GIN 28 cells and C GCE 28 cells were treated with TTFields at 300 kHz, 1V/cm, and 48 hours at NPs concentration of 5 μg/
mL. D GNPs mediated enhanced TTFields effect on GIN 28 cells at 300 kHz, 1V/cm after 48 and 72 hours at a concentration of 25 μg/mL. Error bar
represents mean ± S.E.M. from triplicate or quadruplicate repeats and two independent experiments could be due to the enhanced bipolar electrophoretic
(GNPs and ZnO) and dielectrophoretic effects (SiO2)
mediated by inorganic NPs. Further in vitro studies are
required to elucidate the detailed molecular mecha-
nism underlying the observed NPs mediated TTFields
enhancement. TTFields and NPs Mediated Enhanced EF Effects in GBM
Cells Interestingly, inorganic NPs (4 hours)
+ TTFields (48 hours) treated showed a ~ 40% decrease
in metabolic activity compared to untreated control (p
<0.0001). Importantly, this corresponds to a ~15% higher
decrease in metabolic activity compared to TTFields alone
(p = 0.002 for GNPs; 0.0001 for ZnO, and 0.04 for SiO2). f
To further validate and establish the observed
enhanced EF effect on GBM cells with GNPs regard-
ing the conductivity of cells, we incubated GIN 28
cells with GNPs at a higher concentration (25 μg/mL)
to further increase the intracellular concentration of
GNPs cell conductivity. In GNP + TTFields treated
group a ~52% and ~75% decrease in the metabolic
activity of GIN 28 cells was observed after 48- and 72-h
treatment, respectively, which was found to be signifi-
cantly higher than both untreated and TTFields treated
groups (Fig. 3D). Overall, the obtained data suggest that
all three NPs utilized in this work enhance EF mediated
anticancer effects in patient derived GBM cells. From
the observed in vitro effects, we hypothesise that this Together with our previous observations and the
obtained data we tentatively suggest that the inorganic
NPs irrespective of their dielectric properties, could acts
as EF transducers (Robinson et al., 2021). The observed (2022) 8:17 Jain et al. Bioelectronic Medicine (2022) 8:1 Page 7 of 9 Jain et al. Bioelectronic Medicine could be due to the enhanced bipolar electrophoretic
(GNPs and ZnO) and dielectrophoretic effects (SiO2)
Conclusions
In summary, conducting GNPs, semiconducting ZnO,
Fig. 3 TTFields and inorganic NPs (Gold – GNPs; Zinc oxide – ZnO; Silica oxide – SiO2) mediated enhanced EF effects on patient-derived GBM cells. A Schematic representation of TTFields setup consisting of – a base plate containing 8 ceramic dishes is connected to TTFields generator via a flat
cable. Cells were seeded on a coverslip placed within a ceramic dish. The base plate is placed inside an incubator where the cells were maintained
at 37°C and 5% CO2. B GIN 28 cells and C GCE 28 cells were treated with TTFields at 300 kHz, 1V/cm, and 48 hours at NPs concentration of 5 μg/
mL. D GNPs mediated enhanced TTFields effect on GIN 28 cells at 300 kHz, 1V/cm after 48 and 72 hours at a concentration of 25 μg/mL. Error bar
represents mean ± S.E.M. Conclusions In summary, conducting GNPs, semiconducting ZnO,
and insulating SiO2 with excellent physicochemical
properties, colloidal stability, and biocompatibility,
were investigated as a transducer for enhancing EF
activity in vitro. We demonstrated that inorganic NPs Jain et al. Bioelectronic Medicine (2022) 8:17 Jain et al. Bioelectronic Medicine (2022) 8:17 Page 8 of 9 Page 8 of 9 Page 8 of 9 Jain et al. Bioelectronic Medicine irrespective of dielectric permittivity, enhance the effi-
ciency and efficacy of EFs in patient derived GBM cells
isolated from intra-tumour regions. The in vitro efficacy
was significantly enhanced by GNPs and ZnO treat-
ment, which is attributed to the ability of these NPs to
polarize and act as bipolar nanoelectrodes/ transduc-
ers which can sense external EFs, thereby enhancing
electrophoretic effects. We additionally suggest that
SiO2 NPs may enhance EF effects by increasing the
forces exerted due to DEP. Furthermore, we have dem-
onstrated that the FDA approved inorganic NPs can be
used as nano transducers for enhancing intracellular EF
effects. This work further paves the pathway for future
studies to systemically deliver these NPs across the BBB
to determine the in vivo efficacy. Abbreviations
l
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https://openalex.org/W4306944148
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https://tckh.daihoctantrao.edu.vn/index.php/sjttu/article/download/797/767
|
Vietnamese
| null |
ĐIỀU TRA, ĐÁNH GIÁ VAI TRÒ CỦA CỘNG ĐỒNG TRONG VIỆC BẢO TỒN MỘT SỐ LOÀI CÂY THUỐC QUÝ HIẾM TẠI KHU BẢO TỒN LOÀI HẠT TRẦN QUÝ HIẾM NAM ĐỘNG, TỈNH THANH HÓA
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Khoa học
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cc-by-sa
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Abstract: This paper introduces the research results about the role of the community
in the conservation of some rare medicinal plant species in the Nam Dong
Valuable Gymnosperm Conservation Area, Thanh Hoa province. As a result,
we recorded 21 species belonging to the Vietnam Red Data Book (2007) as
Endangered (five species) and Vulnerable (16 species). The survey evaluated
10 roles of local organizations and identify the advantages and disadvantages,
opportunities and challenges of the community in the conservation and
development of medicinal plants. The study also offers 2 main solutions to
the conservation and development of medicinal plant resources research area. That situ Conservation (Insitu), also known as protected species live where
and Conservation trans (Ex-stu) is the conservation of bonobos in artificial
conditions under the supervision of the people. INVESTIGATION AND ASSESSMENT OF THE ROLE OF THE
COMMUNITY IN THE CONSERVATION OF SOME RARE MEDICINAL
PLANT SPECIES IN THE NAM DONG VALUABLE GYMNOSPERM
CONSERVATION AREA, THANH HOA PROVINCE Nguyen Huu Cuong
National University of Forestry, Vietnam
Email address: nguyenhuucuong.tvr@gmail.com
DOI: 10.51453/2354-1431/2022/797 Vol 8. No.3_ August 2022 Vol 8. No.3_ August 2022 TẠP CHÍ KHOA HỌC ĐẠI HỌC TÂN TRÀO
ISSN: 2354 - 1431
http://tckh.daihoctantrao.edu.vn/ 2.1. Đối tượng, địa điểm nghiên cứu Đối tượng nghiên cứu: Cộng đồng người dân địa
phương, nhà quản lý và một số loài cây thuốc quý hiếm
tại Khu Bảo tồn các loài hạt trần quý hiếm Nam Động,
tỉnh Thanh Hóa. Địa điểm nghiên cứu: Khu Bảo tồn các loài hạt trần
quý hiếm Nam Động, tỉnh Thanh Hóa. Từ khóa: Bảo tồn, cây thuốc, đa dạng
sinh học, khu Bảo tồn các loài
hạt trần quý hiếm Nam Động,
Thanh Hóa. Tóm tắt Bài báo này giới thiệu kết quả nghiên cứu về vai trò của cộng đồng trong
việc bảo tồn một số loài cây thuốc quý hiếm tại Khu Bảo tồn các loài hạt trần
quý hiếm Nam Động, tỉnh Thanh Hóa. Kết quả điều tra đã ghi nhận 21 loài
cây thuốc quý hiếm theo Sách đỏ Việt Nam (2007) với 5 loài mức nguy cấp
(EN) và 16 loài mức sẽ nguy cấp (VU), đánh giá 10 vai trò của các tổ chức
đoàn thể địa phương và nhận định được những thuận lợi, khó khăn, cơ hội
và thách thức của cộng đồng trong công tác bảo tồn và phát triển các loài
cây thuốc. Nghiên cứu cũng đưa ra được 2 giải pháp chính để bảo tồn và
phát triễn nguồn cây thuốc của khu vực nghiên cứu. Đó là Bảo tồn nguyên
vị (Instu) hay còn gọi là bảo vệ tại nơi loài đang sống và Bảo tồn chuyển vị
(Ex-stu) là bảo tồn các cá thể của loài trong điều kiện nhân tạo dưới sự giám
sát của con người. Ngày nhận bài: 21/05/2022
Ngày sửa bài: 15/07/2022
Ngày duyệt đăng: 01/08/2022 Ngày nhận bài: 21/05/2022
Ngày sửa bài: 15/07/2022
Ngày duyệt đăng: 01/08/2022 1. Đặt vấn đề việc bảo tồn cây thuốc quý hiếm có trong Khu bảo tồn. Nhằm tạo lập cơ sở khoa học cho việc đề xuất giải pháp
bảo tồn một số loài cây thuốc quý hiếm trên quan điểm
sinh thái nhân văn, chúng tôi đã tiến hành điều tra, đánh
giá vai trò của cộng đồng trong việc bảo tồn một số loài
cây thuốc quý hiếm tại Khu Bảo tồn các loài hạt trần
quý hiếm Nam Động, tỉnh Thanh Hóa. Do có tầm quan trọng trong công tác bảo tồn đa
dạng sinh học, Khu Bảo tồn các loài hạt trần quý hiếm
Nam Động (thành lập theo quyết định 84/QĐ-UBND
ngày 08 tháng 01 năm 2014 của Chủ tịch Ủy ban nhân
dân tỉnh Thanh Hóa) được xem là một bộ phận của hệ
sinh thái karst và rừng núi đá vôi đất thấp ở miền Bắc
Việt Nam. Kể từ khi được thành lập các nghiên cứu
ở Khu Bảo tồn các loài hạt trần quý hiếm Nam Động
chỉ tập trung vào việc nghiên cứu và đánh giá hiện
trạng 6 loài cây hạt trần quý hiếm: Thông pà cò (Pinus
kwangtungensis), Đỉnh tùng (Cephalotaxus mannii),
Dẻ tùng sọc hẹp (Amentotaxus argotaenia), Dẻ tùng
sọc rộng (Amentotaxus yunnanensis), Thông đỏ đá
vôi (Taxus chinensis), Thông tre lá dài (Podocarpus
neriifolius) và một số nghiên cứu về khu hệ thực vật [2,
4]. Tuy nhiên chưa có bất cứ một công trình nghiên cứu
cụ thể nào đề cập đến các vai trò của cộng đồng trong ĐIỀU TRA, ĐÁNH GIÁ VAI TRÒ CỦA CỘNG ĐỒNG TRONG VIỆC BẢO TỒN
MỘT SỐ LOÀI CÂY THUỐC QUÝ HIẾM TẠI KHU BẢO TỒN
LOÀI HẠT TRẦN QUÝ HIẾM NAM ĐỘNG, TỈNH THANH HÓA Nguyễn Hữu Cường
Trường Đại học Lâm nghiệp, Việt Nam
Địa chỉ email: nguyenhuucuong.tvr@gmail.com
DOI: 10.51453/2354-1431/2022/797 Keywords: Conservation,
medicinal plants,
biodiversity, Nam Dong
Valuable Gymnosperm
Conservation Area,
Thanh Hoa province 72| Vol 8. No.3_ August 2022 TẠP CHÍ KHOA HỌC ĐẠI HỌC TÂN TRÀO
ISSN: 2354 - 1431
http://tckh.daihoctantrao.edu.vn/ 2.2. Phương pháp nghiên cứu Phương pháp phỏng vấn: Sử dụng một số câu hỏi
cho những người được chọn, phỏng vấn tự do có thể hỏi |73 Nguyen Huu Cuong/Vol 8. No.3_ August 2022|p.72-77 bảo tồn tài nguyên cây thuốc theo Sách Đỏ Việt Nam
(2007) [1], Đỗ Tất Lợi (1999) [3] và đề xuất giải pháp
bảo tồn [2, 4, 5, 7] về bất cứ hoạt động nào với những câu hỏi tùy thuộc
vào hoàn cảnh khi đó, thứ tự các nội dung cần hỏi có
thể thay đổi tùy ý dựa trên câu trả lời của câu hỏi trước
đó của người cung cấp thông tin. 3.1. Danh sách các loài cây thuốc quý hiếm tại
Khu Bảo tồn các loài hạt trần quý hiếm Nam Động Quá trình điều tra tại Khu Bảo tồn các loài hạt trần
quý hiếm Nam Động đã xác định được 21 loài cây
thuốc quý hiếm (bảng 1) có tên trong Sách đỏ Việt Nam
(2007), trong đó: 5 loài ở mức nguy cấp (EN), 16 loài
ở mức sẽ nguy cấp (VU). Ngành Hạt kín có số lượng
loài cây thuốc quý hiếm lớn nhất với 17 loài (4 loài mức
EN, 13 loài mức VU) trong khi đó ngành Dương xỉ và
ngành Thông chỉ có 2 đến 3 loài theo tuần tự. Kết quả
này cho thấy sư đa dạng, phong phú của các loài cây
thuốc quý hiếm tại khu vực. Phương pháp xử lí nội nghiệp: Sau mỗi chuyến đi
điều tra thực địa, các mẫu thực vật được thu thập theo
quy định và lưu giữ tại Trường Đại học Lâm nghiệp
Việt Nam (VNF) và Phòng thực vật của Viện Thực vật
Komarov thuộc Viện Hàn lâm Khoa học Nga (LE), các
thông tin từ phiếu điều tra được chúng tôi tổng hợp
thành một bảng kết quả. Tra cứu và đánh giá về giá trị Bảng 1. Danh sách các loài cây thuốc quý hiếm ở Khu Bảo tồn các loài hạt trần quý hiếm Nam Động
Tt
Tên khoa học
Tên phổ thông
SĐVN, 2007
I. Polypodiopyta
Ngành Dương xỉ
1. Polypodiaceae
Họ Dương xỉ
1
Drynaria bonii Christ
Cốt toái bổ
VU
2
Drynaria fortunei (Kunze ex Mett.) J. Sm. Tắc kè đá
EN
II. Pinophyta
Ngành Thông
2. Cephalotaxaceae
Họ Phỉ
3
Cephalotaxus mannii Hook.f. Đỉnh tùng
VU
3. Taxaceae
Họ Thông đỏ
4
Taxus chinensis Pilger
Thông đỏ
VU
III. Magnoliophyta
Ngành Ngọc Lan
A. Magnoliopsida
Lớp Ngọc Lan
4. Annonaceae
Họ Na
5
Xylopia pierrei Hance
Giền trắng
VU
5. Apocynaceae
Họ Trúc đào
6
Rauvolfia verticillata (Lour.) Baill. Ba gạc lá vòng
VU
7
Rauvolfia cambodiana Pierre ex Pit. Ba gạc cam-pu-chia, Ba gạc lá to
VU
6. Balanophoraceae
Họ Dó đất
8
Balanophora laxiflora Hemsl. Dương hoa thưa (Nấm đất)
EN
7. Bignoniaceae
Họ Đinh
9
Markhamia stipulata (Wall.) Seem. Đinh
VU
8. Burseraceae
Họ Trám
10
Canarium tramdenum Dai. & Yakovl. Trám đen
VU
9. Campanulaceae
Họ Hoa chuông
11
Codonopsis javanica (Blume) Hook.f. Đảng sâm
VU
10. Cucurbitaceae
Họ Bầu bí ảng 1. 3. Kết quả nghiên cứu Phương pháp điều tra theo tuyến: Đã tiến hành điều
tra trên 7 tuyến với chiều dài các tuyến từ 2,2– 4,1 km,
trên toàn bộ khu vực nghiên cứu. Tất cả các vị trí đã
được xác định bởi hệ thống định vị Garmin (gpsmap
62s, 78s). Sử dụng các phương pháp nghiên cứu thực
vật truyền thống của Nguyễn Nghĩa Thìn (2007) [6]. 3.1. Danh sách các loài cây thuốc quý hiếm tại
Khu Bảo tồn các loài hạt trần quý hiếm Nam Động 3.1. Danh sách các loài cây thuốc quý hiếm tại
Khu Bảo tồn các loài hạt trần quý hiếm Nam Động Danh sách các loài cây thuốc quý hiếm ở Khu Bảo tồn các loài hạt trần quý hiếm Nam Động 74| Nguyen Huu Cuong/Vol 8. No.3_ August 2022|p.72-77 Tt
Tên khoa học
Tên phổ thông
SĐVN, 2007
12
Gynostemma pentaphyllum (Thunb.) Makino
Giảo cổ lam
EN
11. Fabaceae
Họ Đậu
13
Sophora tonkinensis Gagnep. Hoa hoè bắc bộ (sơn đậu căn)
VU
12. Opiliaceae
Họ Rau sắng
14
Melientha suavis Pierre
Rau sắng
VU
13. Polygonaceae
Họ Rau răm
15
Reynoutria multiflora (Thunb.) Moldenke (Fallopia multiflora
(Thunb.) Haraldson)
Hà thủ ô đỏ
VU
14. Primulaceae (Myrsinaceae)
Họ Anh thảo (Đơn nem)
16
Ardisia silvestris Pit. Lá khôi
VU
B. Liliopsida
Lớp Hành
15. Arecaceae
Họ Cau
17
Calamus nambariensis Becc. (Calamus platyacanthus Warb. ex Becc.)
Song mật (Mây gai dẹt)
VU
16. Asparagaceae (Convalariaceae)
Họ Măng tây (Mạch môn)
18
Disporopsis longifolia Craib
Trúc căn thất, Hoàng tinh cách
VU
19
Peliosanthes teta Andrews
Sâm cau (Sơn mộc đá)
VU
17. Orchidaceae
Họ Lan
20
Anoectochilus setaceus Blume
Lan gấm trung bộ (Lan kim tuyến)
EN
18. Trilliaceae
Họ Trọng lâu
21
Paris polyphylla Smith
Bảy lá một hoa
EN
Ghi chú: EN - Nguy cấp; VU - Sẽ nguy cấp 3.2. Đánh giá vai trò của cộng đồng địa phương
trong công tác bảo tồn một số loài cây thuốc quý hiếm
tại Khu Bảo tồn các loài hạt trần quý hiếm Nam Động STT
Các tổ chức, đoàn thể địa phương có quan tâm
đến các loài cây thuốc trong Khu Bảo tồn các
loài hạt trần quý hiếm Nam Động
7
Cộng đồng khác
8
Chính quyền xã
9
Ban quản lý Khu Bảo tồn các loài hạt trần quý
hiếm Nam Động
10
Hạt Kiểm lâm huyện huyện Quan Hóa Kết quả điều tra, thảo luận với người dân địa phương
trong Khu Bảo tồn các loài hạt trần quý hiếm Nam Động
về việc quản lý và sử dụng tài nguyên thiên nhiên, trong
đó có các loài cây thuốc quý hiếm, đã xác định có 10 tổ
chức đoàn thể địa phương quan tâm (bảng 2). Các tổ chức có quan tâm đến sử dụng tài nguyên cây
thuốc của khu vực được xắp xếp theo thứ tự từ 1 đến 10. Thuận lợi (S) - Đất đai màu mỡ, khí hậu lạnh ẩm, phù hợp cho sự phát triển
của các loài cây thuốc. - Khu Bảo tồn các loài hạt trần quý hiếm Nam Động có diện
tích tự nhiên 647 ha, với trên 502 ha rừng nguyên sinh rất
thuận lợi cho việc quản lý các loài cây thuốc. - Nhà nước đang có nhiều chính sách phát triển kinh tế nông
thôn để nâng cao đời sống nhân dân, từ đó sẽ giảm bớt các tác
động đến nguồn tài nguyên rừng, đặc biệt là các loài cây thuốc
quý hiếm. gạ
- Thường xuyên xảy ra các hoạt động khai thác lâm sản, lâm
sản ngoài gỗ và các loài cây thuốc để buôn bán, - Khu Bảo tồn các loài hạt trần quý hiếm Nam Động khá đa
dạng về thực vật làm thuốc. Bảng 3. Kết quả phân tích thuận lợi, khóa khăn, cơ hội và thách thức Bảng 3. Kết quả phân tích thuận lợi, khóa khăn, cơ hội và thách thức Bảng 3. Kết quả phân tích thuận lợi, khóa khăn, cơ hội và thách thức
Thuận lợi (S)
Khó khăn (W)
- Đất đai màu mỡ, khí hậu lạnh ẩm, phù hợp cho sự phát triển
của các loài cây thuốc. - Khu Bảo tồn các loài hạt trần quý hiếm Nam Động có diện
tích tự nhiên 647 ha, với trên 502 ha rừng nguyên sinh rất
thuận lợi cho việc quản lý các loài cây thuốc. - Nguồn nhân lực sẵn có tại địa phương. - Cộng đồng địa phương có kinh nghiệm trong việc sử dụng
và gây trồng một số loài cây thuốc như: Khôi nhung, Giảo cổ
lam, Thất diệp nhất chi hoa,…
- Có các chương trình, dự án đầu tư và phát triển kinh tế nông
thôn xóa đói giảm nghèo, tại địa phương. - Nhu cầu của thị trường về cây thuốc là rất lớn
- Khu Bảo tồn các loài hạt trần quý hiếm Nam Động nằm trên
huyện vùng sâu vùng xa, kinh tế - xã hội chậm phát triển, dân
trí thấp, tỷ lệ mù chữ còn cao. - Địa hình trong khu vực khá hiểm trở với nhiều núi cao, bị
chia cắt mạnh, giao thông đi lại trong vùng còn gặp nhiều khó
khăn. - Một số cấp ủy, chính quyền còn chưa thật sự quan tâm đến
công tác xóa đói giảm nghèo cho cộng đồng. - Đời sống nhân dân chủ yếu dựa vào sản xuất nông nghiệp,
khai khai thác lâm sản và các loài lâm sản ngoài gỗ. - Nguồn cây thuốc ngoài tự nhiên ngày càng khan hiếm, đặc
biệt là một số loài có giá trị cao và một số loài quý hiếm. Cơ hội (O)
Thách thức (T)
- Hiện nay thị trường về thực vật làm thuốc đang được mở
rộng. - Nhà nước đang có nhiều chính sách phát triển kinh tế nông
thôn để nâng cao đời sống nhân dân, từ đó sẽ giảm bớt các tác
động đến nguồn tài nguyên rừng, đặc biệt là các loài cây thuốc
quý hiếm. 3.4. Đề xuất một số giải pháp bảo tồn và phát
triễn nguồn cây thuốc của khu vực nghiên cứu - Thường xuyên tuyên truyền và giáo dục cho quần
chúng nhân dân, đặc biệt là các thôn bản xa trung tâm
về tầm quan trọng của rừng đối với con người, trong đó
có các loài cây thuốc. Từ những phân tích về điểm mạnh, điểm yếu, cơ hội
và thách thức nêu ở trên, chúng tôi xin đề xuất hai giải
pháp chính nhằm bảo tồn và phát triển nguồn thực vật
làm thuốc tại Khu Bảo tồn các loài hạt trần quý hiếm
Nam Động như sau: - Cần nghiêm cấm hoạt động chăn thả gia súc trong
Khu bảo tồn. Theo điều 18 của quy chế quản lý rừng
(2006) đã chỉ rõ: Nghiêm cấm mọi hoạt động chăn thả
gia súc, gia cầm trong Phân khu bảo vệ nghiêm ngặt
và Phân khu Phục hồi sinh thái của các VQG hoặc các
KBTTN. 3.4.1. Bảo tồn nguyên vị (Instu) hay còn gọi là bảo
vệ tại nơi loài đang sống 3.1. Danh sách các loài cây thuốc quý hiếm tại
Khu Bảo tồn các loài hạt trần quý hiếm Nam Động Trong số đó, chính quyền thôn, cộng đồng thôm, các
hộ gia đình, người khai thác và buôn bán là những tổ
chức và cá nhân quan tâm trước tiên nhất đến các loài
cây thuốc trong khu vực. Khi xét về ảnh hưởng thì cộng
đồng thôn là tổ chức có ảnh hưởng nhiều nhất đến các
loài cây thuốc của khu vực. Tuy nhiên, khi đánh giá về
vai trò của các tổ chức đến việc quản lý, bảo tồn và sử
dụng các loài cây thuốc của khu vực thì Ban quản lý
Khu Bảo tồn các loài hạt trần quý hiếm Nam Động, Hạt
kiểm lâm huyện Quan Hóa, Chính quyền thôn, người
khai thác và buôn bán là các tổ chức và cá nhân có ảnh
hưởng mạnh mẽ nhất. Bảng 2. Các tổ chức, đoàn thể địa phương có quan
tâm đến các loài cây thuốc trong Khu Bảo tồn các
loài hạt trần quý hiếm Nam Động
STT
Các tổ chức, đoàn thể địa phương có quan tâm
đến các loài cây thuốc trong Khu Bảo tồn các
loài hạt trần quý hiếm Nam Động
1
Cộng đồng thôn
2
Chính quyền thôn
3
Tổ an ninh thôn
4
Đoàn thể
5
Hộ gia đình
6
Người khai thác và buôn bán các loài cây thuốc |75 Nguyen Huu Cuong/Vol 8. No.3_ August 2022|p.72-77 3.3. Đánh giá thuận lợi, khó khăn, cơ hội và thách thức của cộng đồng trong công tác bảo tồn và phát triển
các loài cây thuốc quý hiếm Bảng 3. Kết quả phân tích thuận lợi, khóa khăn, cơ hội và thách thức - Ngày nay, xu thế chữa bệnh bằng các loại thuốc có nguồn
gốc từ thực vật và chữa bệnh trực tiếp bằng những cây thuốc
sẵn có đang được nhiều người quan tâm. - Khu Bảo tồn các loài hạt trần quý hiếm Nam Động khá đa
dạng về thực vật làm thuốc. - Thiếu nguồn vốn đầu tư và khoa học kỹ thuật cho việc phát
triển nguồn thực vật làm thuốc của khu vực. - Việc truyền đạt các thông tin, kiến thức khoa học cho họ về
bảo tồn và phát triển các loài cây thuốc gặp nhiều khó khăn,
trở ngại. - Thường xuyên xảy ra các hoạt động khai thác lâm sản, lâm
sản ngoài gỗ và các loài cây thuốc để buôn bán, Thuận lợi (S) 3.3. Đánh giá thuận lợi, khó khăn, cơ hội và thách thức của cộng đồng trong công tác bảo tồn và phát triển
các loài cây thuốc quý hiếm 3.3. Đánh giá thuận lợi, khó khăn, cơ hội và thách thức của cộng đồng trong công tác bảo tồn và phát triển
các loài cây thuốc quý hiếm Để bảo tồn và phát triển các loài cây thuốc quý hiếm ở khu vực này chúng ta cần đánh giá được những điểm
mạnh, điểm yếu, cơ hội và thách thức của vùng để đề xuất các biện pháp bảo tồn và phát triển hợp lý. Kết quả phân
tích, tổng hợp được như sau: Bảng 3. Kết quả phân tích thuận lợi, khóa khăn, cơ hội và thách thức REFERENCES ngoài tự nhiên đặc biệt là các loài quý hiếm, làm giảm
sự tác động vào rừng tự nhiên. [1]. Ban, N.T. (Ed.) 2007. Vietnam Red Data Book,
Part 2 - Plants. Natural Science and Technology
Publishing House, Hanoi, Vietnam. - Áp dụng các tiến bộ khoa học kỹ thuật hiện đại
vào việc nhân giống, gây trồng và chăm sóc các loài
cây thuốc. [2]. Center for Environment and Sustainable
Forestry Development (2006), The result of the
biodiversity survey in Nam Dong Conservation Area,
Thanh Hoa, Vietnam. - Cần đầu tư nguồn vốn, giống và các vật tư cần
thiết cho những HGĐ gây trồng, chăm sóc các loài cây
thuốc đặc biệt là các loài quý hiếm tại các vườn thuốc. [3]. Loi, D.T. (1999), Vietnam medicinal plants
and herbs. Science and Technology Publishing House,
Hanoi. Ngoài ra, sau khi gây trồng và bảo tồn các loài cây
thuốc nhà nước cần phải mở rộng thị trường tiêu thụ,
tăng cường quan hệ hợp tác với mục đích tạo đầu ra cho
các sản phẩm cây thuốc. [4]. Cuong, N.H., Averyanov L., Egorov A., Doi, B.T. (2020), Conservation status of conifers in Nam Dong
Conservation Area (Thanh Hoa Province, northern
Vietnam), IOP Conf. Series: Earth and Environmental
Science 574 (2020) 012012. doi:10.1088/1755-
1315/574/1/012012 3.4.1. Bảo tồn nguyên vị (Instu) hay còn gọi là bảo
vệ tại nơi loài đang sống Với giải pháp bảo tồn này chúng ta cần thực hiện
như sau: 3.4.2. Bảo tồn chuyển vị (Ex-stu) là bảo tồn các cá
thể của loài trong điều kiện nhân tạo dưới sự giám sát
của con người - Ban quản lý Khu Bảo tồn các loài hạt trần quý
hiếm Nam Động và chính quyền các cấp trong khu vực
cần phải thực hiện tốt công tác quản lý, bảo vệ nguồn
tài nguyên rừng, đặc biệt là nguồn cây thuốc quý hiếm. Đối với giải pháp này ta thực hiện như sau: - Cần phải xây dựng các vườn cây thuốc theo quy
mô lớn như Công ty dược liệu và quy mô nhỏ trong các
Hộ gia đình. Việc trông cây thuốc không những đáp
ứng được cho nhu cầu sử dụng tại chỗ của nhân dân, mà
còn làm giảm sự phụ thuộc và nguồn nguyên liệu thuốc - Cần nghiêm cấm các hoạt động khai thác và buôn
bán trái phép tài nguyên cây thuốc trong Khu bảo tồn. - Cần tăng cường các lực lượng bảo vệ rừng: Kiểm
Lâm, Tổ, Đội tuần tra bảo vệ rừng để đảm bảo cho các
loài cây thuốc được phát triển tốt. 76| Nguyen Huu Cuong/Vol 8. No.3_ August 2022|p.72-77 [7]. Cuong,N.H., Egorov A.A. (2021), Medicinal
plants of forests in the mountain areas of Nam Dong
Reserve (Quan Hoa district, Thanh Hoa province,
northern Vietnam). Collection of materials of the
international conference in the natural and human
sciences, St. Petersburg, 2021. 4. Kết luận Kết quả điều tra đã ghi nhận được 21 loài cây thuốc
quý hiếm theo Sách đỏ Việt Nam (2007) với 5 loài mức
nguy cấp (EN) và 16 loài sẽ nguy cấp (VU), đánh giá
được 10 vai trò của các tổ chức đoàn thể địa phương
(Cộng đồng thôn, Chính quyền thôn, Tổ an ninh thôn,
Đoàn thể, Hộ gia đình, Người khai thác và buôn bán các
loài cây thuốc, Cộng đồng khác, Chính quyền xã, Ban
quản lý Khu Bảo tồn các loài hạt trần quý hiếm Nam
Động, Hạt Kiểm lâm huyện Quan Hóa,) và nhận định
được những thuận lợi, khó khăn, cơ hội và thách thức
của cộng đồng trong công tác bảo tồn và phát triển các
loài cây thuốc. Từ đó, nghiên cứu cũng đưa ra được 2
giải pháp chính để bảo tồn và phát triễn nguồn cây thuốc
của khu vực nghiên cứu. Đó là Bảo tồn nguyên vị (Instu)
hay còn gọi là bảo vệ tại nơi loài đang sống và Bảo tồn
chuyển vị (Ex-stu) là bảo tồn các cá thể của loài trong
điều kiện nhân tạo dưới sự giám sát của con người. [5]. Cuong,N.H., Averyanov L., Egorov A., Doi,
B.T., Quyet, P.T. (2021), Traditional knowledge on
non-medicinal plants used by the tribal people in
Nam Dong Commune, Quan Hoa district, Thanh Hoa
provice, nothern Vietnam, IOP Conf. Series: Earth
and Environmental Science 876 (2021) 012053. doi:10.1088/1755-1315/876/1/012053 [6]. Thin, N.N. (1997), Manual for biodiversity
research: Agricultural Publishing House, Hanoi. [7]. Cuong,N.H., Egorov A.A. (2021), Medicinal
plants of forests in the mountain areas of Nam Dong
Reserve (Quan Hoa district, Thanh Hoa province,
northern Vietnam). Collection of materials of the
international conference in the natural and human
sciences, St. Petersburg, 2021. |77
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English
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Correlated Inter-Domain Motions in Adenylate Kinase
|
PLOS computational biology/PLoS computational biology
| 2,014
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cc-by
| 7,524
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Introduction DPQ~- m0cPcQh
8p3rPQ3
X
ij SijcoswPQ
i
coswPQ
j
ð1Þ ð1Þ Conformational heterogeneity as a consequence of dynamics is
an intrinsic feature of proteins linked to biological function.[1] An
important aspect for our understanding of protein dynamics is a
molecular characterization of the structural states that are
populated and of how correlated conformational changes can
mediate biological functions such as allostery and signal transduc-
tion.[2,3] The characterization of correlated conformational
changes requires a description of how local structural heteroge-
neity translates into global conformational changes through
collective motions. Recent developments in the analysis of various
NMR parameters at atomic resolution, such as spin relaxation
rates[4] and residual dipolar couplings (RDCs),[5–7] have enabled
the
description
of
local
structural
heterogeneity. However,
inferences
of
collective
conformational
changes
from
local
correlated motions have relied on force fields or motional
models.[7–9] In Eq. 1, for two nuclei P and Q, Qi
PQ is the angle between the
inter-nuclear vector and axis i of the molecular frame, Sij is an
element of the alignment tensor (S), rPQ is the distance between P
and Q, cX is the gyromagnetic ratio of nucleus X, m0 is the
magnetic susceptibility of vacuum and h is Planck’s constant. The
alignment tensor S is given by the alignment mechanism and,
under steric alignment conditions, can be computed accurately
and is closely related to the shape of the aligned macromole-
cule.[12–14] RDCs measured in steric alignment combine the
local information contained in NMR parameters with the shape
information contained in relaxation rates[15–17] and small angle
X-ray scattering (SAXS).[18] Since inter-domain motions alter the
shape of proteins RDCs measured in steric alignment have, as we
will show, the potential to act as reporters of inter-domain
structural heterogeneity. RDCs are NMR parameters that report on both the local and
global structural properties of weakly aligned macromolecules
and, under the assumption that alignment does not alter the
properties of the protein, can be used to study the amplitude of
dynamics, especially when combined with simulations.[5,10]
Alignment
can
be
induced
by
steric
and/or
electrostatic
interactions of the macromolecule with external media. Specif-
ically,
for
a
given
conformation,
RDCs
depend
on
the
geometrical properties of the environment of the nuclei in the
molecular frame and on the direction and degree of align-
ment[11,12] (Eq. 1, Fig. 1). Abstract Correlated inter-domain motions in proteins can mediate fundamental biochemical processes such as signal transduction
and allostery. Here we characterize at structural level the inter-domain coupling in a multidomain enzyme, Adenylate Kinase
(AK), using computational methods that exploit the shape information encoded in residual dipolar couplings (RDCs)
measured under steric alignment by nuclear magnetic resonance (NMR). We find experimental evidence for a multi-state
equilibrium distribution along the opening/closing pathway of Adenylate Kinase, previously proposed from computational
work, in which inter-domain interactions disfavour states where only the AMP binding domain is closed. In summary, we
provide a robust experimental technique for study of allosteric regulation in AK and other enzymes. Citation: Esteban-Martı´n S, Fenwick RB, A˚de´n J, Cossins B, Bertoncini CW, et al. (2014) Correlated Inter-Domain Motions in Adenylate Kinase. PLoS Comput
Biol 10(7): e1003721. doi:10.1371/journal.pcbi.1003721 Editor: Helmut Grubmu¨ller, Max Planck Institute for Biophysical Chemistry, Germany d February 5, 2014; Accepted May 28, 2014; Published July 31, Copyright: 2014 Esteban-Martı´n et al. This is an open-access article distributed under the terms of the Creative Commons Attribu
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ban-Martı´n et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
tion, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by IRB (RBF, CWB, XS), the BSC (SEM, BC, VG), the Swedish Research Council (621-2010-5247 to MWW), ‘‘Umea˚ University
Young researcher award’’ (MWW), ICREA (XS) MICINN (CTQ2009-08850-BQU to XS), MINECO (FIS2008-00114 to VG and BIO2012-31043 to XS), Marato´ de TV3
(102030 to XS) and the Juan de la Cierva (SEM) and Ramo´n y Cajal (CWB) programs. The authors acknowledge the use of computational resources of the Red
Espan˜ola de Supercomputacio´n (RES). Spanish ministry of education and science MICINN CTQ2009-08850, MINECO FIS2008-00114, MINECO BIO2012-31043
(http://www.mecd.gob.es) Marato´ de TV3 102030. Swedish Research Council (http://www.vr.se/) Grant Number 621-2010-5247. 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. * Email: santiago.esteban.martin@gmail.com (SEM); xavier.salvatella@irbbarcelona.org (XS) Correlated Inter-Domain Motions in Adenylate Kinase Santiago Esteban-Martı´n1*, Robert Bryn Fenwick2, Jo¨ rgen A˚ de´n3, Benjamin Cossins1,
Carlos W. Bertoncini2, Victor Guallar1,4, Magnus Wolf-Watz3, Xavier Salvatella2,4*
1 Joint BSC CRG IRB Research Programme in Computational Biology Barcelona Supercomputing Center BSC Barcelona Spain 2 Joint BSC CRG IRB Research Programme Santiago Esteban-Martı´n1*, Robert Bryn Fenwick2, Jo¨ rgen A˚ de´n3, Benjamin Cossins
Carlos W. Bertoncini2, Victor Guallar1,4, Magnus Wolf-Watz3, Xavier Salvatella2,4* 1 Joint BSC-CRG-IRB Research Programme in Computational Biology, Barcelona Supercomputing Center - BSC, Barcelona, Spain, 2 Joint BSC-CRG-IRB Research Programme
in Computational Biology, Institute for Research in Biomedicine – IRB Barcelona, Barcelona, Spain, 3 Department of Chemistry, Chemical Biological Centre, Umea˚
University, Umea˚, Sweden, 4 Institucio´ Catalana de Recerca i Estudis Avanc¸ats - ICREA, Barcelona, Spain PLOS Computational Biology | www.ploscompbiol.org July 2014 | Volume 10 | Issue 7 | e1003721 Correlated Inter-Domain Motions in Adenylate Kinase An
ensemble size of 4 to 64 accounted for the data and equivalent
distributions were observed regardless of ensemble size and
method used to calculate the RDCs (Figs S3 to S5). It is worth
noting that the LID domain of AKe is in equilibrium with a locally
unfolded state with a population of ca. 5% at 37uC;[32] under the
conditions used in this study (25uC) this state has a low population
(,1%). Similarly, cracking of the AMPbd along the closing
mechanism may play a role.[26,33] However, cracking occurs at
the transition state, and therefore has a very low population. The
states obtained for AKe are shown in Fig. 2b. An analysis of the
results indicated that substrate free AKe samples three major states
corresponding to i) a closed-like state (hLID ,105u, hAMPbd ,60u,
population ,0.560.1), where the LID is closed and the AMPbd
slightly opened ii) the open state (hLID ,146u, hAMPbd ,74u,
population ,0.2560.1), where both nucleotide binding domains
are open, and iii) conformations in which the degree of domain
opening is intermediate between that of the fully closed and fully
open. The estimate of the fraction closed AKe from our analysis is
0.5, which is in agreement with numbers from single molecule
FRET studies where fractions of 0.6[29] and 0.3[28] has been
enumerated. Results and Discussion Our approach is based on the determination of ensembles
that collectively agree with RDCs. The generation of the
ensemble is divided in two steps (see Methods and Supporting
Information). First, an enumeration (ca. 105) of inter-domain
orientations
is
performed
using
unrestrained
simulations. Secondly, the ensemble of minimal size that best fits the RDCs
is identified by a genetic algorithm (see Figs. S1 and S2).[23] To
maximize the coverage of conformational space in the first step
we used PELE, an all atom Monte Carlo simulation algorithm
with a move set designed to explore normal modes (see Methods
and Supporting Information).[24] The RDCs of each conformer
are calculated via Eq. 1 using two independent methods to
compute the S from knowledge of the structure.[12,13] In this
case we used only the steric tensor because it is related to shape
but it is principle possible to use also the electrostatic tensor,
particularly when conformational changes modulate the charge
distribution. An analysis of the conformational ensemble allowed us to assess
the presence and degree of inter-domain correlation, an important
property of AKe. As shown in Fig. 2b, the ATP and AMP domain
movements are correlated, disfavouring conformations where the
LID is open and the AMPbd is closed. Our findings agree with
free energy calculations[34], molecular dynamics simulations[35]
as well as with normal mode analysis.[33,36] Although it is
impossible to derive the closure pathway from equilibrium data,
the conformational states observed favour a step-wise mechanism
in which LID closure takes place before AMPbd closure. Such a
mechanism would be beneficial because the initial closure of the
LID, together with the substantially higher affinity of this domain
for its substrate (ATP) as compared to AMP (50 vs 1700 mM),[30]
would reduce the probability of non-productive binding of AMP to
the LID.[37] AKe is an essential enzyme that catalyses the reversible
conversion of ATP and AMP into two ADP molecules. The role
of AKe is to maintain the energy balance in the cell, which is
essential. AKe is a modular enzyme composed of three sub-
domains: a CORE domain responsible for thermal stability[25,26]
and two flexible substrate-binding domains referred to as LID and
AMPbd (Fig 2a). Author Summary Most proteins contain several domains, and inter-domain
motions play important roles in their biological functions. Describing the various inter-domain orientations that
multi-domain proteins adopt at equilibrium is challenging,
but key for understanding the relationship between
protein structure and function. When more than two
domains are present in a protein, correlated domain
motions can be of fundamental importance for biological
function. This type of behaviour is typical of molecular
machines but is extremely challenging to characterize
both from experimental and theoretical viewpoints. In this
paper, we present a hybrid experimental/computational
approach to address this problem by exploiting the
information on molecular shape contained in nuclear
magnetic resonance experiments to determine accurate
conformation ensembles for the multi-domain enzyme
adenylate
kinase
with
help
of
advanced
simulation
methods. the various conformations in fast exchange that contribute to the
measured RDC can have different alignment tensors.[20,22] Here
we show that for a molecule undergoing conformational changes
in two distal sites the RDCs depend on the degree of correlation of
such changes. We exploit this to characterize the degree of
correlation of the inter-domain conformational changes occurring
in the substrate-free state of E. coli Adenylate Kinase (AKe), an
enzyme that undergoes conformational changes involving shape
changes. We performed additional control simulations to assess the
robustness of the distributions obtained. Scrambling the RDCs
lead to a list of restraints that could not be fit to any distribution
(Fig. S6), showing that the distribution obtained is encoded in the
data and not biased by the population of the conformers in the
pool. The distribution shown in Fig. 2a was found to be robust to
various sources of error, including errors in the experimental
RDCs (Fig. S7) and in the prediction of the alignment tensor (Fig. S8). We also performed computational experiments to ensure that
a single set of RDCs suffices to distinguish between ensembles with
a distinct number of states and identify correlated structural
changes (see Figs. S9 to S13). Correlated Inter-Domain Motions in Adenylate Kinase state was better than that of the free state, which we interpreted as
evidence for inter-domain dynamics in the substrate-free enzyme. To identify major conformational states sampled by substrate-
free AKe we used backbone NH RDCs measured in steric
alignment to select ensembles of conformations from a pool
derived from simulations started from the open and closed X-ray
structures[27],[31] (see Methods). The ensembles collectively
agree with the RDCs (Q<0.26) even though the structures they
contain do not (Q.0.6) when considered individually. An
ensemble size of 4 to 64 accounted for the data and equivalent
distributions were observed regardless of ensemble size and
method used to calculate the RDCs (Figs S3 to S5). It is worth
noting that the LID domain of AKe is in equilibrium with a locally
unfolded state with a population of ca. 5% at 37uC;[32] under the
conditions used in this study (25uC) this state has a low population
(,1%). Similarly, cracking of the AMPbd along the closing
mechanism may play a role.[26,33] However, cracking occurs at
the transition state, and therefore has a very low population. The
states obtained for AKe are shown in Fig. 2b. An analysis of the
results indicated that substrate free AKe samples three major states
corresponding to i) a closed-like state (hLID ,105u, hAMPbd ,60u,
population ,0.560.1), where the LID is closed and the AMPbd
slightly opened ii) the open state (hLID ,146u, hAMPbd ,74u,
population ,0.2560.1), where both nucleotide binding domains
are open, and iii) conformations in which the degree of domain
opening is intermediate between that of the fully closed and fully
open. The estimate of the fraction closed AKe from our analysis is
0.5, which is in agreement with numbers from single molecule
FRET studies where fractions of 0.6[29] and 0.3[28] has been
enumerated. state was better than that of the free state, which we interpreted as
evidence for inter-domain dynamics in the substrate-free enzyme. state was better than that of the free state, which we interpreted as
evidence for inter-domain dynamics in the substrate-free enzyme. To identify major conformational states sampled by substrate-
free AKe we used backbone NH RDCs measured in steric
alignment to select ensembles of conformations from a pool
derived from simulations started from the open and closed X-ray
structures[27],[31] (see Methods). The ensembles collectively
agree with the RDCs (Q<0.26) even though the structures they
contain do not (Q.0.6) when considered individually. Introduction RDCs can be used to study the structural heterogeneity of
globular and disordered proteins by analysing the effect of (sub-ms)
motional averaging on this NMR parameter. The effect of fast
(sub-ns) local motions that do not directly alter the magnitude and
main directions of alignment can be analysed in the molecular
frame defined by the alignment tensor of the average struc-
ture.[19,20] The analysis of motions that change the shape of the
protein, that are typically slower than the timescale of alignment
(0.5 to 5 ns) [21] needs on the contrary to explicitly consider that July 2014 | Volume 10 | Issue 7 | e1003721 1 July 2014 | Volume 10 | Issue 7 | e1003721 PLOS Computational Biology | www.ploscompbiol.org Correlated Inter-Domain Motions in Adenylate Kinase RDC calculation NH RDCs used in this work for free AKe were measured in
stretched polyacrylamide gels.[30] Two independent methods,
PALES [12] and ALMOND[13], [13]were used to calculate the
alignment tensor using Eq. 1. They gave equivalent distributions
as shown in Figs. S3–S5. In Figs. 2 we provide the distributions
that lead the best agreement between calculated and experimental
RDCs [43] (Tab. S1). For AKe it was obtained using ALMOND. The agreement between calculated and experimental (or refer-
ence) RDCs was assessed by the quality factor Q [43] defined in
Eq. 2. Here we have used the dependence of RDCs on global
molecular shape via S to determine ensembles reporting on the
amplitude and degree of correlation of inter-domain motions. Although RDCs are uniquely suited for this, this is a challenging
endeavour due to their intrinsic degeneracies.[39] It is therefore
relevant to discuss the factors that allowed the approach used in
this work to alleviate them. The first factor is that our approach
accounts for how shape changes alter the value of the RDC by
computing S for each inter-domain orientation i.e. two inter-
domain orientations that would have degenerate RDCs when the
tensor is assumed to be constant can be differentiated if they have
sufficiently large differences in shape (Fig. S14).[39] A second
factor is the presence of structural constraints due to the covalent
linkage, where steric clashes between domains restrict the available
conformational space;[40,41] AKe is a well-structured proteins
where this effect is particularly important but for multi-domain
proteins with flexible linker sequences RDCs measured in multiple
alignment media will be required. Alternatively, RDCs could be
complemented with structural restraints derived from smFRET or
SAXS data. Q~ rms(Dexp{Dcalc)
rms(Dexp)
ð2Þ ð2Þ The calculated RDCs were scaled, after averaging across the
ensemble, to minimize Q to account for the difficulty of predicting
the absolute concentration of alignment medium. We used RDCs
corresponding to structured regions in the protein (see Fig. S15). For AKe, we found that RDCs in loop regions were more difficult
to fit and therefore we decided to exclude them. However, we note
that the ensemble derived including RDCs in the loop regions
remains unaffected. We also evaluated the impact of the alignment
media by monitoring changes in chemical shifts induced by the
presence of the stretched polyacrylamide gel. Results and Discussion Crystallographic structures of AKe have been
solved for the open (inactive) and closed (active) states.[27]
Substrate-free AKe has been shown to sample a closed-like state
using single-molecule Fo¨ster resonance energy transfer (smFRET)
experiments.[28,29] We previously reported an analysis of AKe
using RDCs in the substrate-free (open) and substrate-bound
(closed) states where we fit the RDCs to the corresponding
crystallographic structures.[30] We found that the fit of the closed AKe is an essential enzyme that catalyses the reversible
conversion of ATP and AMP into two ADP molecules. The role
of AKe is to maintain the energy balance in the cell, which is
essential. AKe is a modular enzyme composed of three sub-
domains: a CORE domain responsible for thermal stability[25,26]
and two flexible substrate-binding domains referred to as LID and
AMPbd (Fig 2a). Crystallographic structures of AKe have been
solved for the open (inactive) and closed (active) states.[27]
Substrate-free AKe has been shown to sample a closed-like state
using single-molecule Fo¨ster resonance energy transfer (smFRET)
experiments.[28,29] We previously reported an analysis of AKe
using RDCs in the substrate-free (open) and substrate-bound
(closed) states where we fit the RDCs to the corresponding
crystallographic structures.[30] We found that the fit of the closed Single-molecule FRET[28,29] and NMR[28] have shown that
the open and closed states of AKe are in equilibrium and that
nucleotide binding shifts it towards the closed state.[30] Of
particular interest are smFRET experiments, as these can resolve
conformational states and provide a qualitative validation of the
ensemble. Two different reaction coordinates, corresponding to
distances between residues in the LID and the AMPbd (Aquifex July 2014 | Volume 10 | Issue 7 | e1003721 PLOS Computational Biology | www.ploscompbiol.org 2 Correlated Inter-Domain Motions in Adenylate Kinase Figure 1. RDCs report on molecular shape. RDCs depend on the geometry of the relevant nuclei (Qi) and on the degree and direction of
alignment (S) of the molecule with respect to the laboratory frame. S is a 363 symmetric and traceless matrix, which contains information on the
shape of the molecule. Qi is the angle between the inter-nuclear vector and axis i of the molecular reference frame where S is defined. The direction
and magnitude of alignment are shown as eigenvectors (eˆi, i = 1, 2, 3) and eigenvalues (vector size, not in scale) for AKe in the open (left) and closed
(right) states. doi:10.1371/journal.pcbi.1003721.g001 Figure 1. RDCs report on molecular shape. Results and Discussion RDCs depend on the geometry of the relevant nuclei (Qi) and on the degree and direction of
alignment (S) of the molecule with respect to the laboratory frame. S is a 363 symmetric and traceless matrix, which contains information on the
shape of the molecule. Qi is the angle between the inter-nuclear vector and axis i of the molecular reference frame where S is defined. The direction
and magnitude of alignment are shown as eigenvectors (eˆi, i = 1, 2, 3) and eigenvalues (vector size, not in scale) for AKe in the open (left) and closed
(right) states. doi:10 1371/journal pcbi 1003721 g001 g
doi:10.1371/journal.pcbi.1003721.g001 AK)[28] and between residues in the LID and the CORE domain
(AKe),[29] have been studied. In the former the authors resolved
two states in which open conformations were populated to ca. 70%, whereas in the latter the authors found that the open
conformation was disfavoured, with a population of ca. 40%. These results can be rationalized based on the ensemble. Following the approach of Beckstein et al.,[38] where distances
corresponding to the open and closed states are mapped in domain
angle space, we estimated open populations along the LID-
AMPbd and LID-CORE smFRET reaction coordinates .50%
and ,30%, respectively (for LID-AMPbd large uncertainties are
found due to difficulties in assigning open and closed states as
quantified by smFRET). Overall, the ensemble is able to
qualitatively reconcile two apparently contradictory smFRET
studies, which also suggests that steric alignment does not
significantly alter the structural heterogeneity of AKe. AK)[28] and between residues in the LID and the CORE domain
(AKe),[29] have been studied. In the former the authors resolved
two states in which open conformations were populated to ca. 70%, whereas in the latter the authors found that the open
conformation was disfavoured, with a population of ca. 40%. These results can be rationalized based on the ensemble. Following the approach of Beckstein et al.,[38] where distances
corresponding to the open and closed states are mapped in domain
angle space, we estimated open populations along the LID-
AMPbd and LID-CORE smFRET reaction coordinates .50%
and ,30%, respectively (for LID-AMPbd large uncertainties are
found due to difficulties in assigning open and closed states as
quantified by smFRET). Overall, the ensemble is able to
qualitatively reconcile two apparently contradictory smFRET
studies, which also suggests that steric alignment does not
significantly alter the structural heterogeneity of AKe. binding allostery. Results and Discussion Our results, therefore, reinforce that correlated
inter-domain motions in proteins can mediate important bio-
chemical processes. PLOS Computational Biology | www.ploscompbiol.org RDC calculation As shown in the
Supporting Figure S16, no significant chemical shift perturbations
were observed when apo AKe was immersed into the anisotropic
phase. As a reference the chemical shift perturbations induced by
binding of the inhibitor Ap5A to AKe are also displayed. A
thorough analysis of the robustness of the procedure to various
source of error is provided as Supporting Information. To select
the optimum ensemble size we monitored the agreement with
RDCs used to guide the genetic algorithm and to RDCs left out of
this process or free RDCs. Ten sets of randomly removed RDCs
(20%) or free RDCs sets were used and 20 independent
calculations were run for each set of randomly removed RDCs. Correlated
motions
in
proteins
are
thought
to
mediate
biochemical functionality.[42] Recently, we have identified weak
long-range correlated motions in a surface patch of ubiquitin
involved in molecular recognition.[7] Here, we move a step
forward and find correlated domain movements in a representa-
tive multi-domain enzyme. The observed inter-domain correlation
suggests functional roles in allowing ligand access, in adopting the
inter-domain orientation necessary for catalysis as well as in July 2014 | Volume 10 | Issue 7 | e1003721 3 Correlated Inter-Domain Motions in Adenylate Kinase Figure 2. Determination of correlated inter-domain conformational heterogeneity in AKe from RDCs. (A) X-ray structure of substrate
free open AKe (pdb code 4ake). The CORE (yellow), LID (red) and AMPbd (cyan) domains are shown. The angles used to describe inter-domain
conformational changes (see methods) are shown. (B) Inter-domain orientation distribution for AKe. The robustness of the distributions against
various sources of error is discussed in the Supporting Information. (C) Representative conformations for each basin are shown together with angles
and their estimated populations; little structural heterogeneity occurs within basins. The uncertainties in the populations due to estimated
experimental in the measurements of the RDCs and computational errors are estimated to 10%. doi:10.1371/journal.pcbi.1003721.g002 Figure 2. Determination of correlated inter-domain conformational heterogeneity in AKe from RDCs. (A) X-ray structure of substrate
free open AKe (pdb code 4ake). The CORE (yellow), LID (red) and AMPbd (cyan) domains are shown. The angles used to describe inter-domain
conformational changes (see methods) are shown. (B) Inter-domain orientation distribution for AKe. The robustness of the distributions against
various sources of error is discussed in the Supporting Information. Reference pool of conformations An extensive set of inter-domain conformations (ca. 105) was
generated for AKe. The X-ray structures 1ake [31] and 4ake [27]
were used as seeds. Trajectories were generated where either the
AMP or ATP binding domain open (or closed). From each state
along the trajectory a second trajectory was generated where the
other domain was forced to open (or to close). These simulations
were
performed
with
the
molecular
simulation
package
CHARMM c35.[44] A time step of 1.0 fs was used. Simulations
were performed at 300K. A shape-term (biasing) potential on the
backbone atoms was used. The CHARMM commands "CON-
Straint HARmonic BEStfit COMParison" and the IC table
tool (CONStraint IC BOND ANGL IMPR DIHE) were used
to sample inter-domain orientations between open and closed RDC calculation (C) Representative conformations for each basin are shown together with angles
and their estimated populations; little structural heterogeneity occurs within basins. The uncertainties in the populations due to estimated
experimental in the measurements of the RDCs and computational errors are estimated to 10%. doi:10.1371/journal.pcbi.1003721.g002 The ensemble was qualitatively validated against two independent
smFRET experiments (see results and discussion). conformations. The initial force was set to 0.1 and exponentially
increased by a factor of 1.05 during 200 cycles of 100 steps each. The degree of opening ranged between ,30–90u and ,90–160u
for the AMP- and ATP-binding domains (see angle definitions for
AKe). The angles sampled covered the range observed by known
X-ray structures of AKe and related proteins. [38] An advanced
sampling strategy based on the PELE [24] method was used (see
Protein Energy Landscape Exploration, see Supporting Informa-
tion). This allowed identifying high-energy conformations which
are unlikely to be of sufficiently low energy to be present in the
crystalline state or sampled by conventional MD. Searching algorithm An in-house genetic algorithm GA was developed to efficiently
search within the pool of structures used to determine the
experimental inter-domain orientation distributions of T4L and
AKe. Initially 1000 ensembles were generated with structures July 2014 | Volume 10 | Issue 7 | e1003721 PLOS Computational Biology | www.ploscompbiol.org 4 Correlated Inter-Domain Motions in Adenylate Kinase randomly selected from a pool of ca. 105 conformations (see
reference pool of conformations above). Ensembles of size N = 1,
2, 4, 8, 16, 32 and 64 were used. The 103 ensembles of size N were
submitted to evolution through 1000 steps. At each step two new
sets of 103 ensembles were generated by mutation and crossing
operations, leaving 36103 ensembles. At each step the best 103
ensembles, based on the value of Q (see below), were retained. After 1000 iterations the best ensemble was saved and the
complete procedure was repeated 200 times. Because too high
mutation and recombination rates may lead to loss of good
solutions and to premature convergence of the GA, these
parameters evolved during the calculations. Initially, the mutation
and crossing rates were set to 100% and 2%, respectively. At each
step new mutation and crossing rates were determined by
dividing/multiplying them by a factor of 1.001, respectively. In
Figs. S1 and S2 a scheme illustrating the GA and the convergence
of the method are shown. The error in the population of the states
observed were determined from the influence of RDC experi-
mental uncertainty by performing 200 calculations with random
Gaussian error added to each experimental RDC. We used a
standard deviation of 1.0 Hz for AKe, three times the estimated
experimental error. iterations. b, Mutation and crossing operation rates applied at
each step of the ensemble refinement. (TIF) iterations. b, Mutation and crossing operation rates applied at
each step of the ensemble refinement. (TIF) Figure S3
Fitting of NH RDCs for AKe and determina-
tion of the optimum ensemble size by monitoring the
agreement
with
RDCs
used
to
guide
the
genetic
algorithm and RDCs left out of the calculation (20%). The results obtained using two independent methods to calculate
the alignment tensor, ALMOND and PALES, are shown. The
Qfactor for working (Qwork; restrained) and free (Qfree; unrestrained)
RDCs are shown (see Supporting Text S1). For each ensemble size
200 independent calculations were performed and the results
pooled. Inter-domain angle definitions The definition used by Beckstein et al. [38] was adopted in this
work. Briefly, the angle formed between the AMP-binding domain
(AMPbd) and the core domain (CORE) was determined as the
angle formed by two vectors: Vector 1 connects the centers of mass
(Ca atoms) of CORE residues 90–100 with residues 115–125
(‘‘hinge region’’ between the CORE and LID domains). Vector 2
connects the centers of mass of CORE residues 90–100 and
AMPbd residues 35–55. The angle formed between the ATP-
binding domain (LID) and the CORE was defined equivalently
using the angle formed between two vectors: Vector 1 connects the
centers of mass of residues 115–125 (‘‘hinge region’’ CORE–LID
domains) with CORE residues 179–185. Vector 2 connects the
centers of mass for residues 115–125 (‘‘hinge region’’ CORE–LID)
with LID residues 125–153. Figure S5
Experimental AKe inter-domain orientation
distributions obtained for ensembles of several sizes
(see Supporting Text S1). The distributions were obtained
using PALES to calculate the alignment tensor (see Fig. S4 for
distributions obtained using the ALMOND method). For each
ensemble size 200 independent calculations were performed and
the results pooled. The x and y axis correspond to the hAMPbd and
hLID angles (in degrees) shown in Fig. 2a. (TIF) Figure S6
Distributions obtained for AKe after random-
ly scrambling the experimental RDCs to obtain incor-
rect lists of restraints (see Supporting Text S1). The Q
factor obtained in all cases was .0.9, indicating that it was not
possible to fit the incorrect data to any physically possible
distribution of inter-domain orientations; for each ensemble size
a different list of randomly scrambled RDCs was used. For AKe,
the x and y axis correspond to the hAMPbd and hLID angles (in
degrees) shown in Fig. 2a. (TIF) Searching algorithm Figure S4
Experimental AKe inter-domain orientation
distributions obtained for ensembles of several sizes
(see Supporting Text S1). The distributions were obtained
using the ALMOND method to calculate the alignment tensor (see
Fig. S5 for distributions obtained using PALES). For each
ensemble size 200 independent calculations were performed and
the results pooled. The x and y axis correspond to the hLID and
hAMPbd angles (in degrees) shown in Fig. 3a. (TIF) 5. Clore GM, Schwieters CD (2004) How much backbone motion in ubiquitin is
required to account for dipolar coupling data measured in multiple alignment
media as assessed by independent cross-validation? J Am Chem Soc 126: 2923–
2938. Supporting Information Figure
S1
Schematic
flow-chart
showing
the
steps
involved in the calculation of inter-domain conforma-
tional heterogeneity from RDCs. In step 1 a pool of
conformations is generated. In step 2 an algorithm for searching
ensembles of structures that match experimental data is used (see
Methods in the main text). The algorithm randomly selects 103
ensembles of size N from a pool of ca. 105 conformations
generated in the first step. For each value of N the agreement of
the ensemble with the experimental RDCs is optimized by
substituting ensemble members with structures of the pool as well
as by swapping structures between ensembles. The best 103
ensembles generated in the second step are retained and the
process is repeated until agreement between calculated and
experimental RDCs is reached. The optimal value of N is
determined by monitoring the agreement with RDCs used to
guide the genetic algorithm (Qwork) and RDCs left out of the
calculation (free RDCs, Qfree). (TIF) Figure S7
Impact of error in the experimentally mea-
sured RDCs of AKe. The fitting of RDCs (Qwork) as well as the
distributions obtained are shown. Random Gaussian error was
added to the RDCs prior to ensemble calculations. The results
shown were obtained using the ALMOND method to calculate
the alignment tensor (see Supporting Text S1). For AKe, The x
and y axis correspond to the hAMPbd and hLID angles (in degrees)
shown in Fig. 2a. Figure
S8
Experimental
distributions
of
AKe
as
a
function of error in the prediction of the alignment
magnitude (see Supporting Text S1). The RDCs of each
conformation were scaled by a number which depended on its
position along the RC(s) used in this work. The error was
increased exponentially along the RC(s) reaching at the end
points of the RC a value of 2, 3, 10, 20 and 50 fold. The x and
y axis correspond to the hAMPbd and hLID angles (in degrees)
shown in Fig. 2a. Figure
S2
Genetic
algorithm. a,
Convergence
of
the
algorithm developed in this work to select ensembles of structures
that match RDCs. Text S1
Supporting methods.
(DOCX) Text S1
Supporting methods. (DOCX) Figure
S13
Agreement
with
RDCs
left
out
of
the
calculations (20%) for AKe protein for the synthetic
distributions shown in Figs. S11 and S12 (see Supporting
Text S1). The Qfactor for working (Qwork; restrained) and free
(Qfree; unrestrained) RDCs is shown. Distributions rebuilt with
added random Gaussian error with standard deviations of 1% (first
row), 3% (second row) and 6% (third row) of error with respect the
maximum coupling are shown. 6. Lange OF, Lakomek N-A, Fare`s C, Schro¨der GF, Walter KFA, et al. (2008)
Recognition dynamics up to microseconds revealed from an RDC-derived
ubiquitin ensemble in solution. Science 320: 1471–1475. 7. Fenwick RB, Esteban-Martı´n S, Richter B, Lee D, Walter KFA, et al. (2011)
Weak Long-Range Correlated Motions in a Surface Patch of Ubiquitin Involved
in Molecular Recognition. J Am Chem Soc 133: 10336–10339. Author Contributions Conceived and designed the experiments: SEM XS. Performed the
experiments: SEM RBF JA˚ BC. Analyzed the data: SEM RBF VG MWW. Contributed reagents/materials/analysis tools SEM RBF JA˚ BC VG
MWW. Wrote the paper: SEM RBF CWB MWW XS. Conceived and designed the experiments: SEM XS. Performed the
experiments: SEM RBF JA˚ BC. Analyzed the data: SEM RBF VG MWW. Contributed reagents/materials/analysis tools SEM RBF JA˚ BC VG
MWW. Wrote the paper: SEM RBF CWB MWW XS. g
8. Clore GM, Schwieters CD (2004) Amplitudes of protein backbone dynamics and
correlated motions in a small alpha/beta protein: correspondence of dipolar Supporting Information Both data sets, restrained (Qwork) and
unrestrained RDCs (Qfree), reached a plateau region after 400 July 2014 | Volume 10 | Issue 7 | e1003721 PLOS Computational Biology | www.ploscompbiol.org 5 Correlated Inter-Domain Motions in Adenylate Kinase Figure
S9
Reconstruction of a synthetic (computer
designed)
unimodal
distribution
represented
by
a
magenta box with an increasing number of conforma-
tions, N = 1, 8 and 32. Ensembles with 8 or more members fit
equally well the synthetic RDCs and best predicted RDCs (20%)
left out of the calculations (see Supporting Text S1). The x and y
axis correspond to the hAMPbd and hLID angles (in degrees) shown
in Fig. 2a. (TIF) Figure S14
Maximum deviation in the fitted popula-
tions as a function of the alignment tensor prediction
error for AKe protein for computer designed distribu-
tions (see Supporting Text S1). The target distributions
covered closed/open states whose populations varied between 0%
to 100% in steps of 1 percent. For each degree of random error
added 100 independent runs were performed. (TIF) Figure S15
Sequence (first raw) and secondary struc-
ture of AKe in the open (pdb code 4ake; second raw) and
closed (pdb code 1ake; third raw) states. $ indicates that
the RDC was used in the calculations. Residues labeled with *
were excluded. The label – indicates that the RDC was not
available. The AMPbd (residues 28–72) and LID (113–176)
domains are highlighted in red and yellow, respectively. Residues
1–27, 73–112 and 176–214 correspond to the CORE domain. (TIF) Figure S10
Reconstruction of a synthetic (computer
designed)
four
state
distribution
represented
by
magenta boxes of equal population with N = 1, 2, 4,
8, 16 and 32. Ensembles with 16 or more members fit equally
well the synthetic RDCs and best predicted RDCs (20%) left
out of the calculations (see Supporting Text S1). The x and y
axis correspond to the hAMPbd and hLID angles (in degrees)
shown in Fig. 2a. (TIF) 1. Eisenmesser EZ, Millet O, Labeikovsky W, Korzhnev DM, Wolf-Watz M, et al.
(2005) Intrinsic dynamics of an enzyme underlies catalysis. Nature 438: 117–
121. 2. Kern D, Zuiderweg ERP (2003) The role of dynamics in allosteric regulation.
Curr Opin Struct Biol 13: 748–757. (TIF) Table S1
Agreement between the calculated and exper-
imental RDCs for AKe. Scalc refers to the result obtained when
S is predicted based on the atomic coordinates of each protein
conformation, whereas Sfit refers to the result obtained when S was
fit using single value decomposition. The Q values are those of the
final ensemble. (DOCX) Figure S12
Reconstruction of synthetic multi-modal
inter-domain orientation distributions of AKe represent-
ed by magenta boxes of equal population from NH RDCs
(see Supporting Text S1). Distributions rebuilt with added
random Gaussian error with standard deviations corresponding to
1%, 3% and 6% of error respect the maximum coupling are
shown. The best ensemble size was determined by monitoring the
agreement with RDCs used to guide the genetic algorithm (Qwork)
and RDCs left out of the calculation (free RDCS, Qfree) (Fig. S13). The x and y axis correspond to the hAMPbd and hLID angles (in
degrees) shown in Fig. 2a. (TIF) Table S2
Average structural root-mean-squared devia-
tion (RMSD) between the calculated ensemble members
and reference X-ray structures for AKe. (DOCX) Table S3
NH RDCs measured for E. coli AKe. RDCs used
in
ensemble
refinement
are
shown
in
Fig. S15
(average
experimental error ,0.3 Hz). (DOCX) (TIF) Figure S16
Alignment of AKe does not induce significant
chemical shift perturbations. Shown are chemical shift
perturbations to apo AKe induced by the inhibitor Ap5A (red)
and by introduction of the enzyme into the anisotropic stretched
polyacrylamide gel (black). Chemical shift perturbations were
calculated according to: dv = 0.2|D15N|+| D1H |. (TIF) Figure S11
Reconstruction of synthetic unimodal inter-
domain orientation distributions of AKe represented by
a magenta box from NH RDCs (see Supporting Text S1). Distributions rebuilt with added random Gaussian are shown
(standard deviations of 1%, 3% and 6% with respect to the
maximum coupling). The best ensemble size was determined by
monitoring the agreement with RDCs used to guide the genetic
algorithm (Qwork) and RDCs left out of the calculation (free
RDCS, Qfree) (Fig. S13). The x and y axis correspond to the
hAMPbd and hLID angles (in degrees) shown in Fig. 2a. (TIF) Correlated Inter-Domain Motions in Adenylate Kinase Correlated Inter-Domain Motions in Adenylate Kinase coupling and heteronuclear relaxation measurements. Biochemistry 43: 10678–
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Imported inputs and Egyptian exports: Exploring the links
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Kiel Institute for the World Economy – Leibniz Center for Research on Global Economic Challenges
Suggested Citation: Parra, María Dolores; Martínez-Zarzoso, Inmaculada (2015) : Imported inputs
and Egyptian exports: Exploring the links, Economics: The Open-Access, Open-Assessment E-
Journal, ISSN 1864-6042, Kiel Institute for the World Economy (IfW), Kiel, Vol. 9, Iss. 2015-38, pp. 1-31,
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https://hdl.handle.net/10419/122198 Article
Imported inputs and Egyptian exports: Exploring the links Economics: The Open-Access, Open-Assessment E-Journal Imported Inputs and Egyptian Exports: Exploring
the Links María Dolores Parra and Inmaculada Martínez-Zarzoso Vol. 9, 2015-38 | November 12, 2015 | http://dx.doi.org/10.5018/economics-ejournal.ja.2015-38 Vol. 9, 2015-38 | November 12, 2015 | http://dx.doi.org/10.5018/economics-ejournal.ja.2015-38 Received April 15, 2015 Published as Economics Discussion Paper April 27, 2015
Revised October 28, 2015 Accepted October 28, 2015 Published November 12, 2015
© Author(s) 2015. Licensed under the Creative Commons License - Attribution 3.0 Received April 15, 2015 Published as Economics Discussion Paper April 27, 2015
Revised October 28, 2015 Accepted October 28, 2015 Published November 12, 2015 Abstract This paper is the first to explore the links between exporting and importing activities of
Egyptian firms using panel data over the period from 2003 to 2007. The main aim is
twofold. Firstly, the authors report regression results indicating that firms that both export and
import are the most productive, followed by importing-, exporting-only firms and non-traders. Secondly, they estimate the determinants of the extensive and intensive margins of exports and
imports using dynamic panel-Probit and panel-Tobit models in combination with the method
proposed by Rabe-Hesketh and Skrondal (Avoiding biased versions of Wooldridge’s simple
solution to the initial conditions problem, 2013) to tackle the initial conditions problem. The
results show that both activities present a high degree of hysteresis, which is higher for imports
than for exports pointing to the existence of sunk costs in both activities. Moreover, past
productivity does affect the extensive margin of imports, but not of exports and the initial
condition status is also only relevant for the import side. Similar outcomes are obtained for
the intensive margin of trade. (Published in Special Issue Micro-econometric Analyses of International Firm Activities) EL
F14
Keywords
Firm level data; Egypt; internationalization; imports; exports; intermediates © Author(s) 2015. Licensed under the Creative Commons License - Attribution 3.0 Citation María Dolores Parra and Inmaculada Martínez-Zarzoso (2015). Imported Inputs and Egyptian Exports:
Exploring the Links. Economics: The Open-Access, Open-Assessment E-Journal, 9 (2015-38): 1—31. http://
dx.doi.org/10.5018/economics-ejournal.ja.2015-38 1 There is only a working paper, Kiendrebeogo (2014), investigating the learning by exporting and
selecting into exporting hypotheses for the Egyptian case, but importing activities are disregarded. Authors María Dolores Parra,
Research Group of Industry and Territory, Department of
Economics – CREIP, Universitat Rovira i Virgili, 43204 Reus, Catalonia, Spain, and
Institute of International Economics, Universitat Jaume I, Castellón, 12071, Spain,
mparra@eco.uji.es
Inmaculada Martínez-Zarzoso, Department of Economics, University of Göttingen,
37001 Göttingen, Germany, and Institute of International Economics, Universitat Jaume I,
Castellón, 12071, Spain María Dolores Parra,
Research Group of Industry and Territory, Department of
Economics – CREIP, Universitat Rovira i Virgili, 43204 Reus, Catalonia, Spain, and
Institute of International Economics, Universitat Jaume I, Castellón, 12071, Spain,
mparra@eco.uji.es Inmaculada Martínez-Zarzoso, Department of Economics, University of Göttingen,
37001 Göttingen, Germany, and Institute of International Economics, Universitat Jaume I,
Castellón, 12071, Spain Inmaculada Martínez-Zarzoso, Department of Economics, University of Göttingen,
37001 Göttingen, Germany, and Institute of International Economics, Universitat Jaume I,
Castellón, 12071, Spain Citation María Dolores Parra and Inmaculada Martínez-Zarzoso (2015). Imported Inputs and Egyptian Exports:
Exploring the Links. Economics: The Open-Access, Open-Assessment E-Journal, 9 (2015-38): 1—31. http://
dx.doi.org/10.5018/economics-ejournal.ja.2015-38 1
Introduction 1 In recent years, there has been a growing interest in the study of the
internationalization strategies of small- and medium-size firms in developing
countries. According to the related trade literature, a high proportion of trading firms
are engaged in both importing and exporting activities. Kasahara and Lapham
(2013) show that this is due to the presence of cost complementarities in both
activities. Once one of the activities is carried out, the second becomes easier. These
cost complementarities have motivated a new strand of research that further
investigates the relationship between import and export activities at the firm level,
especially those focused on the use of imported intermediates and their role in
enhancing exports (Muûls and Pisu 2009; Bas 2012; Aristei et al. 2013; Kasahara
and Lapham 2013; Lo Turco and Maggioni 2013). While most of the existent literature on the relationship between productivity
and import and export activities has focused on developed countries, the literature
concerning developing countries is still scarce. In particular, a relevant question is
whether importing intermediates generates productivity gains that add to the gains
arising from learning-by-exporting. It is yet to be established to what extent this is
also a source of gains for developing countries, which may profit more than others
from having access to intermediates from abroad. Therefore, we aim to extend the
existing evidence by investigating export and import activities of firms located in
Egypt, a developing country that to the best of our knowledge has not yet been
investigated.1 Atiyas (2011) summarizes the research that uses firm-level data in MENA
(Middle East and North African Countries) countries to analyse productivity and its
relation to trade, trade policy and financial constraints. The author emphasizes the
fact that researchers have scarcely utilized the recently available firm-level data
covering MENA countries provided by the World Bank Enterprise Survey (WBES)
to investigate the relationship between trade and productivity. We focus our analysis
on Egypt because is one of the most important countries in the MENA (Middle East
and North African) region in terms of population and gross domestic product (GDP),
and it is a developing country. According to Smeets and Warzynski (2010) and Bas 2 2 www.economics-ejournal.org and Strauss-Kahn (2011), developing countries are able to profit more than
developed countries from the benefits of importing intermediate inputs, which they
cannot always produce due to the existence of supply side restrictions. 1
Introduction In this paper, we estimate the determinants of the decision to export/import by
using a dynamic panel-Probit model applied to data from approximately 500
Egyptian industrial companies. To analyse the extensive margin of trade, we employ
a novel technique based on Rabe-Hesketh and Skrondal (2013) that is able to deal
with the endogeneity problem of the lagged dependent variable and that controls for
initial conditions in dynamic models. We also test whether the same determinants
are important in determining the intensive margin of trade; in this case a tobit
procedure is employed. The period analysed spans the years from 2003 to 2007, during which the
country experienced reductions in tariff barriers and important changes in trade
policy. More specifically, the bilateral interim agreement between the EU and
Egypt, signed in 2004, will gradually eliminate tariffs on imported products from
the EU and eventually increase competition, thus forcing some firms to exit the
market. Simultaneously, decreases in trade costs generated by more flexible rules of
origin (RoO) for products traded with the EU had a positive effect on Egyptian
exports (Bensassi et al. 2011). The main results show that export and import activities have common sunk costs
and that those are higher for import than for export activities. Past Total Factor
Productivity (TFP) levels explain the decision to import, but not the decision to
export and firms with foreign ownership are more prone to export. Our results also
show that past experience in both exporting and importing activities are the most
important factors determining internationalization strategies and similar outcomes
are found for the extensive and intensive margins of trade. This paper is organized as follows: Section 2 describes the theoretical
framework and related empirical literature. Section 3 presents the data and some
descriptive statistics. Section 4 reports the importer and exporter premia. Section 5
includes the empirical strategy and outlines the main results, and Section 6
concludes. 3 www.economics-ejournal.org 2
Theoretical framework and literature review The empirical investigations focused on explaining the links between
productivity and international trade are rich, where they find evidence confirming
the self-selection hypothesis (only firms with high productivity levels become
exporters), others support the learning-by-exporting hypothesis (firm productivity www.economics-ejournal.org 4 www.economics-ejournal.org increases after they start exporting). Although most investigations focus on the
export side, a few recent papers also consider an import perspective.2 Among the studies that focus on the export side, Bernard and Jensen (1999),
Delgado et al. (2002), Arnold and Hussinger (2005) and Aw et al. (2000) find
support for the self-selection hypothesis for exports, finding that only the most
productive firms are able to start exporting, whereas De Loecker (2007), Bustos
(2011), Van Biesebroeck (2005), Rizov and Walsh (2009) and Clerides et al. (1998)
find evidence of learning-by-exporting. Nevertheless, the results remain mixed and
mainly depend on the characteristics of the countries considered in the analysis. A few authors have investigated the self-selection and learning hypotheses from
an import perspective and have analysed the role played by intermediate imports in
increasing productivity. On the one hand, Halpern et al. (2011), Amiti and Konings
(2007) and Kasahara and Rodrigue (2008) find support for a learning-by-importing
effect. On the other hand, Wagner (2007) analyses both hypotheses, and only finds
evidence to support the self-selection hypothesis. Most of the studies focusing on foreign intermediates find different channels
through which imported inputs affect firm productivity. Some authors find that firms
that import have access to a wider variety of inputs than firms that only use domestic
providers; this in turn leads to firms easily adapting their products to the foreign
market. Indeed, Kugler and Verhoogen (2009) show that access to imports increases
the availability of different types of inputs. They find that plants which are more
productive purchase higher-quality inputs, and that despite import prices being
higher than domestic prices for the same input category in the same plant and year,
firms still use foreign inputs due mainly to their higher quality. Halpern et al. (2011)
find that firms that import all of their inputs have a 12 percent higher productivity
in comparison to firms that import only part of them. Access to foreign inputs also
means that firms are able to use inputs that are cheaper and of higher quality than
domestic inputs, especially in developing countries. Goldberg et al. 2
Theoretical framework and literature review 2 With the introduction of firm heterogeneity in models of international trade by the
seminal paper of Melitz (2003), the empirical literature studying the link between
trade and productivity has dynamically evolved over time. According to Melitz
model there is a fixed cost of exporting and firms can enter in a foreign market by
paying it. They then select their level of productivity and if it is too low to be
profitable, they are forced to leave the market. This seminal theory has been
extended in several directions, one of this works is Kashara and Lapham (2013) that
introduces the importance of importing activities in the internationalization process
of the firm. These authors extended Melitz (2003) model introducing imported
inputs and showed the existence of some productivity gains stemming from
importing inputs, which allow importers to start exporting. As a result, a cost
complementarity effect emerges between import and export activities. In order to
produce final goods, firms can use imported inputs, domestic inputs or a
combination of both, and their decision to import/export is linked to the associated
import/export fixed costs in which they have to incur. The model is based in an open
economy with heterogeneous final goods producers, where the firm makes
simultaneously the decision to export their output and the decision to use imported
intermediates and firms have to pay a fixed cost to enter into the foreign market in
order to import and export. The authors also introduce firm’s productivity, transport
costs for importing intermediates and for exporting final goods, and take into
account the trade status of the firm in order to capture the observed changes in the
firm’s trade status over time. In particular, they consider whether a firm is import-
only, export-only, both or only sells in the domestic market. They assume that two-
way traders necessarily face higher trade costs, and for this reason only the most
productive firms are able to operate as such. The model predict that if there is a
common fixed cost for both activities, the firms that are one-way traders are more
likely to start exporting and, in due course, become two-way traders. 2 See Silva et al. (2012) for a survey of the learning-by exporting literature and Singh (2010) for a
detailed literature review about the effects of international trade on productivity and economic growth
at the macro- and micro-levels. 2
Theoretical framework and literature review (2010) show
how the combined use of foreign and domestic inputs increases the product scope
of Indian firms, and that better access to foreign inputs after trade liberalization is 5 5 www.economics-ejournal.org more important than the price reduction effect produced by the decrease in trade
costs. Another important aspect worth mentioning is that the diffusion of modern
technologies through the use of foreign intermediate goods appears especially
beneficial for developing countries, which benefit the most from these technological
spillovers. Meanwhile, the origin of the imported inputs and their impact on
productivity have also been analysed in order to understand the technology transfer
linked to imported intermediates. In their analysis, Smeets and Warzynski (2010)
distinguish between inputs from the OCDE and those from low-income economies,
analysing their impact on total factor productivity (TFP). The authors find that both
affect productivity in a similar way. However, Bas and Strauss-Kahn (2011),
compare imported inputs from developed and developing countries for French firms,
and find that foreign intermediates from developed countries increase TFP 20
percent more than inputs from developing countries. They also find that importing
more varieties of intermediate inputs increases TFP and also the number of exported
varieties of French firms. Other authors have focused their attention on analysing how trade liberalization
in intermediate inputs affects productivity. Amiti and Konings (2007) was one of
the first studies to estimate the relationship between productivity and the effects of
trade liberalization on imported inputs. Using Indonesian data, they analyse the
productivity gains that result from reducing tariffs on final goods and on
intermediate inputs separately, showing that a ten percent reduction in input tariffs
led to a productivity gain of 12 per cent for firms that use imported inputs, and that
this gain was twice as large as gains from reducing tariffs on final goods. Bas (2012)
studies the impact of input-trade liberalization on Argentinian firms’ export
decisions, finding that a reduction in input-tariff on foreign intermediates enhances
Argentinian firms' performance in the export market and also increases the
percentage of exports. Goldberg et al. (2010) provide evidence indicating that trade
liberalization increases productivity not only due to the access to cheap inputs but
also due to the opportunity to access new intermediate inputs that allow firms to
create new varieties in the domestic market. The literature that directly links both international activities is scarce. Bernard
et al. www.economics-ejournal.org 2
Theoretical framework and literature review (2007) were the first authors to consider jointly both activities and they find
that two-way traders are more productive than only exporters or importers. Altomonte and Békés (2010) highlight that the previous literature that analyses the 6 6 www.economics-ejournal.org export-productivity link without taking import decisions into account overestimates
the export gains. There are a few papers that focus in particular on how imports affect exports. For example, Sjöholm (2003) estimates a static panel model using data for
Indonesian manufacturing firms and finds, that the probability to export in the
current year are positively affected by the past firm import status. Sjöholm and Takii
(2008) estimate instead a dynamic binary model using the approach proposed by
Wooldridge (2005). The authors obtain a high degree of hysteresis on the export
activity, where past import status does not increase the probability to export. Lo
Turco and Maggioni (2013) analyse how imports affect the probability to export for
Italian manufacturing firms. They find that importing form low-income countries
affects the probability to export and that past export status positively affects the
probability to continue with this activity. To our knowledge, only two recent papers analyse the link between both
activities in a dynamic framework. Firstly, Muûls and Pisu (2009) test the existence
of sunk costs of imports in addition to sunk cost of exports using a dynamic panel
probit and taking the coefficient of the lagged dependent variable as a measure of
sunk costs. Their results show that exports and imports show a high degree of
hysteresis, meaning that past status explain the current status. Also they find that
sunk costs decrease when the complementary activity was carried out the past year,
meaning that common fixed costs exist, and obtain a higher sunk cost for imports
than for exports. Secondly, Aristei et al. (2013) using data for Eastern European and
Central Asian firms obtain a high degree of hysteresis for both activities, but higher
for exports than for imports and find that past imports affect the probability to export
in the current year but not the other way around. The numbers of studies focused on MENA countries are few. Related to the role
that imported intermediates could play in technological diffusion, Brach (2010)
assesses the role of technological readiness in the MENA region and the
implications for Egypt. www.economics-ejournal.org 3 The data comes from a firm-level survey based on a representative sample of manufacturing Egyptian
firms classified using ISIC codes 15-37, 45, 50-52, 55, 60-64, and 72 (ISIC Rev.3.1). Formal
(registered) companies with 5 or more employees are targeted for interviews and firms with 100%
government/state ownership are not eligible to participate in the Enterprise Survey. Business owners
and top managers answer the Enterprise Survey from the World Bank. Sometimes the survey
respondent calls company accountants and human resource managers into the interview to answer
questions concerning the sales and labour sections of the survey, which covers a broad range of
business environment topics including access to finance, corruption, infrastructure, crime, competition,
and performance measures. Typically, 1200-1800 interviews are conducted in larger economies, 360
interviews in medium-sized economies, and only 150 interviews in small economies. See World Bank
(2012) for more details. In our empirical application we have 519 firms for which panel data is
available, i.e. they are interviewed in all three years. Hence we have around 50 percent of the original
sample. 2
Theoretical framework and literature review The author takes a closer look at the technological progress
and innovative activities in the MENA region and within this context investigates
the implications for economic development and job creation, as well as the main
economic policy recommendations. She finds that one of the major constraints to
improving economic performance and sustainable job creation is a general lack of
technological capabilities of the MENA countries. Innovation in these countries is
mainly linked to the adaptation and modification of existing technologies and the 7 7 www.economics-ejournal.org low level of technological readiness negatively impacts innovation and productivity. Hence, the use of foreign intermediates can be a good way to transfer modern
technologies from foreign markets to MENA countries. To the best of our
knowledge, only Kiendrebeogo (2014) analyses the Egyptian manufacturing sector
and how Egyptian firms perform depending on their export activity using WBES. He finds that exporter firms are larger, more capital-intensive and more productive
than domestic-only firms. He examines the self-selection and learning-by-exporting
hypotheses, showing that although exporting has a positive impact on firm
productivity, supporting the learning by exporting hypothesis of Egyptian firms, the
pre-entry differences in productivity do not explain firms’ export decisions. However, the author does not consider importing activities in his analysis. For this
reason, in our paper we want to extend this analysis by considering exporting and
importing activities, taking advantage of the raw data characteristics. By focusing
on the relationship between exporting and importing activities in Egyptian firms, we
aim at producing some policy recommendations for this country concerning their
participation in regional integration processes and their industrial policies after the
Arab Spring revolution. 3
Data and descriptive statistics Data on Egyptian firms are obtained from the World Bank Enterprise Survey
dataset.3 The dataset includes 3,129 firms for the years 2004, 2005 and 2007. For
some variables, namely sales, exporting and importing status we are able to use 8 www.economics-ejournal.org information for an additional year per questionnaire, since each firm is asked in the
questionnaire about the current year of the questionnaire and the previous year. Some firms are only included in one or two years, whereas 554 firms are included
in the three questionnaires. Therefore, using the available information for these
firms and after data cleaning, we build a panel dataset from 2003 to 2007 keeping
519 firms obtaining around 1,890 observations. Table 1 shows the evolution over time of the exporting and importing status of
Egyptian firms in our sample, distinguishing between firms that only sell products
in the country (domestic firms), firms that sell in the domestic market and only carry
out one international activity (export-only) (import-only) and firms that sell in the
domestic market and are involve in both international activities, (two-way traders). The results show that the majority of Egyptian manufacturing firms are focused on
the domestic market, results are in line with the empirical literature which highlight
that international-trading firms are fairly scarce (Bernard et al 2007). The
percentages of export-only and import-only firms remain quite stable over time,
around 8 and 11 percent on average, respectively. We observe that only 7 percent of
all firms in our sample are involved in both importing and exporting activities in
2003. This number has increased over time and has reached 16 per cent of the
number of total firms in 2007. The last rows of Table 1 show the percentage of
imported inputs used by import-only firms and two-way traders, showing that on
average, more than half of the inputs used in production are imported. In addition,
the share has not increased over time and it is relatively stable for both types of
firms. The second part of Table 1 displays the relative importance of each industry by
status. Firms are classified into nine industrial categories, of which Garments, Non-
metal industries and Other industries mainly sell their products in the domestic
market, whereas almost half of the two-way traders belong to the Chemical and
Machinery and equipment industries. 3
Data and descriptive statistics Despite the fact that Egyptian firms are
mainly focused on the domestic market, those that are involved in international
activities tend to engage in both import and export activities, rather than in only one
of them. There are only a few exceptions in some industries in which one of the
international activities is more important than the other. This is the case in the
electronics industry, where import of intermediate goods is the only 9 www.economics-ejournal.org www.economics-ejournal.org Table 1: Sample composition by trade status and percentage of imported input
Year
Import-only
Export-only
Two-way traders
Domestic
2003
13%
7%
7%
73%
2004
12%
7%
13%
68%
2005
10%
6%
15%
69%
2006
9%
10%
10%
71%
2007
11%
9%
16%
64%
Average
11%
8%
12%
69%
Industries
Import-only
Export-only
Two-way traders
Domestic
Agro industries
16%
11%
13%
60%
Chemicals
22%
9%
26%
43%
Electronics
36%
0%
0%
64%
Garments
5%
7%
8%
80%
Machinery and equipment
22%
11%
20%
47%
Metal industries
13%
8%
11%
68%
Non-metal industries
9%
7%
8%
76%
Other industries
8%
8%
13%
71%
Textiles
12%
7%
13%
68%
% imported intermediates
Import-only
Two-way
traders
2003
54%
49%
2004
57%
48%
2005
50%
46%
2006
48%
49%
2007
51%
48%
Average
52%
48%
Note: Authors’ elaboration using data from the World Bank Enterprise Survey. Export-only firms
denotes firms that sell in the local market and also export, Import-only firms denotes firms that sell
into domestic market and also import, Two-way traders refers to firms that sell into the domestic market
and also export and import and Domestic indicate firms that only sell in the local market and are not
engaged in international activities. Table 1: Sample composition by trade status and percentage of imported input Note: Authors’ elaboration using data from the World Bank Enterprise Survey. Export-only firms
denotes firms that sell in the local market and also export, Import-only firms denotes firms that sell
into domestic market and also import, Two-way traders refers to firms that sell into the domestic market
and also export and import and Domestic indicate firms that only sell in the local market and are not
engaged in international activities. international activity. It seems that firms in this industry import intermediate goods
to produce products for the local market; especially the majority are larger factories
assembling products for international brands. 3
Data and descriptive statistics Also, the chemical and machinery and
equipment industries show a higher share of importers than exporters. This
descriptive analysis shows that the nature of the different industries might influence
the decision to import/export; indeed, some industries are more likely to participate
in international markets. For this reason, we need to take industry effects into
account in our analysis. 10 10 www.economics-ejournal.org www.economics-ejournal.org Table 2: Descriptive statistics by trade status
Variable
Obs
Mean
Std. Min
Max
Export-only firms
TFP i,t
182
7.12
1.68
0.95
10.35
work i,t
188
251.45
478.84
8.00
2800
foreignowner i,t
191
3.00
15.38
0.00
1.00
px i,t
191
39.92
32.91
0.50
100
pm i,t
191
0.00
0.00
0.00
0.00
capital i,t
180
20229.64
53644.10
50.00
531419
investment i,t
185
129047.50
1601345
0.00
2.18e+07
Import-only firms
TFP i,t
258
6.98
1.61
0.95
11.39
work i,t
281
250.75
907.84
8.00
13,695
foreignowner i,t
281
0.06
0.23
0.00
1.00
px i,t
281
0.00
0.00
0.00
0.00
pm i,t
281
50.84
31.19
1.00
100
capital i,t
253
192808.40 1446639.00
0.00
1.57e+07
investment i,t
262
128012.50 1323347.00
0.00
1.52e+07
Two-way traders
TFP i,t
297
7.83
1.76
0.98
14.37
work i,t
314
634.40
1206.94
0.00
13,15
foreignowner i,t
316
0.11
0.31
0.00
1.00
px i,t
316
39.02
33.81
0.90
100
pm i,t
316
47.25
29.08
2.00
100
capital i,t
298
129055.70
698418.30
5.00
9,800,000
investment i,t
297
175131.00
1902239
0.00
2.99e+07
Domestic firms
TFP i,t
1646
5.44
1.48
1.41
12.93
work i,t
1770
69.11
427.99
0.00
10,500
foreignowner i,t
1783
0.02
0.12
0.00
1.00
px i,t
1783
0.00
0.00
0.00
0.00
pm i,t
1783
0.00
0.00
0.00
0.00
capital i,t
1639
33258.00
476477.50
0.00
1.22e+07
investment i,t
1686
9237.41
165149.10
0.00
6550000 Table 2: Descriptive statistics by trade status Notes: Obs denotes number of observations; Std. Dev denotes standard deviation and Min and Max
are the minimum and maximum value of each variable. TFP i,t is total factor productivity, obtained
using the Levinsohn-Petrin (2003) procedure. 3
Data and descriptive statistics We explain the choice of this methodology and the
estimation in Appendix A.2; work i,t is the average number of workers; foreignowneri,t is a dummy
variable that takes the value of 1 if the firm is owned by foreigners and 0 otherwise; pxi,t is the share
of exports over total sales and pmi,t is the share of imports over total sales; capital i,t is the total
fixed tangible assets value of machinery and investment i,t is the net book value of machinery. 11 www.economics-ejournal.org www.economics-ejournal.org Table 2 presents summary statistics of the main variables used in the analysis. The figures show that firms involved in international activities perform better than
domestic-only firms in terms of total factor productivity (TFP)4 and size (measured
with number of workers: work). Among the three types of international firms, we
observe that firms with higher productivity are more often two-way traders than
export-only firms or import-only firms, and domestic firms have the lowest average
productivity. It is also worth noting that two-way traders are not only bigger in size
than import-only and export-only firms but also invest more. We also observe that
firms owned by foreigners are more focused on international activities. 4 TFP has been obtained using the Levinsohn-Petrin (2003) methodology. See Appendix A.2. How different are Egyptian traders? Firm-level
characteristics of traders vs. non-traders Following studies that analyse how firm trade status affects firm characteristics
(Bernard et al. 2007; Muuls and Pisu 2009; Castellani et al. 2010; Seker 2012;
Sharma and Mishra 2015), we start this section by computing the exporter and
importer premia for Egyptian firms. Exporter/importer premia are conventionally
determined estimated by regressing the dependent firm-performance variable
indicators, usually expressed as TFP, labour productivity, wages, number of workers
or capital, among others, on an exporter/importer dummy and other a number of
control variables as explanatory variables using OLS estimations. The estimated
coefficients of the dummy trade variables show the exporter/importer premia
meaning or simple correlations between the dependent variable and the trade
dummy variables used. At this point, a causal interpretation of the results is not
possible. Clearly the results cannot be interpreted as causal effects. The main idea
aim is to confirm whether an export/import premium for Egyptian international
firms is present, which will be in accordance with the related empirical literature. Since some exporters are also importers —as in Bernard et al (2007), Altomonte and
Bekés (2010), Muûls and Pisu (2009)—we distinguish between import-only firms,
export-only firms and two-way traders to better understand the characteristics of
international Egyptian firms compared with domestic-only firms. The estimated equation is: 12 www.economics-ejournal.org 𝑙𝑛𝐹𝑖,𝑡= 𝛼0 + 𝛼1 𝑑𝑖,𝑡
𝑥𝑜+ 𝛼2 𝑑𝑖,𝑡
𝑚𝑜+ 𝛼3𝑑𝑖,𝑡
𝑥𝑚+ β1𝑓𝑜𝑟𝑒𝑖𝑔𝑛𝑜𝑤𝑛𝑒𝑟 𝑖,𝑡+ 𝛽2𝑙𝑛𝑤𝑜𝑟𝑘𝑖,𝑡
+𝛾𝑘+ 𝛿𝑡+ 𝜀𝑖,𝑡 (1) (1) Where ln denotes natural logs; Fi,t denotes a given firm performance indicator5
(TFP, size, sales, capital and investment) used as dependent variable. TFPi,t is
obtained using the Levinsohn-Petrin (2003) methodology, worki,t denotes firm size
proxied by the average number of workers, salesi,t denotes the total sales of the firm,
capitali,t is the total fixed tangible assets deflated using the production price index
for manufactures and investmenti,t is the net book value of machinery and equipment,
all variables are in natural logs. As explanatory variables we include 𝑑𝑖,𝑡
𝑥o, a dummy
variable taking the value of 1 if the firm only exports and zero otherwise. di,t
mo takes
the value of 1 if the firm only imports and di,t
xm takes the value of 1 if the firms are
two-way traders, zero otherwise. As control variables, we include the percentage of
the firm owned by a foreigner and when the dependent variable is not employment,
we include also firm size. 5See Appendix A.1. www.economics-ejournal.org How different are Egyptian traders? Firm-level
characteristics of traders vs. non-traders We also include industry dummies and year dummies to
take into account any unobserved effects that are industry specific and time invariant
and those that are common across industries and time variant. The former could be
a proxy of specific comparative advantages and the later controls for the business
cycle. Both type of effects also control for potential measurement errors. Table 3 presents the estimated trade-status premia using fixed effects obtained
from estimating Equation (1). 13 13 www.economics-ejournal.org Table 3. Exporter and importer premia
(1)
(2)
(3)
(4)
(6)
Dependent Variable
lnTFPi,t
lnworki,t
lnsales i,t
lncapital i,t
lninvestment i,t
Export-only firms
1.107***
0.896***
1.329***
0.213
1.295***
(0.194)
(0.142)
(0.217)
(0.234)
(0.269)
Import-only firms
0.889***
0.955***
1.130***
0.367*
0.976***
(0.165)
(0.119)
(0.180)
(0.215)
(0.217)
Two-way traders
1.407***
1.767***
1.855***
0.116
1.608***
(0.199)
(0.143)
(0.219)
(0.198)
(0.246)
foreignowner i,t
0.322***
0.448***
0.787***
0.376***
(0.0362)
(0.0396)
(0.0655)
(0.0478)
lnwork i,t
0.000123
-0.00145
-0.000321
0.00627
-0.0135
(0.00243)
(0.00383)
(0.00278)
(0.00673)
(0.0127)
Constant
4.785***
3.143***
5.387***
4.406***
4.572***
(0.135)
(0.0408)
(0.145)
(0.257)
(0.189)
Observations
1,978
2,547
1,985
1,963
1,968
Number of firms
518
519
518
519
519
Year dummies
yes
yes
yes
yes
yes
Industry dummies
yes
yes
yes
yes
yes
Rho
0.238
0.297
0.298
0.186
0.180
Note: Robust standard errors (clustering at firm level) in parentheses *** p<0.01, ** p<0.05, * p<0.1. Note: Robust standard errors (clustering at firm level) in parentheses *** p<0.01, ** p<0.05, * p<0. We observe that firms involved in international trade, irrespective of their trade
pattern, are more productive, are larger in size, sell more, own more capital, and
invest more than domestic-only firms. In particular, two-way traders are the best
performers in all cases, and only exporters have higher premia than only importers
in TFP, sales and investment. These results are in line with those obtained by
Bernard et al (2007), Muûls and Pisu (2009), Castellani et al (2010) Seker (2012)
and Sharma and Mishra (2015) for other countries. Some recent studies for other
developing countries, for example, Seker (2012) using data for 43 developing
countries obtain that two-traders are best performers in all measures, followed by
only exporters and only importers in comparison to domestic-only firms. Also
Sharma and Mishra (2015) using data for Indian manufacturing firms over the
period 1994–2006 finds the same pattern. 14 www.economics-ejournal.org www.economics-ejournal.org 5.1.1
Extensive margin of trade In order to estimate the determinants of export and import decisions and analyse
how both activities are related, we model the probability of exporting/importing as
a function of TFP, size of the firm and ownership structure. In order to account for
correlations between exporting and importing activities, we include in the models
past import-status in the export equation and past export-status in the import
equation. We also add the lagged left hand side variables as explanatory variables. In this dynamic framework we will be able to investigate the existence of state
dependence, also termed hysteresis, in export and import activities. In other words,
we assume that there is some sort of persistence affecting the decision to export final
outputs and import intermediates, and we would like to disentangle the effect of past
status from the firm’s initial condition as exporter/importer. The inclusion in the
model of the lagged values of the dependent variables has been considered by
several authors as a way to introduce a measure of the sunk costs (Bernard and
Jensen, 2004; Muûls and Pisu, 2009; and Roberts and Tybout, 1997). The proposed equations for exports and imports are given by, Pr(𝑥𝑖,𝑡= 1) = Pr (𝛽0 + 𝛽1𝑥𝑖,𝑡−1 + 𝛽2 𝑚𝑖,𝑡−1 + 𝛽3 ln(𝑤𝑜𝑟𝑘𝑖,𝑡−1) +
𝛽4𝑓𝑜𝑟𝑒𝑖𝑔𝑛𝑜𝑤𝑛𝑒𝑟𝑖,𝑡 + 𝛽5 ln(𝑇𝐹𝑃𝑖,𝑡−1) + 𝛾𝑘+ 𝛿𝑡+ 𝑢𝑖,𝑡 >0) (2) (2) Pr(𝑚= 1) = Pr (𝛽0 + 𝛽1𝑚𝑖,𝑡−1 + 𝛽2 𝑥𝑖,𝑡−1 + 𝛽3 + 𝛽4𝑓𝑜𝑟𝑒𝑖𝑔𝑛𝑜𝑤𝑛𝑒𝑟𝑖,𝑡 +
𝛽5 ln(𝑇𝐹𝑃𝑖,𝑡−1) + 𝛾𝑘+ 𝛿𝑡+ 𝑢𝑖,𝑡 >0) (3) where ln denotes natural logarithms, the subscript i indexes firms; t, indexes time. The dependent variable in Equation (2), Pr(xi,t=1), denotes the probability of exports
and is a dummy variable that takes the value of 1 if firm i exports in year t, and 0
otherwise and the dependent variable in Equation (3), Pr(mi,t=1), is the probability
of importing, which takes the value of 1 if firm i imports in year t, and 0 otherwise. As explanatory variables we include mi,t-1 is a dummy variable reflecting the import 15 www.economics-ejournal.org www.economics-ejournal.org status of the firm in year t–1 and xi,t-1 is a dummy variable indicating the exporting
status of the firm in year t–1, TFPt-1 is total factor productivity of the firm obtained
by using the Levinsohn-Petrin (2003) methodology. 6 We also used alternatively the percentage of the firm owned by a foreigner, but since high and a low
percentage of foreign ownership have approximately the same effect, we decided to create a 1/0
dummy. 7 Results are obtained using xtprobit command in Stata11. 5.1.1
Extensive margin of trade workit-1 denotes the average
number of workers in t–1, and foreigowneri.t is a dummy variable that takes the
value of 1 if the firm is owned by foreigners and 0 otherwise.6 𝑙𝑛𝑇𝐹𝑃
̅̅̅̅̅̅̅𝑖 and 𝑤𝑜𝑟𝑘𝑡
̅̅̅̅̅̅̅̅𝑖
are respectively the average of each variable. Industry (𝛾k) and time dummies (δt)
have also been included in the model to proxy for factors that are industry specific
and time-invariant and for those that vary over time and are common to all firms. These variables have been commonly included as control variables in models used
to estimate the determinants of the decision to export; see for example Greenaway
et al (2007) and Muûls and Pisu (2009). The main difficulty of explicitly allowing for lagged effects is that the
correlation between the unobserved heterogeneity and the lagged dependent variable
in the dynamic binary choice model makes the lagged dependent variable
endogenous. Hence, the estimators used before will not be consistent. A familiar
alternative approach is based on Wooldridge (2005), which builds on the random
effects specification and basically adds the initial condition and the averages over
time of the time-variant variables as additional regressors. The solution proposed by
Wooldridge (2005) has been improved by Rabe-Hesketh and Skrondal (2013), who
exclude the first period when calculating the averages over time of the regressors. Therefore, we follow a similar strategy to Aristei et al (2013) and Muûls and
Pisu (2009) but use instead a more reliable estimation technique that will enable us
to disentangle the effect of the initial conditions from the effect of the past
export/import status of the firm on the decision to export/import. To deal with the so-called “initial condition” problem (basically, we cannot
observe the first dependent observation in the data-generating process, hence we
cannot treat the stochastic process from its starting point and consequently we
cannot treat it as fixed) previous literature used Wooldridge’s auxiliary model. However, as stated by Rabe-Hesketh and Skrondal (2013), Wooldridge’s (2005)
method performs poorly for short panels, mainly because if the means are based on
all periods, the initial conditions are also used to compute those means, and this
induces endogeneity. The authors suggest including the initial-period as explanatory 16 www.economics-ejournal.org variable and calculate the mean only using the remaining periods, which is t+1 until
n. variable and calculate the mean only using the remaining periods, which is t+1 until
n. Columns (1) and (2) of Table 4 include only the lagged value of the dependent
variable. A high degree of hysteresis is obtained for both activities, indicating that
past trade status increase the probability to continue with the same activity. We
obtain that the sunk costs that firms face to import intermediates are higher than
those needed to export. Indeed, liberalization of imports with the EU started in 2004
with the entry into force of the FTA and imports from EU were progressively
liberalised during a period of 10 years, whereas exports were already liberalized in
1972 with the bilateral cooperation agreements. Results in Columns (3) and (4) in Table 4 show that the combination of both
export and import activities affect the probability of importing/exporting. It can be
observed that past export and import participation have a high degree of hysteresis,
and firms face higher sunk costs for imports than for exports. The results are
similar to those obtained in Muûls and Pisu (2009), in that exporter/importer status
in the previous year has a positive effect on the probability of exporting/importing
in the current year and the magnitude of the effect is also higher for imports than
for exports. Contrary to Aristei et al. (2013) we find that foreign ownership affects positively
both the export and import status of the firm, remaining only significant for exports
when the imports variable dummy is included in the regression. However, past TFP
affects the probability of importing but not of exporting, and the size of the firm
affects only the probability to export when the import dummy variable is not
included. Both, Aristei et al. (2013) and Muûls and Pisu (2009) find that past TFP
influences export and import activities, however in our case, the results show that
past TFP only affect the extensive and intensive margin of imports only, but not of
exports. There is also a different effect of the variable firm size on the probability
of exporting and importing. Firm size has a positive effect only on the decision to
start exporting. This could be explained by the fact that larger firms are able to serve
the domestic and the foreign market because they have a higher production capacity
than smaller firms. 5.1.1
Extensive margin of trade As in Wooldridge (2005), we assume that 𝑢𝑖, (𝜀𝑖), the firm specific effects are
determined by, 𝑢𝑖 = 𝛽0 + 𝛽1𝑥|𝑚𝑖0 + 𝛽2𝑙𝑡𝑓𝑝
̅̅̅̅̅𝑖+ 𝛽3𝑙𝑤𝑜𝑟𝑘𝑡
̅̅̅̅̅̅̅̅𝑖+ 𝜇𝑖,𝑡
(4 (4) where 𝜇𝑖 is an independently and normally distributed error term and the control
variables are now the firm-level average of each variable over time. However, as
Rabe-Hesketh and Skrondal (2013) suggest, the firm-level average must be obtained
excluding the initial period and then adding a dummy in the regression capturing
whether the firm exports ( 𝑖𝑒𝑖)/imports ( 𝑖𝑖𝑖) in the first period of the sample. If we
now substitute Equation (4) into Equations (2) and (3) we obtain: Pr(𝑥𝑖,𝑡= 1) = Pr (𝛽0 + 𝛽1𝑥𝑖,𝑡−1 + 𝛽2𝑚𝑖,𝑡−1 + 𝛽3 ln(𝑤𝑜𝑟𝑘𝑖,𝑡−1) +
𝛽4𝑓𝑜𝑟𝑒𝑖𝑔𝑛𝑜𝑤𝑛𝑒𝑟𝑖,𝑡 +
𝛽5𝑙𝑇𝐹𝑃𝑖+ 𝛽6𝑙𝑇𝐹𝑃
̅̅̅̅̅̅𝑖+ 𝛽7𝑤𝑜𝑟𝑘𝑡
̅̅̅̅̅̅̅̅𝑖+ 𝜇𝑖 + 𝑖𝑒𝑖+ 𝑖𝑖𝑖+ 𝛾𝑘+
𝛿𝑡+ 𝑢𝑖,𝑡>0)
(5) (5) Pr(𝑚𝑖,𝑡= 1) = Pr (𝛽0 + 𝛽1𝑚𝑖,𝑡−1 + 𝛽2 𝑥𝑖,𝑡−1 + 𝛽3 ln(𝑤𝑜𝑟𝑘𝑖,𝑡−1) +
𝛽4𝑓𝑜𝑟𝑒𝑖𝑔𝑛𝑜𝑤𝑛𝑒𝑟𝑖,𝑡 + 𝛽5𝑙𝑇𝐹𝑃𝑖+ 𝛽6𝑙𝑇𝐹𝑃
̅̅̅̅̅̅𝑖+ 𝛽7𝑤𝑜𝑟𝑘𝑡
̅̅̅̅̅̅̅̅𝑖+ 𝜇𝑖 + 𝑖𝑒𝑖+ 𝑖𝑖𝑖+ 𝛾𝑘+
𝛿𝑡+ 𝑢𝑖,𝑡>0)
(6) (6) In order to test the existence of sunk costs in import and export activity and to
measure the importance of these sunk costs, we estimate the parameters of Equations
(5) and (6) using a panel-Probit model with random effects7 based on maximum
likelihood estimation techniques for the period 2003–2007, and we interpret the
estimated coefficients for the dependent lagged variable as a measure of the
importance of sunk costs, as the authors cited above. We argue that sunk costs
generate hysteresis in the export and import market participation. The results from
estimating Equations (5) and (6) are shown in Table 4. 17 www.economics-ejournal.org www.economics-ejournal.org TFPi,t denotes total factor productivity, it is obtained using the
Levinsohn-Petrin (2003) procedure; TFPi,t -1 are lagged values of TFPi,t;; worki,t denotes the average
number of workers and work i,t–1 are aged value of the variable; xt.i are a dummy variable that takes
value 1 if the firm is exporting and 0 otherwise, xt.i–1 are the corresponding lagged value and foreign
owneri,t is a dummy variable that takes the value of 1 if the firm is owned by foreigners and 0 otherwise. The number of observations differ between column (1) and (2) and between columns (3) and (4) due
to the inclusion of lagged imports and initial conditions for which 22 and 19 observations are
respectively missing data. However, firm size does not affect the probability of importing
indicating that firms import intermediates probably because these are not available
in the domestic market, independently of the scale of production 18 www.economics-ejournal.org www.economics-ejournal.org Table 4: Dynamic panel-Probit model controlling for initial conditions (Exports and
Imports)
(1)
(2)
(3)
(4)
P(xit=1)
P(mit=1)
P(xit=1)
P(mit=1)
x i, t-1
1.489***
1.408***
1.342***
(0.112)
(0.136)
(0.140)
m i, t-1
1.591***
1.219***
1.427***
(0.114)
(0.135)
(0.140)
ln work i,t-t
0.130**
0.057
0.071
–0.003
(0.056)
(0.055)
(0.062)
(0.064)
foreign owner i,t
0.625***
0.319*
0.600***
0.047
(0.175)
(0.177)
(0.202)
(0.218)
ln TFP i, t-1
0.041
0.063*
0.034
0.059
(0.036)
(0.036)
(0.039)
(0.041)
ln TFP mean i, t-1
0.097
0.094
0.012
0.067
(0.060)
(0.061)
(0.069)
(0.075)
ln work mean i,t-t
0.072
0.070
0.096
0.046
(0.073)
(0.074)
(0.083)
(0.088)
iei
–0.060
0.162
(0.122)
(0.157)
iii
0.029
0.347**
(0.118)
(0.170)
Year dummies
yes
yes
yes
yes
Industry dummies
yes
yes
yes
yes
Number of observations
1889
1867
1882
1863
Notes: The dependent variable P(x=1) is a dummy variable for the exporter status and P(m=1) for the
importer status. t–1 denotes lagged values of these variables. iei denotes initial exporter dummy. iii
means initial importer dummy. Standard errors are in brackets, where *** p<0.01, **p<0.05, * p<0.1. Industrial and year dummies included. TFPi,t denotes total factor productivity, it is obtained using the
Levinsohn-Petrin (2003) procedure; TFPi,t -1 are lagged values of TFPi,t;; worki,t denotes the average
number of workers and work i,t–1 are aged value of the variable; xt.i are a dummy variable that takes
value 1 if the firm is exporting and 0 otherwise, xt.i–1 are the corresponding lagged value and foreign
owneri,t is a dummy variable that takes the value of 1 if the firm is owned by foreigners and 0 otherwise. The number of observations differ between column (1) and (2) and between columns (3) and (4) due
to the inclusion of lagged imports and initial conditions for which 22 and 19 observations are
respectively missing data. Table 4: Dynamic panel-Probit model controlling for initial conditions (Exports and
Imports) Notes: The dependent variable P(x=1) is a dummy variable for the exporter status and P(m=1) for the
importer status. t–1 denotes lagged values of these variables. iei denotes initial exporter dummy. iii
means initial importer dummy. Standard errors are in brackets, where *** p<0.01, **p<0.05, * p<0.1. Industrial and year dummies included. 5.1.2
Intensive margin of trade In this section we analize whether the results are analogous for the impact on the
intensive margin of trade. The estimated model is given by Equations (7) and (8),
similar to Equations (2) and (3) used for the extensive margin; the difference is that 19 www.economics-ejournal.org the dependent variable is proxied by the percentage of exports over total sales of
firm i in year t, and by the percentage of total purchases of materials inputs imported
from firm i in year t. We propose a dynamic model following the method proposed
by Rabe-Hesketh and Skrondal (2013). 𝑒𝑥𝑝𝑖,𝑡= 𝛽0 + 𝛽1𝑒𝑥𝑝𝑖,𝑡−1 + 𝛽2 𝑖𝑚𝑝𝑖,𝑡−1 + 𝛽3 ln(𝑤𝑜𝑟𝑘𝑖,𝑡−1) +
𝛽4𝑓𝑜𝑟𝑒𝑖𝑔𝑛𝑜𝑤𝑛𝑒𝑟𝑖,𝑡 + 𝛽5 ln(𝑡𝑓𝑝𝑖,𝑡−1) + 𝛽6𝑙𝑡𝑓𝑝
̅̅̅̅̅𝑖+ 𝛽7𝑤𝑜𝑟𝑘𝑡
̅̅̅̅̅̅̅̅𝑖+
𝜇𝑖 + 𝑖𝑒𝑖+ 𝑖𝑖𝑖+ 𝛾𝑘+ 𝛿𝑡+ 𝑢𝑖
(7) (7) 𝑖𝑚𝑝𝑖,𝑡= 𝛽0 + 𝛽1𝑖𝑚𝑝𝑖,𝑡−1 + 𝛽2 𝑒𝑥𝑝𝑖,𝑡−1 + 𝛽3 ln(𝑤𝑜𝑟𝑘𝑖,𝑡−1) +
𝛽4𝑓𝑜𝑟𝑒𝑖𝑔𝑛𝑜𝑤𝑛𝑒𝑟𝑖,𝑡 + 𝛽5 ln(𝑡𝑓𝑝𝑖,𝑡−1) + 𝛽6𝑙𝑡𝑓𝑝
̅̅̅̅̅𝑖+ 𝛽7𝑤𝑜𝑟𝑘𝑡
̅̅̅̅̅̅̅̅𝑖+
𝜇𝑖 + 𝑖𝑒𝑖+ 𝑖𝑖𝑖+ 𝛾𝑘+ 𝛿𝑡+ 𝑢𝑖
(8) (8) In this case, the parameters of the model are estimated using a panel-Tobit
procedure. The election of this estimation technique is justified by the fact that our
dependent variable is continuous and positively distributed, taking censored values
from 0 to 100. As stated in Wooldridge (2010), the use of linear models is not
recommended in this case where corner solutions are present, and a censored re-
gression model is more recommended in this setting. In our sample approximately
80 percent of the observations in the dependent variable take the value of 0. The results are presented in Table 5, and they are similar to those obtained for
the extensive margin of trade. Aristei et al. (2013) also used a Tobit model to analyse
the relationship between both activities, nevertheless the authors find that only past
imports are positively correlated with current exports, but not the other way round. As we can observe in columns (1) and (2) we find a high degree of hysteresis, since
the past percentage of exports and imported intermediates explains the current levels
of each activity, been more important for imports. When both activities are included
in the estimation, sunk costs decrease for both activities, the reduction being more
important for imports than for exports (20 percent versus 15 percent), showing the
existence of common costs in both activities. 20 www.economics-ejournal.org Conclusions 6 According to the recent literature investigating the relationship between productivity
and international trade, firms involved in international activities are larger and more
productive than domestic-only firms. The bulk of the literature has mainly focused
on the export side, disregarding the importance of importing activity. It has been
only recently that more attention has been paid to the import side of
internationalization strategies (Muûls and Pisu 2009; Bas 2012; Aristei et al. 2013;
Kasahara and Lapham 2013; Lo Turco and Maggioni 2013). These studies show the
presence of cost complementarities in both activities, indicating that importing
intermediates is crucial to start exporting and to stay in foreign markets. These cost
complementarities have motivated a new strand of research that further investigates
the relationship between firm’s import and export activities, especially those
focused on the use of imported intermediates and their role in enhancing exports. In
this paper we contribute to this research by providing new evidence for Egypt. More
specifically, using firm-level data for 519 manufacturing companies in Egypt, we
first estimate the two-way traders and importer- and exporter-only premia with
respect to non-traders. Our findings show that firms involved in international
activities have higher productivity, are larger, own more capital and invest more
than domestic-only firms. In particular, two-way traders are the best performers in
terms of all outcome variables, followed by exporter-, importer-only and non-
traders. Secondly, we investigate the relationship between exporting and importing
activities by estimating dynamic panel-Probit models for the extensive margin of
exports (imports) and panel-Tobit models for the intensive margin of exports
(imports). We use the solution proposed by Rabe-Hesketh and Skrondal (2013) that
improve Wooldridge (2005) for initial condition problem in dynamic models. The results indicate that both activities are significantly interrelated and that
sunk costs are higher for import than for export activities, with both activities
showing a high degree of hysteresis. Moreover, past productivity does affect the
extensive and intensive margin of imports, but not the export side and the initial
condition status is also only relevant for the import side. 8 Results including lagged values of exp, t and imp, t where the dependent variable is exp i, t-1 and imp
i,t-1, are available upon request. Acknowledgements: We would like to thank Antonello Maruotti for his comments and useful
suggestions and we acknowledge the financial support obtained from the Universitat Jaume I,
P1.1B2013-06, PREDOC/2009/55 and E-2012-09. Conclusions 21 www.economics-ejournal.org Table 5: Dynamic panel-Tobit model controlling for initial conditions (Exports and
Imports)8
(1)
(2)
(3)
(4)
exp i, t
imp i, t
exp i, t
imp i, t
exp i, t_1
0.493***
0.430***
0.306***
(0.025)
(0.031)
(0.021)
imp i, t-1
0.525***
0.286***
0.436***
(0.026)
(0.020)
(0.032)
ln work i,t-t
0.035***
0.019
0.018
0.002
(0.011)
(0.012)
(0.011)
(0.011)
foreign owner i,t
0.179***
0.080**
0.153***
0.013
(0.039)
(0.039)
(0.038)
(0.039)
ln TFP i, t-1
0.011
0.016**
0.007
0.010
(0.007)
(0.007)
(0.007)
(0.007)
ln TFP mean i, t-1
0.011
0.012
-0.005
0.006
(0.011)
(0.012)
(0.011)
(0.012)
ln work mean i,t-t
0.016
0.012
0.016
0.004
(0.015)
(0.014)
iei
-0.015
0.022
(0.026)
(0.015)
(0.028)
(0.015)
iii
0.018
0.061**
(0.026)
(0.028)
Year dummies
yes
yes
yes
yes
Industry dummies
yes
yes
yes
yes
Observations
1889
1867
1882
1863 Table 5: Dynamic panel-Tobit model controlling for initial conditions (Exports and
Imports)8 Notes: The dependent variables are the natural logs of the percentage of exports over total sales of a
firm and the percentage of material purchases imported. t–1 means lagged values of these variables. Standard errors are in brackets, where *** p<0.01, **p<0.05, * p<0.1. iei denotes initial exporter
dummy. iii means initial importer dummy. Industrial and year dummies included. TFP i,t denotes total
factor productivity, it is obtained using the Levinsohn-Petrin (2003) procedure; TFP i,t -1 are lagged
values of TFP i,t;; work i,t denotes the average number of workers and work i,t-1 are aged value of the
variable; xt.i are a dummy variable that takes value 1 if the firm is exporting and 0 otherwise, xt.i–1 are
the corresponding lagged value and foreignowner i,t is a dummy variable that takes the value of 1 if
the firm is owned by foreigners and 0 otherwise. The number of observations differ between column
(1) and (2) and between columns (3) and (4) due to the inclusion of lagged imports and initial
conditions for which 22 and 19 observations are respectively missing data. 22 www.economics-ejournal.org The main economic policy implication is that governments in developing
countries willing to support the international strategy of the firm should be aware of
the fact that facilitating importing activities is as much important as facilitating
exporting business. As an example, governments, as a way of promoting importing
activities, should support trade negotiations that reduce tariffs in importing inputs
and reduce the number of documents needed to import. www.economics-ejournal.org Conclusions Acknowledgements: We would like to thank Antonello Maruotti for his comments and useful
suggestions and we acknowledge the financial support obtained from the Universitat Jaume I,
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Table A1: Variables description
Variable
Description
Dependent
Variables
xi,t
Dummy variable that take value 1 if firm export in year t
mi,t
Dummy variable that take value 1 if firm import inputs in
year t
expi,t
Percentage of total sales exported in t
pmi,t-1
Percentage of purchases of materials inputs imported
Firm characteristics
work i,t
Average number of workers in t
foreign owneri, t
Percentage of the firm owned by a foreign Arabic owner and
by other foreign owner
capitali,t
Total fixed tangible assets
salesi, t
Total sales in t. Value in thousands of Egyptian pounds. Not
defalted
investmenti, t
Net book value of machinery and equipment
tfpi,t
Levinsohn and Petrin (2003) TFP
Total Factor Productivity
capitaldefi,t
Total fixed tangible assets deflated by the Production price
index for manufactures
materialsde i,t
Total purchases of raw material and intermediate goods
deflated by the Production price index for manufactures
salesdefi, t
Total sales in t. Value in thousands of Egyptian pounds. We
deflate sales using the Production price index for
manufactures using 2005 year as a base years. Table A1: Variables description Total fixed tangible assets deflated by the Production price
index for manufactures Total purchases of raw material and intermediate goods
deflated by the Production price index for manufactures Total sales in t. Value in thousands of Egyptian pounds. We
deflate sales using the Production price index for
manufactures using 2005 year as a base years. 29 www.economics-ejournal.org Appendix A2: Total Factor Productivity (TFP) Estimation To calculate TFP estimates of a traditional Cobb-Douglas production are obtained. The Cobb-Douglas production function is given by: 𝑙𝑛𝑠𝑎𝑙𝑒𝑠i,t = 𝛽0 + 𝛽𝑙 ln (𝑤𝑜𝑟𝑘𝑖,𝑡) + βmln (𝑚𝑎𝑡𝑒𝑟𝑖𝑎𝑙𝑠𝑑𝑒𝑓i,t) +
βk ln (𝑐𝑎𝑝𝑖𝑡𝑎𝑙𝑑𝑒𝑓i,t) + ωi,t + ηi,t (9) (9) where all the variables are in natural logarithms, salesi,t is total sales of firm in year
t, in thousands of Egyptian pounds. As independent variables we include worki,t
defined as the average number of workers, materialsi,t denotes the total purchases
of raw material and intermediates goods, capitali,t denotes the total fixed tangible
assets of the firm and the error term id discomposed into ωi,t, which indicates
productivity socks and an i.i.d. error term given by ηi,t. We deflate firm level sales
and input expenditures using the industry level production price index for
manufactures using 2005 as a base year, the data comes from the International
Financial Statistics (IFS and UN) for manufacturing. When researchers estimate TFP using firm level data they have to deal with
different bias associated with input and output firm level data. The existing literature
proposes a number of avenues to overcome these problems. The first bias is caused
by simultaneity between firm inputs choice and the unobserved productivity level. A second bias emerge when the firm makes its input choice conditional on its
survival, which means that there is a correlation between the unobserved
productivity and the firm’s capital, conditional to being in the dataset. A third bias
appears because industry-level price data are used to deflate output and inputs and
to proxy these variables as quantities. The problem is that under imperfect
competition in input or output markets, inputs and output prices used as deflators,
must be correlated with firm level deviations of inputs or outputs. The literature has
not yet provided a formal solution to deal with this bias. Finally, a fourth bias is
related to the fact that firms can produce different products with different in
production technologies, to deal with this aspect Bernard et al (2009) propose use
the number and type of products of the firm and use them to allow for varying
technology parameters in the production function. An alternative solution is to
estimate different regressions for firms that produce a single product and for multi-
product firms. Appendix A2: Total Factor Productivity (TFP) Estimation Also a measurement problem arises because the true value of the 30 www.economics-ejournal.org capital stock is difficult to mesure, in particular due to the fact that the depreciation
rate and the initial stock of the firm are unknown. Given that the available
methodologies deal with different biases, in what follows we present a number of
alternative estimates of the coefficients of the production function used to obtain
TFP, as proposed by Van Beveren (2012). Table A.2 shows several estimates that
overcome the abovementioned biases. Column 1 in Table A.2 shows the classical OLS estimates that are subject to
endogeneity and selection biases. In Column 2 the model is estimated with firm
fixed effects, controlling for time-invariant unobserved heterogeneity which is firm-
specific. The third alternative, (Column 3), was proposed by Levinsohn and Petrin
(2003), who proposed to estimate the production function using inputs as control. Finally, Column 4 shows the coefficients estimated by using Olley and Pakes (1996)
method. Olley and Pakes (1996) propose a three steep procedure. In the first steep
the unobserved productivity is obtained for each firm using their level of investment,
in the second step we obtain the survival probability of the firm and the last steep
employs the outcomes of the previous two steps to control for simultaneity and
selection biases. Consistent and unbiased estimates of the production function are
used to obtain unbiased estimates of TFP, which is computed as the residual of the
estimated production function. We decide to use TFP estimated using the Levinsohn
and Petrin (2003) as independent variable in the export and import models because
this methodology control for two important biases, namely, simultaneity and self-
selection and our data availability do not allow use obtain accurate values of the
firm investments. Table A.2. Product function estimates
OLS
FE
LP reg
OP reg
lncapitaldefi.t
0.085***
0.081***
0.055***
0.081***
(0.011 )
(0.012)
0.010
(0.029)
lnwork I,t
0.350***
0.372***
0.363***
0.606***
(0.020)
(0.025)
0.019
(0.042)
lnmaterialsdefi,t
0.603***
0.611***
0.608***
0.315***
(0.012)
(0.014)
0.012
(0.043)
Number of observations
2429
2429
2429
2480
Note: where OLS denotes Ordinary Least Squares, FE denotes OLS fixed effects, LP denotes,
Levinsohn and Petrin, and OP denotes Olley and Pakes. *** p<0.01, ** p<0.05, * p<0.1. Robust standard errors in parentheses. Table A.2. Product function estimates Table A.2. Note: where OLS denotes Ordinary Least Squares, FE denotes OLS fixed effects, LP denotes,
Levinsohn and Petrin, and OP denotes Olley and Pakes. *** p<0.01, ** p<0.05, * p<0.1.
Robust standard errors in parentheses. © Author(s) 2015. Licensed under the Creative Commons Attribution 3.0. Appendix A2: Total Factor Productivity (TFP) Estimation Product function estimates Note: where OLS denotes Ordinary Least Squares, FE denotes OLS fixed effects, LP denotes,
Levinsohn and Petrin, and OP denotes Olley and Pakes. *** p<0.01, ** p<0.05, * p<0.1. Robust standard errors in parentheses. 31 31 www.economics-ejournal.org Please note: You are most sincerely encouraged to participate in the open assessment of this article. You
can do so by either recommending the article or by posting your comments. Please go to: http://dx.doi.org/10.5018/economics-ejournal.ja.2015-38 The Editor The Editor The Editor
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To cite this version: Jérôme Darmont, Cécile Favre, Sabine Loudcher, Camille Noûs. Data Lakes for Digital Humanities. 2nd International Digital Tools & Uses Congress (DTUC 2020), Oct 2020, Hammamet, Tunisia. pp.38-
41, 10.1145/3423603.3424004. hal-02889487 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-02889487
https://hal.science/hal-02889487v1
Submitted on 3 Dec 2020 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés. HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License ABSTRACT Moreover, most source information exploited by humanities
and social sciences comes in textual format. Again, such textual
documents are difficult to manage without substantial transforma-
tions: digitization, encoding and tagging, e.g., via the Text Encoding
Initiative (TEI), and even lowercasing, stemming, lemmatization,
stopword removal or normalization when it comes to text mining
and natural language processing.f Traditional data in Digital Humanities projects bear various for-
mats (structured, semi-structured, textual) and need substantial
transformations (encoding and tagging, stemming, lemmatization,
etc.) to be managed and analyzed. To fully master this process, we
propose the use of data lakes as a solution to data siloing and big
data variety problems. We describe data lake projects we currently
run in close collaboration with researchers in humanities and social
sciences and discuss the lessons learned running these projects. Another important methodological issue is the black box effect
that occurs when resorting to computer scientists only “as a ser-
vice”. How can DH researchers work without mastering the whole
process? Furthermore, designing and managing such processes also
lead to research issues for computer scientists. CCS CONCEPTS • Information systems →Data management systems; Data-
base administration; Information integration; • Applied com-
puting; • Digital Humanities; To leverage the above-mentioned issues, we propose the use of
data lakes, a concept introduced by Dixon in 2010 as a solution to
data siloing and big data variety problems [2]. Even if data exploited
by DH are not always big data in terms of volume, they can bear
considerable variety, i.e., including structured and semi-structured
data, as well as unstructured data such as texts, various types of
images, sounds and videos. Traditional data management tends to
manage such heterogeneity with different systems, thus separating
data into so-called silos. Permission to make digital or hard copies of all or part of this work for personal or
classroom use is granted without fee provided that copies are not made or distributed
for profit or commercial advantage and that copies bear this notice and the full citation
on the first page. Copyrights for components of this work owned by others than the
author(s) must be honored. Abstracting with credit is permitted. To copy otherwise, or
republish, to post on servers or to redistribute to lists, requires prior specific permission
and/or a fee. Request permissions from permissions@acm.org.
ddh20, October 15–17, 2020, Hammamet, Tunisia
© 2020 Copyright held by the owner/author(s). Publication rights licensed to the
Association for Computing Machinery. KEYWORDS Data Lakes, Digital Humanities, Metadata ACM Reference Format:
Jérôme Darmont, Cécile Favre, Sabine Loudcher, and Camille Noûs. 2020. Data Lakes for Digital Humanities. In ddh20: Data and Digital Humanities
2020, October 15–17, 2020, Hammamet, Tunisia. ACM, New York, NY, USA,
4 pages. https://doi.org/ A data lake is a scalable storage and analysis system for data of
any type, retained in their native format and used mainly (but not
only) by data specialists (statisticians, data scientists or analysts)
for knowledge extraction [10]. Data Lakes for Digital Humanities Camille Noûs
camille.nous@cogitamus.fr
Université de Lyon, Lyon 2, Laboratoire Cogitamus
Bron, France Camille Noûs
camille.nous@cogitamus.fr
Université de Lyon, Lyon 2, Laboratoire Cogitamus
Bron, France Jérôme Darmont
Cécile Favre
Sabine Loudcher
jerome.darmont@univ-lyon2.fr
cecile.favre@univ-lyon2.fr
sabine.loudcher@univ-lyon2.fr
Université de Lyon, Lyon 2, ERIC UR 3083
Bron, France © 2020 Copyright held by the owner/author(s). Publication rights licensed to the
Association for Computing Machinery. 2.1
HyperThesau The “Hyper thesaurus and data lakes: Mine the city and its archaeo-
logical archives” (HyperThesau) project involves a multidisciplinary
team consisting of two research laboratories of archaeology and of
computer science, a digital library, two archaeological museums
and a private company. This project has two main objectives: (3) (3) The data storage layer is core to a data lake. It must have the
capacity to store all data in any format. (3) The data storage layer is core to a data lake. It must have the
capacity to store all data in any format. (4) The data distillation layer provides a set of tools for data
cleaning (eliminating errors such as duplicates and type
violations) and encoding formalization (converting various
data and character encoding). (1) the design and implementation of an integrated platform to
host, search, share and analyze archaeological data;i (5) The data insights layer provides a set of tools for data trans-
formation (e.g., into models) and exploratory data analysis
(e.g., pattern discovery). Note that transformed data may
also be stored into the lake for later reuse. (2) the design of a domain-specific thesaurus taking the whole
archaeological data lifecycle into account, from data creation
to publication.f Archaeological data may bear many different types, e.g., tex-
tual documents, images (photographs, drawings...), sensor data, etc. Moreover, similar documents, e.g., excavation reports, are often
created by various software tools that are not compatible with each
other. The description of an archaeological object also differs with
respect to users, usages and time. Such a variety of archaeological
data induces many scientific challenges related to storing heteroge-
neous data in a centralized repository, guaranteeing data quality,
cleaning and transforming the data to make them interoperable,
finding and accessing data efficiently and cross-analyzing the data
with respect to their spatial and temporal dimensions. (6) The data application layer provides applications that allow
users extracting value from data, e.g., through an interactive
query system, reports or dataviz.l (7) The workflow manager layer provides tools to automate the
flow of data processes. (8) The communication layer provides tools that allow the other
layers to communicate with each other. It must provide syn-
chronous and asynchronous communication capability. (9) The data governance layer provides a set of tools to establish
and execute plans and programs for data quality control [4]. 1
INTRODUCTION One of the main advantages of data lakes is that data are stored
in their initial form, and are thus recognizable by their producers,
such as DH researchers. A data lake does not propose a new data
model nor new data formats for data archiving. Moreover, when
data are transformed for processing, the data lineage is stored as
metadata, thus enforcing traceability.fi Traditional data management has long been adopted by many re-
searchers involved in Digital Humanities (DH). However, it requires
a substantial investment in data modeling, including, at the physical
level, technologies such as relational and semi-structured Database
Management Systems (DBMSs), various data formats, e.g., XML
and JSON for semi-structured data, RDF for linked data, and query
languages such as SQL and XQuery. This investment in computer
science and the fact that initial data are inevitably transformed are
presumably impediments to the adoption of DBMSs and related
digital tools for DH. However, a drawback is that unprepared data are difficult to
process and require data specialists who can program. Yet, we
strongly advocate, with other researchers, for the “industrialization”
of data lakes, i.e., providing a software layer that allows non-data
scientists such as DH researchers to transform and analyze their
own data in autonomy, just as dynamic reports are prepared on top
of data warehouses for the use of business (i.e, non technical) users. The remainder of this paper is organized as follows. In Section 2,
we describe data lake projects we currently run in close collabora-
tion with researchers in social sciences and humanities. In Section 3,
we conclude this paper by discussing the lessons learned running
these projects. Darmont & al. ddh20, October 15–17, 2020, Hammamet, Tunisia Darmont & al. Darmont & al. 1https://atlas.apache.org
2hhttps://hadoop.apache.org/docs/r1.2.1/hdfs_user_guide.html
3https://hive.apache.org
4https://www.openldap.org
5http://spark.apache.org
6https://lucene.apache.org/solr/ 2
EXAMPLE DH PROJECTS INVOLVING DATA
LAKES choose the right tools and plans to ingest data into the data
lake with respect to data source properties and the lake’s ca-
pacity. During ingestion, metadata provided by data sources,
e.g., name of excavation sites or instruments, must be gath-
ered as much as possible. 2.1
HyperThesau Each of the above layers is implemented with one or more frame-
works of the Apache Hadoop ecosystem, e.g., Atlas1, HDFS2, HIVE3,
OpenLdap4, Spark5, etc. This prototype is operational and currently
hosts the data of two archaeological research facilities. The meta-
data management system instantiates the MEtadata model for Data
Lakes (MEDAL), which adopts a graph model [10]. It is implemented
with Apache Atlas, which can host not only descriptive metadata,
but also several thesauruses. With the help of a search engine, i.e.,
Solr6, users can find data through descriptive metadata, a thesaurus
or the data lineage. To overcome all these challenges, we implement a data lake. Our
approach aims to collect all types of archaeological data, save them
inside the data lake and propose metadata for better organizing
data and for allowing users to easily find data for analysis purposes. Our data lake prototype is architectured in nine layers (Figure 1)
[5, 6]. 2.2
Bretez/STRATEGE Bretez [7] is a multidisciplinary project aiming at a visual and
sonorous restitution of the XVIIIth-century Paris. It is also an ex-
ploratory project constituted of successive, interlinked modules
that are (and must be) interoperable and open. The historical urban
restitution is achieved with video game engines that bear their
own respective characteristics, of course related to gaming. Yet,
here, they are used for specific management and traceability needs. Moreover, Bretez’ documentation is a voluminous corpus of hetero-
geneous and multimedia data. Within project Bretez, the “Traceability and information manage-
ment system for multimedia data” (STRATEGE) aims at designing,
storing, querying and analyzing all the project’s data. To master
data heterogeneity, manage data quality and volume, warrant data
interoperability and an efficient access while keeping data in their Figure 1: HyperThesau data lake’s layered architecture [5] Figure 1: HyperThesau data lake’s layered architecture [5] (1) The data source layer gathers the basic properties of data
sources, e.g., volume, format, velocity, connectivity, etc. Based
on these properties, data engineers can determine how to
import data into the lake. p
(2) The data ingestion layer provides a set of tools for perform-
ing batch or real-time data integration. Data engineers can Data Lakes for Digital Humanities ddh20, October 15–17, 2020, Hammamet, Tunisia Figure 2: CODAL example screenshot [9] Figure 2: CODAL example screenshot [9] original form so that they remain usable reference for the project’s
researchers, we resort to a data lake.i they are quite similar in terms of data: a corpus of various textual
documents (e.g., annual reports from companies and organizations,
interviews of senior or top executives, press articles; all in French
or English) and data from various sources, including the Web, cu-
rated and inferred by researchers in management sciences from
company legal information and performance indicators such as
workforce, annual revenue, stock-exchange price and perceived
level of digitization and servicization. STRATEGE is in its first stages: we catalogued all existing data,
which included a database, textual documents, sounds, images and
a 3D Unity7 (a game engine) model, and more. The database is
particularly interesting, for it contains both data and metadata. While retaining it, we also restructured it so as to allow its metadata
to interoperate with specific data lake metadata. In short, there are
“business” metadata and technical metadata. With such data handy, the objective is to cross-analyze the terms
and expressions found in textual resources with structured, qual-
itative and quantitative data, in order to discover new insights
regarding how companies communicate vs. their actual customer
relationship management strategy (for COREL) and how digitiza-
tion and servicization impact economic performance (for AURA
PMI). The challenges here are to: The remaining tasks include fully designing and integrating the
metadata system, on the basis of MEDAL [10]; make the data from
the Unity model accessible into the lake; formalize analysis needs;
and design tools that must jointly handle textual, visual and audio
content, as well as the heterogeneity of data sources. Such software
tools must be accessible to all researchers involved in the Bretez
project. (1) leverage metadata that allow querying the whole corpus; 7https://unity.com
8http://coactis.org 9https://www.elastic.co/elasticsearch/
10http://basex.org
11https://neo4j.com ACKNOWLEDGEMENTS (2) proximity analyses such as similarity (what documents are
similar or different [8]) and centrality (to identify the docu-
ments bearing a specific or common vocabulary, hinting at
its importance [3]) analyses; Projects HyperThesau and Bretez/STRATEGE are funded by the
Laboratory of Excellence “Intelligence of Urban Worlds” (IMU)12. Project COREL was funded by the University of Lyon 2. Project Projects HyperThesau and Bretez/STRATEGE are funded by the
Laboratory of Excellence “Intelligence of Urban Worlds” (IMU)12. Project COREL was funded by the University of Lyon 2. Project Projects HyperThesau and Bretez/STRATEGE are funded by the
Laboratory of Excellence “Intelligence of Urban Worlds” (IMU)12. Project COREL was funded by the University of Lyon 2. Project AURA PMI is funded by the Auvergne-Rhône-Alpes Region. (3) custom highlights of the context of terms and, optionally,
their synonyms, in textual documents. 2.3
COREL and AURA PMI (2) jointly analyze structured and unstructured data; (3) allow management science researchers to perform analyses
by themselves. Both the projects “At the heart of customer relationship” (COREL)
and “Digital transformation, servicization and mutations of indus-
trial SME business models” (AURA PMI) relate to management
sciences and are carried out in collaboration with the Coactis lab-
oratory8. Although their respective focus and scope are different, To complete these tasks for the COREL project, we designed a
metadata system that prefigured MEDAL [10] and proposed the
lightweight COREL Data Lake architecture (CODAL) [9], which is
composed of: ddh20, October 15–17, 2020, Hammamet, Tunisia Darmont & al. Darmont & al. (1) a storage layer that notably includes Elasticsearch9 for in-
dexing textual contents; (1) a storage layer that notably includes Elasticsearch9 for in-
dexing textual contents; In consequence, we take great care of accompanying DH users in
their appropriation of our analysis tools, not only by training, but
especially by interweaving research methodologies from computer
science and other disciplines by design, in close collaboration with
partner researchers. (2) a metadata layer leveraging and extending the Metadata
Encoding & Transmission Standard (METS) [11], stored in
the BaseX XML DBMS10; (3) an analysis layer, i.e., an intuitive Web-based graphical inter-
face that allows management science researchers to perform
analyses in autonomy, thus enforcing the “industrialization”
of CODAL. Moreover, the possibility of having both access to the raw data
and the entire possible processing chain is necessary, because black
boxes are seldom compatible with a sound methodological approach
aiming at producing scientific knowledge. Data lakes precisely allow
this much needed transparency. The analysis layer features three kinds of analyses: The analysis layer features three kinds of analyses: (1) data exploration akin to On-Line Analytical Processing (OLAP)[1 All three types of analyses come with various dataviz (Figure 2). All three types of analyses come with various dataviz (Figure 2). [1] E.F Codd, S.B Codd, and C.T Salley. 1993. Providing OLAP (On-line Analytical
Processing) to User-Analysts: An IT Mandate. Technical Report. E.F. Codd &
Associates. [1] E.F Codd, S.B Codd, and C.T Salley. 1993. Providing OLAP (On-line Analytical
Processing) to User-Analysts: An IT Mandate. Technical Report. E.F. Codd &
Associates. The AURA PMI Data Lake (AUDAL) is currently being devel-
oped, and builds upon CODAL. Its metadata system will notably
be a substantial evolution of MEDAL supported by the Neo4J11
graph DBMS. Moreover, the AUDAL analysis layer, which lays on
an Application Programming Interface (API), will be much more
elaborate and efficient than CODAL’s. [2] James Dixon. 2010. Pentaho, Hadoop, and Data Lakes. (October 2010). https:
//jamesdixon.wordpress.com/2010/10/14/pentaho-hadoop-and-data-lakes/ [2] James Dixon. 2010. Pentaho, Hadoop, and Data Lakes. (October 2010). https:
//jamesdixon.wordpress.com/2010/10/14/pentaho-hadoop-and-data-lakes/ [3] Ashley Farrugia, Rob Claxton, and Simon Thompson. 2016. Towards Social
Network Analytics for Understanding and Managing Enterprise Data Lakes. In
Advances in Social Networks Analysis and Mining (ASONAM 2016), San Francisco,
CA, USA (IEEE). 1213–1220. (
)
[4] Vijay Khatri and Carol Brown. 2010. Designing data governance. Commununica-
tions of the ACM 53 (January 2010), 148–152. [4] Vijay Khatri and Carol Brown. 2010. Designing data governance. Commununica-
tions of the ACM 53 (January 2010), 148–152. 12https://imu.universite-lyon.fr 3
CONCLUSION [5] Pengfei Liu, Sabine Loudcher, Jérôme Darmont, and Camille Noûs. 2020. A data
lake architecture and implementation for archaeological data management and
analytics. Technical Report. Université de Lyon, Lyon 2, ERIC UR 3083, France. In all four data lake projects summarized in Section 2, we use differ-
ent versions of the MEDAL metadata system, which is designed to
be generic. However, although MEDAL is quite flexible, we do not
believe in a single model for data lakes. There are indeed significant
differences in data in only four projects, in terms of volume, variety
and velocity, which imply different architectures and technologies. Thus, we think that much needed methodological tools for data
lakes should be instantiated for each project rather than applied
“as is”. [6] Pengfei Liu, Sabine Loudcher, Jérôme Darmont, Emmanuelle Perrin, Jean-Pierre
Girard, and Marie-Odile Rousset. 2020. Metadata model for an archeological
data lake. Digital Humanities (DH 2020), Ottawa, Canada. (July 2020). https:
//dh2020.adho.orgfi g
[7] Mylène Pardoën. Last accessed: June 2020. Bretez site officiel. (Last accessed:
June 2020). https://sites.google.com/site/louisbretez/home [8] Pascal Pons and Matthieu Latapy. 2006. Computing Communities in Large
Networks Using Random Walks. Journal of Graph Algorithms and Applications
10, 2 (2006), 191–218. [9] Pegdwendé-Nicolas Sawadogo, Tokio Kibata, and Jérôme Darmont. 2019. Meta-
data Management for Textual Documents in Data Lakes. In 21st International
Conference on Enterprise Information Systems (ICEIS 2019), Heraklion, Crete-Greece. INSTICC, 72–83. Vol. 1. Furthermore, the software layer we add to “industrialize” our data
lakes might become yet another black box, while there is a strong
stake for researchers in humanities and social sciences involved
in DH projects not to be dispossessed of data by an analysis layer
that would adopt a “click and go” approach. Data are indeed often
partly constructed by said researchers themselves as a product of
scientific work that takes time, thus giving a significant value to
datasets. [10] Pegdwendé-Nicolas Sawadogo, Etienne Scholly, Cecile Favre, Eric Ferey, Sabine
Loudcher, and Jérôme Darmont. 2019. Metadata Systems for Data Lakes: Models
and Features. In 1st International Workshop on BI and Big Data Applications (BBI-
GAP@ADBIS 2019), Bled, Slovenia (Communications in Computer and Information
Science), Vol. 1064. Springer, Heidelberg, Germany, 440–451. p
g
g
y
[11] The Library of Congress. 2017. METS: An Overview and Tutorial. (March 2017). http://www.loc.gov/standards/mets/METSOverview.v2.html
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Primary sclerosing epithelioid fibrosarcoma of kidney with variant histomorphologic features: report of 2 cases and review of the literature
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* Correspondence: dertoy@hacettepe.edu.tr
1Department of Pathology, Hacettepe University Hospital, Ankara, Turkey
Full list of author information is available at the end of the article Ertoy Baydar et al. Diagnostic Pathology (2015) 10:186
DOI 10.1186/s13000-015-0420-z © 2015 Ertoy Baydar et al. 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. Background supported by MUC4 immunostaining and fluorescence
in situ hybridization (FISH). To our knowledge, these
are the sixth and seventh genetically confirmed cases
of SEF reported in the kidney following very recently
described primary renal SEF(s) by Arbajian et al., Argani
et al. and Ohlmann, et al. [1, 8, 9]. Some of their morpho-
logic features are unique and deserve to be noted for full
characterization of this entity in a new environment. Sclerosing epithelioid fibrosarcoma (SEF) is a rare malig-
nant mesenchymal tumor of soft tissues composed of
cords, nests or sheets of relatively monotonous epithelioid
cells within a collagenous background. It has recently been
characterized by recurrent FUS-CREB3L1, FUS-CREB3L2
or EWS1-CREB3L1 translocations and immunohistochemi-
cal MUC4 expression [1–3]. The tumor occurs over a wide
age spectrum at initial presentation without sex predilec-
tion. Most reported cases are in the soft tissue of extrem-
ities and limb girdles [4–7]. Primary SEF in visceral organs
is exceedingly rare, with only a few cases reported. Abstract The authors present two cases of primary sclerosing epithelioid fibrosarcoma (SEF) of the kidney. Both patients had
a mass in the upper part of the left kidney without any primary extrarenal neoplastic lesions. Grossly, the tumors
were solid masses both measuring 7.5 cm in the greatest diameter. Histologically, one of the lesions exhibited a
predominantly lobular growth of round or oval small uniform epithelioid cells in variable cellularity. Circular zones
of crowded tumor cells alternating with hypocellular collagenous tissue in a concentric fashion around entrapped
native renal tubules were distinctive. The second case was distinctive with significant cytological atypia in the
neoplastic cells and prominent reactive proliferations in the trapped renal tubules. Immunohistochemically,
vimentin, bcl-2 and MUC4 were diffusely positive in both. They were negative for S-100 protein, CD34, and desmin,
whereas CD99 were positive in one lesion. Fluorescence in situ hybridization assay using dual staining probes
detected EWSR1-CREB3L1 fusion in each lesion, which is characteristic molecular findings of SEF. One patient
presented widespread distant metastases at the time of diagnosis. In the other, no tumor deposits were detected
other than primary. Both patients have been alive with 30 and 10 month follow-ups, respectively. These tumors are
6th and 7th cases of primary renal SEF in the literature confirmed by FISH study, which exhibit unique and
remarkable histomorphologic features. Keywords: Sclerosing epithelioid fibrosarcoma, Kidney, EWSR1, CREB3L1, Translocation Keywords: Sclerosing epithelioid fibrosarcoma, Kidney, EWSR1, CREB3L1, Translocation Case presentation Two cases of SEF that were found to harbor EWSR1-
CREB3L1 fusion were encountered in the diagnostic
practice of the authors. The clinical records were retrieved
for analysis, and all available pathologic materials were
reviewed. Here, we present two cases of SEF with primary
renal origin bearing a EWS1-CREB3L1 gene fusion, as Ertoy Baydar et al. Diagnostic Pathology (2015) 10:186 Page 2 of 12 Table 1 Features of the antibodies used for the
immunohistochemical stains
Antigen
Clone, dilution, source
Pan-Cytokeratin
AE1/AE3, 1:200, Leica, Newcastle/UK
Epithelial Membrane Antigen
E29, 1:300, Biocare, Concord/CA
PAX8
PAX8, 1:100, Biocare, Concord/CA
WT1
6 F-H2, 1:40, DBS, Pleasenton/CA
ER
EP1, 1:100, Genemed, San Francisco/CA
PR
SP2, 1:1000, Thermo, Fremont/CA
CD34
QBEND/10, 1:100, DBS, Pleasenton/CA
SMA
1A4, 1:1000, NeoMarkers, Fremont/CA
Desmin
D33, 1:50, Biocare, Concord/CA
S100 protein
Z0311, 1:6000, DAKO, Glostrup/Denmark
GFAP
GA-5, 1:50, Thermo, Fremont/CA
Bcl2
Bcl-2-100, 1:80, Invitrogen, Paisley/UK
CD99
HO36-1.1, 1:100, Thermo, Fremont/CA
MUC4
1G8, 1:50, Invitrogen, Paisley/UK
HMB45
HMB45, 1:25, DBS, Pleasenton/CA
Melan-A
A103, 1:100, Thermo, Fremont/CA
INI1
25, 1:50, Zeta, Arcadia/CA Fig. 2 Case #1. Solid white tumor filling out the upper half of the kidney Fig. 2 Case #1. Solid white tumor filling out the upper half of the kidney Fluorescence In Situ Hybridization (FISH) Fluorescence In Situ Hybridization (FISH)
FISH analysis was performed on representative 4–5 μm
thick unstained formalin-fixed, paraffin-embedded tissue
sections of the tumor samples of each case. For characte
rization of the possible underlying gene rearrangement
or fusion gene events, the following FISH probe sets
were utilized on both cases: Vysis LSI EWSR1 (22q12) and
Vysis LSI FUS (16p11) Dual Color Break Apart Probes Immunohistochemistry For immunohistochemical labeling, a polymer detection
system (Leica, DS9800) and the BOND-MAX automated
immunostainer was used. The standard antibodies con-
sumed, vendors, and dilutions were summarized in Table 1. Fig. 3 Case #1. Alternating hypercellular and a hypocellular areas of
neoplastic cells with monomorphic ovoid nuclei and indistinct pale
to clear cytoplasm (H&E x 200; inset: H&E x 400) Fig. 3 Case #1. Alternating hypercellular and a hypocellular areas of
neoplastic cells with monomorphic ovoid nuclei and indistinct pale
to clear cytoplasm (H&E x 200; inset: H&E x 400)
Fig. 1 Case #1. Computerized tomography scan showing a large tumor
in the left kidney Fig. 3 Case #1. Alternating hypercellular and a hypocellular areas of
neoplastic cells with monomorphic ovoid nuclei and indistinct pale
to clear cytoplasm (H&E x 200; inset: H&E x 400) Fig. 1 Case #1. Computerized tomography scan showing a large tumor
in the left kidney Ertoy Baydar et al. Diagnostic Pathology (2015) 10:186 Ertoy Baydar et al. Diagnostic Pathology (2015) 10:186 Page 3 of 12 Fig. 4 Case #1. a Lobular/micronodular pattern (H&E x 40). b Concentric hypocellular inner, hypercellular outer zone around entrapped renal
tubules (H&E x 100) Fig. 4 Case #1. a Lobular/micronodular pattern (H&E x 40). b Concentric hypocellular inner, hypercellular outer zone around entrapped renal
b l
(H&E
100) Fig. 4 Case #1. a Lobular/micronodular pattern (H&E x 40). b Concentric hypocellular inner, hypercellular outer zone around entrapped renal
tubules (H&E x 100) (Abbott Molecular, Inc., Des Plaines, IL) and EWSR1 and
CREB3L1 spanning probe sets using cocktails of BAC
clones (RP11-945 M21 and RP11-1126O13, and RP11-
1014A16, RP11-1106 J11 and RP11-481I24 respectively)
selected on the basis of their location per the UCSC
Human Genome Browser [http://genome.ucsc.edu/cgi-
bin/hgGateway]
and
obtained
from
BAC/PAC
Re-
sources Center (Children’s Hospital Oakland Research
Institute, Oakland, CA, USA). Subsequently, the slides were incubated for 15 min at 37 °C
in protease solution [25 mg of protease in 50 ml of protease
solution (Abbott Molecular, Inc., Des Plaines, IL), washed
in 1 × PBS at room temperature for 5 min and then dehy-
drated in gradient ethanol (75, 85, and 100 %) at room
temperature for 1 min each and air-dried. Immunohistochemistry After the cells
and probes were co-denatured at 80 °C for 10 min and
incubated overnight at 37 °C using the HYBrite™system
(Abbott Molecular, Inc., Des Plaines, IL), post-hybridization
washing was performed in 2 × SSC/0.1 % NP-40 at 72 °C
for 2 min, followed by 2 × SSC/0.1 % NP-40 at room
temperature for 1 min. The slides were then counter-
stained with DAPI II (Abbott Molecular, Inc., Des Plaines,
IL). To confirm correct mapping, optimal signal strength,
and lack of cross-hybridization, each probe set was also
hybridized to metaphase cell preparations of karyotypically
normal peripheral blood lymphocytes before proceeding
with analysis of the patient samples. Hybridization studies using the Vysis LSI Dual Color
Break Apart Probes for the assessment of rearrangement of
the FUS and EWSR1 loci were performed following the
manufacturer’s instructions (Abbott Molecular, Inc., Des
Plaines, IL). With respect to the custom spanning probes,
each BAC clone was directly labeled by nick translation
with either Spectrum Green- or Spectrum Orange-dUTP
per the manufacturer’s protocol (Abbott Molecular, Inc.,
Des Plaines, IL). An amount of 3 ug of DNA for each
probe or 1.5 ug for each of two probes were combined. All nick translation reagents were then multiplied by
the total ug of DNA used in the cocktail. Amounts of
200 ng of each probe were hybridized to the target
DNA and blocked with approximately 15 fold excess
of a combination of Human Cot-I DNA (Invitrogen,
Carlsbad, CA) and human placental DNA. The cutoff level for scoring a specimen as positive for a
rearrangement of the FUS or EWSR1 locus or as positive
for an EWSR1/CREB3L1 fusion was >15 % of the cells eval-
uated. Images were prepared using the Cytovision Image
Analysis System (Applied Imaging, Santa Clara, CA). For
each probe set, 100–200 interphase nuclei with strong and
well-delineated signals were examined. Prior to hybridization, the slides were pretreated at
room temperature in 0.2 N HCl for 20 min, washed in
water for 3 min, incubated at 80 °C for 25 min in VP
2000 Pretreatment Reagent (Abbott Molecular, Inc., Des
Plaines, IL) and then washed again in water for 3 min. Case 1 The patient was a 16-year-old girl who presented at
the urology clinics with pain on the left side of the abdo-
men radiating to the back. Her past medical history was Page 4 of 12 Ertoy Baydar et al. Diagnostic Pathology (2015) 10:186 Fig. 5 Case #1. Strong bcl-2 and weak EMA expression by neoplastic cells (Left: Immunohistochemistry, anti-bcl-2 Ab x 200; Right: Immunohistochemistry,
anti-EMA Ab x 200) bcl-2 and weak EMA expression by neoplastic cells (Left: Immunohistochemistry, anti-bcl-2 Ab x 200; Right: Immunohistochemistry, Fig. 5 Case #1. Strong bcl-2 and weak EMA expression by neoplastic cells (Left: Immunohistochemistry, anti-bcl-2 Ab x 200; Right: Immunohistochemistry,
anti-EMA Ab x 200) noted in many regions where a renal tubule in the center
was surrounded by concentric inner hypo and outer
hypercellular zones of neoplastic cells (Fig. 4a and b). These lobules were separated from each other by myo-
fibroblasts. Tubules entrapped in the tumor were lined
by single layered Pax-8 positive cuboidal cells without
atypia, some showed shallow papillary hyperplasia and
rare mitosis. Mitotic rate was 1/10 hpf in the neoplasm
and there were occasional areas of necrosis in the tumor. Hypercellular areas occasionally contained vague nodules
of collagen mimicking those seen in hyalinizing spindle cell
tumor with giant rosettes (HSCTGR). The surrounding
kidney showed no specific pathologic changes. By im-
munohistochemistry, neoplastic cells were immunore-
active diffusely and strongly for vimentin, bcl-2 and CD99;
EMA labelled them in a weak and patchy fashion (Fig. 5). They were negative for pan-cytokeratin, Pax-8, WT-1,
CD34, S-100, GFAP, Melan-A, HMB-45, desmin, and
estrogen and progesterone receptors. Smooth muscle
actin (SMA) stained myofibroblastic cells in-between the
neoplastic lobules (Fig. 6). INI-1 was preserved. Then, an insignificant. Computerized axial tomography scans of the
chest, abdomen, and pelvis revealed a 70 × 70 × 60 mm left
renal mass (Fig. 1), bilateral pulmonary nodules (the
largest being 16 mm in diameter), and widespread bone
metastases in vertebrae, sacrum and left femoral head. A
biopsy from the tumor in the kidney through laparotomy
was performed followed by left radical nephrectomy after
the diagnosis of malignancy. Tumor occupied the entire upper half of the kidney,
extended into the renal sinus, was a solid white unencapsu-
lated lesion with sharp borders, measuring 7.5 × 7 × 7 cm in
size (Fig. 2). Case 1 Microscopical sections showed diffuse infiltra-
tion of the neoplastic tissue in kidney parenchyma separat-
ing normal renal elements from each other. The neoplastic
cells were small, monotonous and epithelioid with clear to
pale eosinophilic cytoplasm, and were arranged in single
files, cords, nests or irregular aggregates in collagenous
matrix (Fig. 3). Nuclei were generally round to oval, with
indistinct nucleoli. Hypercellular areas alternated randomly
with hypocellular densely hyalinized or at times myxoid
stroma. Additionally, a peculiar lobular organization was Page 5 of 12 Ertoy Baydar et al. Diagnostic Pathology (2015) 10:186 Page 5 of 12 Fig. 6 Case #1. SMA stains myofibroblasts between neoplastic lobules and MUC4 stains neoplastic cells (Left: Immunohistochemistry, anti-SMA Ab x 100;
Right: Immunohistochemistry, anti-MUC4 Ab x 400) Fig. 6 Case #1. SMA stains myofibroblasts between neoplastic lobules and MUC4 stains neoplastic cells (Left: Immunohistochemistry, anti-SMA Ab x 100;
Right: Immunohistochemistry, anti-MUC4 Ab x 400) immunohistochemical stain for MUC4 was performed
which showed strong positivity throughout the tumor
(Fig. 6). Finally, FISH analysis with the EWSR1 Break
Apart probe revealed loss of one copy of the Spectrum
Green labeled probe flanking the 3’ (telomeric) side of the
EWSR1 gene as well as the presence of a single fused
EWSR1/CREB3L1 signal (represented by a juxtaposed or-
ange signal and green signal) consistent with the presence
of an unbalanced der(22)t(11;22)(p11;q12) (Fig. 7), con-
firming the diagnosis of sclerosing epithelioid fibrosar-
coma. FISH study was negative for a rearrangement of the
FUS gene locus. 6 cm in the largest diameter. Her past medical history was
unremarkable except for hypertension, congestive heart fail-
ure and arrhythmia. Serum and urine tests were within the
normal limits. The patient subsequently underwent open
left partial nephrectomy. On gross examination, the specimen had an unencap-
sulated, solid - white firm tumor with irregular borders,
measuring 7.5 × 5.5 × 4 cm (Fig. 8). Histologic examin-
ation revealed that tumor contained abundant entrapped
native renal tubules throughout as in the first case, mim-
icking a biphasic neoplasm (Fig. 9). These tubules were
hyperplastic and proliferating, were of various size and
shapes, some being cystic or leaf-like, and lined by single
layered cuboidal or occasionally flattened cells, all express-
ing nuclear Pax-8. Intraluminal papillary projections were
common, but without cytologic atypia. Neoplastic compo-
nent had a variable cellularity (Fig. 10). Hypocellular re-
gions contained abundant hyalinized collagen and some
showed myxoid change. Case 1 Hypercellular areas were divided
into anastomosing compartments by sclerotic collagen
bands. Neoplastic cells were polygonal epithelioid or plump Patient was given chemotherapy with multiple agents. She is alive with disease after 30 month follow-up. Case 2 The patient was a 57-year-old woman who was investigated
for cholelithiasis due to dyspeptic complaints. During ab-
dominal ultrasonography, she was found to have a left renal
mass incidentally. The CT scan showed that the tumor was
located at the upper pole of the kidney and was measured Ertoy Baydar et al. Diagnostic Pathology (2015) 10:186 Page 6 of 12 Fig. 7 Case #1. An EWSR1 Break Apart probe set and a custom probe set spanning the EWSR1 (Spectrum Orange) and the CREB3L1 (Spectrum Green)
loci demonstrating loss of the Spectrum Green labeled probe signal that flanks the 3’ (telomeric) side of the EWSR1 locus (short arrows, left panel) and
a single fusion signal (long arrows, right panel) Fig. 7 Case #1. An EWSR1 Break Apart probe set and a custom probe set spanning the EWSR1 (Spectrum Orange) and the CREB3L1 (Spectrum Green)
loci demonstrating loss of the Spectrum Green labeled probe signal that flanks the 3’ (telomeric) side of the EWSR1 locus (short arrows, left panel) and
a single fusion signal (long arrows, right panel) spindle type forming short fascicles (Fig. 11). Cytoplasm
was clear or eosinophilic. Cellular pleomorphism was
prominent with scattered bizarre and hyperchromatic
nuclei, intranuclear inclusions were evident in some. Tumor abutted lining urothelium of renal pelvis with-
out ulceration and formed large hypocellular sheets of
short spindle cells in myxoid matrix encircling pelvic wall. There were rare foci of necrosis. Mitotic rate ranged 5–10/
10 hpf. Immunohistochemical profile of tumor was similar
to the first case including strong and diffuse MUC4, vimen-
tin and bcl-2, weak EMA expression (Fig. 12) except nega-
tive immunoreactivity for CD99. SMA showed frequently
scattered myofibroblasts between the tumor cells. FISH
demonstrated an unbalanced der(22)t(11;22)(p11;q12), as in
the case #1 (Fig. 13). A FUS gene rearrangement was not
identified. showed 1 cm residual mass. No adjuvant treatment was
given. Patient is alive without local or distant recurrence
10 months after surgery. Discussion Sclerosing epithelioid fibrosarcoma (SEF) was first de-
scribed by Meis-Kindblom et al. in 1995 [6]. The most
SEFs are deep seated lesions with a wide age range, but
typically occurring in middle aged adults [4, 6]. The
most frequent sites of involvement are deep soft tissues
of lower extremities or limb girdle followed by trunk,
upper extremities, and head and neck region, although
primary SEF of unexpected locations such as bone, retro-
peritoneum, and pelvis were also described as case reports
or small series [10–13]. Primary SEF in visceral organs is
exceedingly rare, with only a single case reports in the liver
[12], the lower gastrointestinal tract [11], the ovary [14],
and the pancreas [10]. Occurrence of primary renal SEF
was proven by Argani et al. who have recently reported 2
well-documented
cases
with
rearrangement
involving The lesion occupied both renal cortex and medulla, and
invaded into peripelvic and perirenal fat tissue. As one of
the surgical margins was in continuation with the tumor,
the patient underwent left radical nephrectomy which Page 7 of 12 Ertoy Baydar et al. Diagnostic Pathology (2015) 10:186 Fig. 8 Case #2. Gross photograph of the kidney in patient no. 2
shows a well-defined solid mass that extends to surgical margin
of the partial nephrectomy specimen EWSR1 and CREB3L1 genes [8] (Table 2). There are also 3
additional cases in the literature described as primary renal
SEFs which were shown to express MUC4 [1, 9]. Two of
these had rearranged EWSR1 with undocumented fusion
gene partner. EWSR1 and CREB3L1 genes [8] (Table 2). There are also 3
additional cases in the literature described as primary renal
SEFs which were shown to express MUC4 [1, 9]. Two of
these had rearranged EWSR1 with undocumented fusion
gene partner. SEF is a difficult diagnosis in visceral organs because of
its rarity and its epithelioid appearance, closely mimicking
carcinomas. It is characterized by a proliferation of epitheli-
oid cells arranged in nests and cords in a densely hyalinized
stroma (Table 3). Neoplastic cells are positive for vimentin,
bcl-2, MUC4, weakly and focally for epithelial membrane
antigen (EMA), and are negative for broad spectrum cyto-
keratins, smooth muscle actin, desmin, CD34, S-100 pro-
tein, HMB45 and melan-A [2, 4, 15]. MUC4 itself has
been recently reported as highly specific for the diagnosis
of SEF [2]. Strong CD99 expression is seen in some cases. Discussion Most common genetic alteration described in pure SEFs is
EWSR1-CREB3L1 fusion [1]. Basic histomorphological
differentials of SEF will be a variety of tumors with epithe-
lioid and sclerotic features, mainly primary or metastatic
carcinoma. Epithelioid angiomyolipoma, metanephric stro-
mal tumor and synovial sarcoma are the other consider-
ations in the differential diagnosis, as well as sclerosing
clear cell sarcoma of the kidney (CCSK) being the most
challenging. A battery of immunohistochemical stains will
help for further characterization in most circumstances, Fig. 8 Case #2. Gross photograph of the kidney in patient no. 2
shows a well-defined solid mass that extends to surgical margin
of the partial nephrectomy specimen Fig. 9 Case #2. Proliferating native renal tubules inside the tumor giving an appearance of a biphasic lesion (H&E x 100). Inset shows that hyperplastic
tubules maintain nuclear pax-8 (H&E x 40) Fig. 9 Case #2. Proliferating native renal tubules inside the tumor giving an appearance of a biphasic lesion (H&E x 100). Inset shows that hyperplastic
tubules maintain nuclear pax-8 (H&E x 40) Page 8 of 12 Ertoy Baydar et al. Diagnostic Pathology (2015) 10:186 Fig. 10 Case #2. Hyper and hypocellular regions (Left: H&E x 200; Right: H&E x 200) Fig. 10 Case #2. Hyper and hypocellular regions (Left: H&E x 200; Right: H&E x 200) CREB3L1 gene fusion with t(11;22)(p11;q12) chromosome
translocation was also found [18]. There have been 5 cases
of primary LGFMS described in kidney or renal pelvis so
far, one in a 6 year old child, others in adults [19–23]. however SEF and sclerosing type CCSK reveal both similar
morphology and immunohistochemical findings Argani et
al. suggest that some cases reported as sclerosing clear cell
sarcoma of the kidney (CCSK) in the literature might in
fact represent SEF [8]. We report 2 distinctive, clinically malignant, renal SEF
bearing EWSR1-CREB3L1 fusions through unbalanced
translocation with unique histomorphologic features. The common finding in both was the diffuse infiltration
of the tumor among renal tubules and glomeruli filling
out the space in-between them. Numerous tubules and
also glomeruli were seen entrapped throughout the
tumors. In case #1, neoplastic cells surrounded these
tubules in a concentric onion-skin like pattern giving a
lobular or micronodular architecture to the lesion in
many areas. Additionally, the neoplastic sleeves around
tubules revealed a zonation pattern with inner hypo-
cellular and outer hypercellular appearance. Discussion This was a
wide-spread occurrence involving also the deeper parts
of the tumor, so differed from what is seen in CCSK or
metanephric stromal tumor where neoplasm encircles
native tubules at the tumor infiltration borders in a SEF and low-grade fibromyxoid sarcoma (LGFMS) are
thought to be related members in the fibrosing fibrosar-
coma family [4]. Classic variants of LGFMS with or without
giant rosettes constitute one end of the spectrum, charac-
terized by a protracted clinical course and a low metastatic
rate, whereas SEF or cellular variants of LGFMS constitute
the other end which appears to be more aggressive. LGFMS
may have SEF-like areas, and vice versa occurs in SEF
lesions. Both tumors are labelled by immunohistochemical
MUC4 staining, 99–100 % in LGFMS and 78 % in SEF,
respectively [2, 16]. Furthermore, a genetic link between
sclerosing epithelioid fibrosarcoma and low-grade fibro-
myxoid sarcoma has been suggested [17]. Although major-
ity (>90 %) of LGFMS and hybrid LGFMS/SEF harbor
t(7;16)(q33;p11) chromosome translocation resulting in
FUS-CREB3L2 gene fusion, rare LGFMS carrying EWSR1- Page 9 of 12 Page 9 of 12 Ertoy Baydar et al. Diagnostic Pathology (2015) 10:186 Fig. 11 Case #2. Spindle cells forming short fascicles vaguely (H-E x 200). Inset highlights marked pleomorphism among the neoplastic cells (H-E x 400) le cells forming short fascicles vaguely (H-E x 200). Inset highlights marked pleomorphism among the neoplastic cells (H-E x 400) atypia in a gradual transition to small round cells. Immu-
nohistochemically, tumor was bcl-2 and CD99+, SMA
and S-100 -. SYT-SSX1 and SYT-SSX2 chimeric transcript
were not identified. We suspect that at least this published
case may represent the entity reported herein. limited extent. No angiodysplasia or juxtaglomerular
hyperplasia was found in our case. The second case presented in this study varied from
the first one in that the entrapped renal tubules exhib-
ited prominent papillary hyperplasia and small gland
budding which gave a biphasic appearance to the
lesion. They were lined by cuboidal or flattened single
layered epithelium devoid of atypia, some were cystic,
and some looked fibroadenomatous and leaf-like due
to compression by the interstitial neoplastic cells. It is
quite likely that we would have diagnosed this lesion
as malignant mixed epithelial and stromal tumor of
kidney (MEST) only a few years ago when typical genetic
alterations or MUC4 staining characteristic of this tumor
type were unknown yet. Discussion In fact, we suspect that other
examples of renal SEF may have been reported in the lit-
erature with an assignment as malignant MEST. Suzuki et
al. [24] reported a malignant MEST in a male with a pros-
tatic adenocarcinoma under anti-androgen treatment, that
was composed of atypical small round cells with a high
nuclear cytoplasmic ratio without necrosis and pleo-
morphism, and also focal spindle cells without cytologic Another feature of the second case of this study that var-
ied from the first one and from 5 cases reported previously
was the presence of high grade cytologic atypia with note-
worthy pleomorphism and scattered bizarre tumor cells. Most SEFs are cytologically uniform tumors with scanty
mitoses; however, it is stated that foci of pleomorphism
-particularly in hybrid SEF and LGFMS cases- and high
mitotic activity can rarely be encountered [4, 7, 17]. SEF is frequently characterized by aggressive clinical
behavior [4, 6]. One or more local recurrences occur in
approximately 50 % of cases, with metastatic spread being
reported in more than 40 % of cases, most often affecting
the pleura, lungs, bone and central nervous system. All of
the cases reported previously except one and our first case
were already metastatic at the time of presentation. As
accurate recognition of SEF is important for appropriate
patient management, we believe more cases need to be Page 10 of 12 Ertoy Baydar et al. Diagnostic Pathology (2015) 10:186 Fig. 12 Case #2. SMA stains interspersed myofibroblasts and MUC4 labels neoplastic cells (Left: Immunohistochemistry, anti-SMA Ab x 200; Right:
Immunohistochemistry, anti-MUC4 Ab x 200) Fig. 12 Case #2. SMA stains interspersed myofibroblasts and MUC4 labels neoplastic cells (Left: Immunohistochemistry, anti-SMA Ab x 200; Right:
Immunohistochemistry anti MUC4 Ab x 200) Fig. 12 Case #2. SMA stains interspersed myofibroblasts and MUC4 labels neoplastic cells (Left: Immunohistochemistry, anti-SMA Ab x 200; Right:
Immunohistochemistry, anti-MUC4 Ab x 200) Fig. 13 Case #2. FISH patterns similar to Case 1. EWSR1 break apart with loss of one copy of the Spectrum Green labeled probe flanking
the 3’ (telomeric) side (left panel) and EWSR1/CREB3L1 fusion seen by a juxtaposed orange signal and green signal (right panel) Fig. 13 Case #2. FISH patterns similar to Case 1. Discussion EWSR1 break apart with loss of one copy of the Spectrum Green labeled probe flanking
the 3’ (telomeric) side (left panel) and EWSR1/CREB3L1 fusion seen by a juxtaposed orange signal and green signal (right panel) Ertoy Baydar et al. Diagnostic Pathology (2015) 10:186 Page 11 of 12 Table 2 Summary of reported cases of primary renal SEF
Authors
Age
(years)
Gender Largest
Diameter
/Laterality
Clinical presentation
FISH
Findings
Therapy Metastases
Outcome
(mo)
Arbajian, et al. [1]
41
Female 9 cm/NS
NS
EWSR1 Del
3′
NS
Bone and lung
DOD (22)
Argani, et al. [8]
17
Male
25 cm/L
Left flank, back and abdominal pain,
weight loss, dysuria, and decreased
appetite
EWSR1-
CREB3L1
fusion
Surgery
+ RT
Rib, vertebrae, epidural
spinal cord and liver
DOC (1)
Argani, et al. [8]
61
Female 5 cm/L
Rib pain
EWSR1-
CERB3L1
fusion
Surgery
Ribs, bone, lung and
lymph nodes
AWD (6)
Ohlmann, et al. [9]
24
Female 22 cm/R
NS
No results
Surgery
+ RCT
Lungs and vertebrae
DOD (82)
Ohlmann, et al. [9]
43
Male
4.2 cm/R
Incidental
EWSR1
split
Surgery
None
ANED (8)
Ertoy Baydar, et
al. (present
case)
16
Female 7.5 cm/L
Abdominal pain radiating to back on
the left
EWSR1-
CREB3L1
fusion
Surgery
+ CT
Lungs, vertebrae,
sacrum and left
femoral head
AWD (30)
Ertoy Baydar, et
al. (present
case)
57
Female 7.5 cm/L
Incidental
EWSR1-
CREB3L1
fusion
Surgery
None
ANED
(10)
NS Not specified, L Left kidney, R Right kidney, RT Radiotherapy, CT chemotherapy, RCT Radiochemotherapy, DOD Died of disease, DOC Died of complications
related to disease treatment, AWD Alive with disease, ANED Alive with no evidence of disease documented so that its full histomorphologic spectrum is
uncovered. are better known to develop in the kidney is a strong
possibility as it has been only recently described and is
unfamiliar to the pathologists. SEF is a neoplasm with
variant morphological features that may overlap with
many lesions confusingly. Immunohistochemistry and
molecular studies that disclose the characteristic genetic
alterations are crucial for accurate recognition. Conclusions Sclerosing epithelioid fibrosarcoma (SEF) is a rare soft
tissue tumor that can occur in the kidney as a primary
malignancy. Its misdiagnosis with other entities which Table 3 Morphology of SEF Table 3 Morphology of SEF
Typical features
Macroscopy
Large, homogeneously white or white–tan, lobulated, and hard tumors
Cell size and shape
Small to medium-sized plump to epithelioid cells
Cytoplasm
Scant clear or eosinophilic cytoplasm
Nuclei
Oval to slightly elongate angulated nuclei with finely speckled chromatin
Cellularity
Variable within the neoplasm
Atypia
Mild
Matrix
Densely sclerotic, areas of metaplastic bone in some
Pattern of cellular arrangement
Small clusters, nests and anastomosing cords
Hybrid morphology
Areas of LGFMS or nodules of collagen reminiscent of HSCTGR in some
Immunohistochemistry
MUC4 ++, EMA +/−, CD99 +/−, bcl2 +/−, pan-keratin -, S100/HMB45/MelanA -
FISH
EWSR1 (or rarely FUS) rearrangements
Previously unrecognized features in renal SEF
Current case 1
A lobular or micronodular architecture due to neoplastic cells surrounding entrapped renal tubules in a concentric fashion
Zonation in the neoplastic lobules around tubules with inner hypocellular and outer hypercellular appearance
Current case 2
Exuberant epithelial hyperplasia and small gland budding in the entrapped native renal tubules, mimicking MEST
High grade cytologic atypia
LGFMS Low grade of fibromyxoid sarcoma, HSCTGR Hyalinizing spindle cell tumor with giant rosettes, MEST Mixed epithelial and stromal tumor of kidney A lobular or micronodular architecture due to neoplastic cells surrounding entrapped renal tubules in a concentric fashion
Zonation in the neoplastic lobules around tubules with inner hypocellular and outer hypercellular appearance
Exuberant epithelial hyperplasia and small gland budding in the entrapped native renal tubules, mimicking MEST
High grade cytologic atypia HSCTGR Hyalinizing spindle cell tumor with giant rosettes, MEST Mixed epithelial and stromal tumor of kidney LGFMS Low grade of fibromyxoid sarcoma, HSCTGR Hyalinizing spindle cell tumor with giant rosettes, MEST Mixed epithelial and stromal tumor of kidney Page 12 of 12 Page 12 of 12 Ertoy Baydar et al. Diagnostic Pathology (2015) 10:186 Authors’ contribution DEB, KK, OA and BB performed the histological examination. FTA and OA
analyzed and interpreted the patients’ data. JAB performed FISH analysis and
figures. DEB drafted the manuscript. KK made substantial contribution to
analysis. All authors read and approved the final manuscript. 16. Doyle LA, Moller E, Dal Cin P, Fletcher CD, Mertens F, Hornick JL. MUC4 is a
highly sensitive and specific marker for low-grade fibromyxoid sarcoma. Am
J Surg Pathol. 2011;35(5):733–41. doi:10.1097/PAS.0b013e318210c268. The authors declare that they have no competing interest. The authors declare that they have no competing interest. 15. Goldblum JR, Folpe AL, Weiss SW. Borderline and Malignant Fibroblastic/
Myofibroblastic Tumors. In: Goldblum JR, Folpe AL, Weiss SW, editors. Enzinger and Weiss's soft tissue tumors. 6th ed. Philadelphia, PA: Saunders/
Elsevier; 2014. p. 288–340. Acknowledgment 17. Guillou L, Benhattar J, Gengler C, Gallagher G, Ranchere-Vince D, Collin F,
et al. Translocation-positive low-grade fibromyxoid sarcoma:
clinicopathologic and molecular analysis of a series expanding the
morphologic spectrum and suggesting potential relationship to sclerosing
epithelioid fibrosarcoma: a study from the French Sarcoma Group. Am J
Surg Pathol. 2007;31(9):1387–402. doi:10.1097/PAS.0b013e3180321959. The authors thank Jonathan I. Epstein, M.D. and Pedram Argani, M.D. (The
Johns Hopkins Medical Institutions, Baltimore, MD) who reviewed first case
and provided a significant diagnostic input. The authors also would like to thank Dr. Irina Vaysertreyger, Dr. Dali Huang,
and Dr. Yimin Sun for their expert technical assistance. 18. Lau PP, Lui PC, Lau GT, Yau DT, Cheung ET, Chan JK. EWSR1-CREB3L1 gene
fusion: a novel alternative molecular aberration of low-grade fibromyxoid
sarcoma. Am J Surg Pathol. 2013;37(5):734–8. doi:10.1097/
PAS.0b013e31827560f8. Consent 9. Ohlmann CH, Brecht IB, Junker K, van der Zee JA, Nistor A, Bohle RM, et al. Sclerosing epithelioid fibrosarcoma of the kidney: clinicopathologic and
molecular study of a rare neoplasm at a novel location. Ann Diagn Pathol. 2015. doi:10.1016/j.anndiagpath.2015.04.005. Written informed consent was obtained from the parent
(case #1) and the patient (case #2) for publication of this
Case Report and any accompanying images. A copy of the
written consent is available for review by the Editor-in-
Chief of this journal. 10. Bai S, Jhala N, Adsay NV, Wei S. Sclerosing epithelioid fibrosarcoma of the
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1543-2165(2007)131[1825:SEFOTC]2.0.CO;2. Author details
1
f 1Department of Pathology, Hacettepe University Hospital, Ankara, Turkey. 2Department of Pathology, Ondokuz Mayis University Hospital, Samsun,
Turkey. 3Departments of Pathology/Microbiology, Pediatrics and Orthopaedic
Surgery, Nebraska Medical Center, Omaha, NE, USA. 4Department of Urology,
Hacettepe University Hospital, Ankara, Turkey. 1Department of Pathology, Hacettepe University Hospital, Ankara, Turkey. 2Department of Pathology, Ondokuz Mayis University Hospital, Samsun,
3 1Department of Pathology, Hacettepe University Hospital, Ankara, Turkey. 2Department of Pathology, Ondokuz Mayis University Hospital, Samsun, Turkey. 3Departments of Pathology/Microbiology, Pediatrics and Orthopaedic
Surgery, Nebraska Medical Center, Omaha, NE, USA. 4Department of Urology,
Hacettepe University Hospital, Ankara, Turkey. Turkey. 3Departments of Pathology/Microbiology, Pediatrics and Orthopaedic
Surgery, Nebraska Medical Center, Omaha, NE, USA. 4Department of Urology,
Hacettepe University Hospital, Ankara, Turkey. 19. Alevizopoulos A, Mygdalis V, Tyritzis S, Stravodimos K, Constantinides CA. Low-grade fibromyxoid sarcoma of the renal pelvis: first report. Case Rep
Nephrol Urol. 2012;2(2):87–91. doi:10.1159/000341191. 20. Arancio M, Ranzoni S, Delsignore A, Maffei N, Landi G, Mina A, et al. Fibromyxoid sarcoma of the kidney. A case report. Urologia. 2010;77(4):263–6. Received: 1 August 2015 Accepted: 1 October 2015 20. Arancio M, Ranzoni S, Delsignore A, Maffei N, Landi G, Mina A, et al. Fibromyxoid sarcoma of the kidney. A case report. Urologia. 2010;77(4):263–6. 21. del Valle GN, Santos Largo J, Martinez-Sagarra Oceja J, Rodriguez TV. Fibromyxoid sarcoma of kidney. Actas Urol Esp. 2009;33(9):1036–9. Received: 1 August 2015 Accepted: 1 October 2015 21. del Valle GN, Santos Largo J, Martinez-Sagarra Oceja J, Rodriguez TV. Fibromyxoid sarcoma of kidney. Actas Urol Esp. 2009;33(9):1036–9. 21. del Valle GN, Santos Largo J, Martinez-Sagarra Oceja J, Rodriguez TV. Fibromyxoid sarcoma of kidney. Actas Urol Esp. 2009;33(9):1036–9. Abbreviations
l SEF: Sclerosing epithelioid fibrosarcoma; FISH: Fluorescence in situ
hybridization; HSCTGR: Hyalinizing spindle cell tumor with giant rosettes;
CT: Computerized tomography; CCSK: Clear cell sarcoma of the kidney;
LGFMS: Low-grade fibromyxoid sarcoma; MEST: Mixed epithelial and stromal
tumor of kidney. 12. Tomimaru Y, Nagano H, Marubashi S, Kobayashi S, Eguchi H, Takeda Y, et al. Sclerosing epithelioid fibrosarcoma of the liver infiltrating the inferior vena
cava. World J Gastroenterol. 2009;15(33):4204–8. 12. Tomimaru Y, Nagano H, Marubashi S, Kobayashi S, Eguchi H, Takeda Y, et al. Sclerosing epithelioid fibrosarcoma of the liver infiltrating the inferior vena
cava. World J Gastroenterol. 2009;15(33):4204–8. 13. Wojcik JB, Bellizzi AM, Dal Cin P, Bredella MA, Fletcher CD, Hornicek FJ, et al. Primary sclerosing epithelioid fibrosarcoma of bone: analysis of a series. Am
J Surg Pathol. 2014;38(11):1538–44. doi:10.1097/PAS.0000000000000265. 13. Wojcik JB, Bellizzi AM, Dal Cin P, Bredella MA, Fletcher CD, Hornicek FJ, et al. Primary sclerosing epithelioid fibrosarcoma of bone: analysis of a series. Am
J Surg Pathol. 2014;38(11):1538–44. doi:10.1097/PAS.0000000000000265. References 22. Rubinstein JC, Visa A, Zhang L, Antonescu CR, Christison-Lagay ER, Morotti
R. Primary Low-Grade Fibromyxoid Sarcoma of the Kidney in a Child with
the Alternative EWSR1-CREB3L1 Gene Fusion. Pediatr Dev Pathol. 2014;17(4):321–6. doi:10.2350/14-05-1487-CR.1. 1. Arbajian E, Puls F, Magnusson L, Thway K, Fisher C, Sumathi VP, et al. Recurrent
EWSR1-CREB3L1 gene fusions in sclerosing epithelioid fibrosarcoma. Am J Surg
Pathol. 2014;38(6):801–8. doi:10.1097/PAS.0000000000000158. 1. Arbajian E, Puls F, Magnusson L, Thway K, Fisher C, Sumathi VP, et al. Recurrent
EWSR1-CREB3L1 gene fusions in sclerosing epithelioid fibrosarcoma. Am J Surg
Pathol. 2014;38(6):801–8. doi:10.1097/PAS.0000000000000158. 2. Doyle LA, Wang WL, Dal Cin P, Lopez-Terrada D, Mertens F, Lazar AJ, et al. MUC4 is a sensitive and extremely useful marker for sclerosing epithelioid
fibrosarcoma: association with FUS gene rearrangement. Am J Surg Pathol. 2012;36(10):1444–51. doi:10.1097/PAS.0b013e3182562bf8. 23. Silverman JF, Nathan G, Olson PR, Prichard J, Cohen JK. Fine-needle
aspiration cytology of low-grade fibromyxoid sarcoma of the renal capsule
(capsuloma). Diagn Cytopathol. 2000;23(4):279–83. 24. Suzuki T, Hiragata S, Hosaka K, Oyama T, Kuroda N, Hes O, et al. Malignant
mixed epithelial and stromal tumor of the kidney: report of the first male
case. Int J Urol. 2013;20(4):448–50. doi:10.1111/j.1442-2042.2012.03155.x. 3. Prieto-Granada C, Zhang L, Chen HW, Sung YS, Agaram NP, Jungbluth AA,
et al. A genetic dichotomy between pure sclerosing epithelioid
fibrosarcoma (SEF) and hybrid SEF/low-grade fibromyxoid sarcoma: A
pathologic and molecular study of 18 cases. Genes Chromosomes Cancer. 2014. doi:10.1002/gcc.22215. 4. Antonescu CR, Rosenblum MK, Pereira P, Nascimento AG, Woodruff JM. Sclerosing epithelioid fibrosarcoma: a study of 16 cases and confirmation of
a clinicopathologically distinct tumor. Am J Surg Pathol. 2001;25(6):699–709. 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 Competing interest g
14. Watanabe K, Suzuki T. Epithelioid fibrosarcoma of the ovary. Virchows Arch. 2004;445(4):410–3. doi:10.1007/s00428-004-1068-3. Submit your next manuscript to BioMed Central
and take full advantage of: 5. Chow LT, Lui YH, Kumta SM, Allen PW. Primary sclerosing epithelioid
fibrosarcoma of the sacrum: a case report and review of the literature. J Clin Pathol. 2004;57(1):90–4. 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 • Convenient online submission 6. Meis-Kindblom JM, Kindblom LG, Enzinger FM. Sclerosing epithelioid
fibrosarcoma. A variant of fibrosarcoma simulating carcinoma. Am J Surg
Pathol. 1995;19(9):979–93. • Thorough peer review 7. Ossendorf C, Studer GM, Bode B, Fuchs B. Sclerosing epithelioid
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Cases With EWSR1-CREB3L1 Gene Fusion. Am J Surg Pathol. 2014. doi:10.1097/PAS.0000000000000338.
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https://openalex.org/W2021777081
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https://publications.goettingen-research-online.de/bitstream/2/61421/1/Journal_Chemical_Sciences_Mukherjee.pdf
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English
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Phenalenyl-based ligand for transition metal chemistry: Application in Henry reaction
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Journal of chemical sciences/Proceedings of the Indian Academy of Sciences. Chemical sciences
| 2,011
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cc-by
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J. Chem. Sci. Vol. 123, No. 2, March 2011, pp. 139–144.
c⃝Indian Academy of Sciences. J. Chem. Sci. Vol. 123, No. 2, March 2011, pp. 139–144. c⃝Indian Academy of Sciences. ARUP MUKHERJEEa, TAMAL K SENa, SWADHIN K MANDALa,∗,
DANIEL KRATZERTb, DIETMAR STALKEb, ALEXANDER DÖRINGb and
CAROLA SCHULZKEc aDepartment of Chemical Sciences, Indian Institute of Science Education and Research (I
Mohanpur 741252, India
bGeorg-August-Universität Göttingen, Tammannstr. 4, 37077 Göttingen, Germany
cSchool of Chemistry, Trinity College Dublin, Dublin 2, Ireland
e-mail: swadhin.mandal@iiserkol.ac.in aDepartment of Chemical Sciences, Indian Institute of Science Education and Research
Mohanpur 741252, India
bGeorg-August-Universität Göttingen, Tammannstr. 4, 37077 Göttingen, Germany
cSchool of Chemistry, Trinity College Dublin, Dublin 2, Ireland
e-mail: swadhin.mandal@iiserkol.ac.in Abstract. We report the synthesis and characterization of the first transition metal complex of a phenalenyl-
based ligand. The reaction of Cu(OAc)2.H2O with 9-N-methylamino-1-N′-methylimino-phenalene (LH) in 1:1
stoichiometric ratio results in the formation of a mononuclear copper complex [LCu(OAc)] (1). The molecular
structure of 1 was established by X-ray crystallography. The electrochemistry of 1 indicates the formation of
an anionic radical by one electron reduction into the non-bonding molecular orbital of the phenalenyl system. The complex 1 efficiently catalyses the C–C bond forming Henry reaction. Keywords. Cu-complex; phenalenyl; radical; X-ray crystallography; Henry reaction. ∗For correspondence X = X' = O, R = lone pair of electrons, R' = H or Me;
X= O, X' = N, R = alkyl or aryl, R' = H;
X = X' = N, R = alkyl, R' = H or Me 2.1
Materials and methods It was filtered and kept at 25◦C. Red-brown
crystals appeared in one day (332 mg, yield 95%). M.pt. 175◦C (decomposes to black mass). IR (KBr, 600–
4000 cm−1): 3032(w), 2925(w), 2896(w), 2792(w),
2353(w), 1637(s), 1587(s), 1512(w), 1477(w), 1430(w),
1395(m), 1346(w), 1279(s), 1254(w), 1192(m), 1164(s),
1018(m), 927(m), 908(w), 830(s), 812(s), 776(m),
746(w), 720(w), 690(w). Elemental analysis: Anal. Calcd. for CuC17H16N2O2: C, 59.47%; H, 4.66%; N,
8.16%; Found: C, 58.44%; H, 4.42%; N, 7.83%. 9-N-methylamino-1-N′-methylimino-phenalene (222 mg,
1.0 mmol) and Cu(OAc)2.H2O (199 mg, 1.0 mmol) were
mixed in dichloromethane (DCM)/ethanol mixture
(30 mL, 1:1) and stirred at 25◦C overnight. The starting
materials slowly dissolved and resulted in a brown-red
colour. Finally, the reaction mixture was refluxed for
30 min. It was filtered and kept at 25◦C. Red-brown
crystals appeared in one day (332 mg, yield 95%). M.pt. 175◦C (decomposes to black mass). IR (KBr, 600–
4000 cm−1): 3032(w), 2925(w), 2896(w), 2792(w),
2353(w), 1637(s), 1587(s), 1512(w), 1477(w), 1430(w),
1395(m), 1346(w), 1279(s), 1254(w), 1192(m), 1164(s),
1018(m), 927(m), 908(w), 830(s), 812(s), 776(m),
746(w), 720(w), 690(w). Elemental analysis: Anal. Calcd. for CuC17H16N2O2: C, 59.47%; H, 4.66%; N,
8.16%; Found: C, 58.44%; H, 4.42%; N, 7.83%. 1.
Introduction systems has not yet been explored for designing tran-
sition metal catalysts. Herein we report the synthesis
and characterization of a copper complex bearing a
symmetrical phenalenyl-based N,N-ligand (LH). The
reaction of Cu(OAc)2.H2O with 9-N-methylamino-1-
N′-methylimino-phenalene (LH) in 1:1 stoichiometric
ratio results in the formation of a mononuclear copper
complex [LCu(OAc)] (1). The molecular structure of
1 was established by X-ray crystallography. The elec-
trochemistry of 1 reveals the presence of a reversible The chemistry of phenalenyl-based ligand systems has
not yet been explored except on the development of
neutral boron containing free radical conductors.1,2
Phenalenyl is well-known odd alternant hydrocarbon
with high symmetry (D3h) which has the ability to form
three redox species: cation, radical, and anion.3 The
formation of this redox triad involves the use of the
nonbonding molecular orbital (MO) of the phenalenyl
moiety and hence does not affect the stability of the
resulting species greatly. The neutral radical state is sta-
bilized by the extended electronic delocalization and
this way a number of phenalenyl-based organic conduc-
tors (chart 1) have been reported exhibiting the high-
est room temperature conductivity among any neutral
radical solids.1,2 Such characteristic features have been
widely utilized for exploring new conjugated electronic
systems, such as multifunctional electronic and mag-
netic materials.2 The chemistry so far developed in
this system is limited to the spiro-conjugated boron
system.1,2 The chemistry of phenalenyl-based ligand X'
X
R'
B
X
X'
X = X' = O, R = lone pair of electrons, R' = H or Me;
X= O, X' = N, R = alkyl or aryl, R' = H;
X = X' = N, R = alkyl, R' = H or Me
R'
R
R
R
R
+
Chart 1. Phenalenyl-based neutral radical conductors. X'
X
R'
B
X
X'
R'
R
R
R
R
+ R' Chart 1. Phenalenyl-based neutral radical conductors. Chart 1. Phenalenyl-based neutral radical conductors. 139 Arup Mukherjee et al. 140 one electron reduction process. The complex 1 exhibits
high catalytic activity in C–C bond forming Henry
reaction.4–15 gen atoms were refined isotropically on calculated posi-
tions using a riding model with their Uiso values con-
strained to 15 times the Ueq of their pivot atoms for
terminal sp3 carbon atoms and 1.2 times for all other
carbon atoms. 2.1
Materials and methods All manipulations were carried out under dry nitro-
gen atmosphere using standard Schlenk line tech-
nique. Solvents were dried using conventional proce-
dure. 9-N-methylamino-1-N ′-methylimino-phenalene
was prepared according to the literature procedure.1a All manipulations were carried out under dry nitro-
gen atmosphere using standard Schlenk line tech-
nique. Solvents were dried using conventional proce-
dure. 9-N-methylamino-1-N ′-methylimino-phenalene
was prepared according to the literature procedure.1a
Copper acetate monohydrate was purchased from
Merck, India and was used as received. Aldehydes
were purchased from Aldrich and were used with-
out further purifications. Triethylamine (Et3N) was
dried over KOH and nitromethane (CH3NO2) was
dried over CaCl2. Elemental analyses were performed
at the Institut für Anorganische Chemie, Universität
Göttingen. The melting point was measured on a Buchi
B-545 melting point apparatus. Infrared spectra were
recorded on a Perkin Elmer FT-IR spectrometer. 1H
and
13C NMR spectra were recorded on a Bruker
500 MHz spectrometer. Electrochemical measure-
ments were performed with an AUTOLAB PGSTAT12
potentiostat/galvanostat using a platinum disc electrode
with a reaction surface of 1 mm2 as working elec-
trode. A platinum rod electrode (together with internal
referencing versus ferrocene/ferrocenium) was used as
reference electrode and a platinum knob electrode as
auxiliary electrode. The scan rate of the cyclic voltam-
metric experiment was 100 mV/s. The sample was
dissolved in acetonitrile (0.1 mmol/L) together with
the electrolyte tetrabutylammonium hexafluorophos-
phate (0.1 mol/L). The electrochemical experiments
were conducted inside a glovebox under argon atmo-
sphere at 25◦C. The crystallographic data for 1 was col-
lected from shock-cooled crystals at 100 K mounted on
a glass fibre. The crystals were cooled and protected
from air with a low temperature mounting device16 in
inert oil. The data of 1 was collected on a INCOATEC
Mo Microsource with Quazar mirror optics equipped
with a low temperature device using monochromatic
MoKα radiation (λ = 0.71073 Å). The data was inte-
grated with SAINT,17 and an empirical absorption cor-
rection with SADABS18 was applied. The structure
was solved by direct methods (SHELXS-97)19 and
refined by full-matrix least-squares methods against F2
(SHELXL-97).18 All non-hydrogen atoms were refined
with anisotropic displacement parameters. The hydro- 9-N-methylamino-1-N′-methylimino-phenalene (222 mg,
1.0 mmol) and Cu(OAc)2.H2O (199 mg, 1.0 mmol) were
mixed in dichloromethane (DCM)/ethanol mixture
(30 mL, 1:1) and stirred at 25◦C overnight. The starting
materials slowly dissolved and resulted in a brown-red
colour. Finally, the reaction mixture was refluxed for
30 min. 2.3
General procedure for the catalytic Henry
reaction Complex 1 (0.066 mmol) was dissolved in ethanol
(2 mL) and aldehyde (0.66 mmol) was added followed
by the addition of nitromethane (3.3 mmol) and triethy-
lamine (0.066 mmol). The mixture was allowed to stir
at 25◦C for 15 h. The volatiles were removed in vacuo,
and the residue was purified by flash chromatography
to afford the nitroaldol adduct. 2.3a
1-(2-nitrophenyl)-2-nitroethanol:
1H
NMR
(500 MHz, CDCl3, ppm)20: δ 3.33 (br, 1H, OH), 4.55
(dd, 1H, J = 9 Hz and 13.8 Hz, CHHNO2), 4.87 (dd,
1H, J = 2.5 Hz and 13.8 Hz, CHHNO2), 6.05 (d, 1H,
J = 9 Hz, CHOH), 7.48 (t, 1H, J = 7.5 Hz, ArH), 7.68
(t, 1H, J = 7.5 Hz, ArH), 7.95 (d, 1H, J = 7.8 Hz,
ArH), 8.08 (d, 1H, J = 7.8 Hz, ArH). 13C NMR
(125 MHz, CDCl3, ppm): δ 66.6, 80.0, 125.2, 128.6,
129.6, 134.2, 134.6, 147.0. 2.3a
1-(2-nitrophenyl)-2-nitroethanol:
1H
NMR
(500 MHz, CDCl3, ppm)20: δ 3.33 (br, 1H, OH), 4.55
(dd, 1H, J = 9 Hz and 13.8 Hz, CHHNO2), 4.87 (dd,
1H, J = 2.5 Hz and 13.8 Hz, CHHNO2), 6.05 (d, 1H,
J = 9 Hz, CHOH), 7.48 (t, 1H, J = 7.5 Hz, ArH), 7.68
(t, 1H, J = 7.5 Hz, ArH), 7.95 (d, 1H, J = 7.8 Hz,
ArH), 8.08 (d, 1H, J = 7.8 Hz, ArH). 13C NMR
(125 MHz, CDCl3, ppm): δ 66.6, 80.0, 125.2, 128.6,
129.6, 134.2, 134.6, 147.0. 2.3b
1-(4-nitrophenyl)-2-nitroethanol:
1H
NMR
(500 MHz, CDCl3, ppm)20: δ 3.3 (br, 1H, OH), 4.61
(m, 2H, CH2NO2), 5.6 (m, 1H, CHOH), 7.63 (d, 2H,
J = 8.6 Hz, ArH), 8.27 (d, 2H, J = 8.6 Hz, ArH). Phenalenyl-based ligand for transition metal chemistry: application in Henry reaction 141 Figure 1. Perspective ORTEP view of the molecular struc-
ture of 1. Thermal ellipsoids are drawn with 50% probability. Hydrogen atoms have been omitted for the sake of clarity. 13C (125 MHz, CDCl3, ppm): δ 69.9, 80.6, 124.1,
126.9, 145.0, 148.1. 3.1
Synthesis of complex 1 The title compound was synthesized by the treatment
of the ligand 9-N-methylamino-1-N ′-methylimino-
phenalene (LH) with Cu(OAc)2.H2O in ethanol and
dichloromethane mixture (1:1) (scheme 1). The reac-
tion mixture was refluxed for 30 min at the end to ensure
complete conversion of the starting materials into the
title compound. The compound 1 was characterized by
IR spectroscopy, elemental analysis and single crystal
X-ray crystallography. The reddish-brown single crys-
tals of 1 were grown from the reaction mixture at 25◦C
under slow evaporation of the solvents. 3.2
X-ray crystal structures of 1 The structure of 1 was established by X-ray diffrac-
tion study. The molecular structure of 1 is shown in
figure 1, and the selected bond lengths and angles
are presented in table 1. The X-ray structure reveals
a distorted square-planar geometry around the copper
(II) centre with two nitrogen atoms of the phenalenyl
ligand and two oxygen atoms of the acetate group
coordinated to the metal centre. The two Cu–N bond
lengths are not equal revealing an inherent asym-
metry in the structure. However, the two bond angles
N(1)–Cu(1)–O(1) and N(2)–Cu(1)–O(2) are equal
(162.7◦). The Cu–N bond lengths [Cu(1)–N(2) =
1.907 Å and Cu(1)–N(1) = 1.912 Å] in complex 1
fall in the range reported for other Cu(II) square-
planar complexes. For example, the Cu–N bond lengths Figure 1. Perspective ORTEP view of the molecular struc-
ture of 1. Thermal ellipsoids are drawn with 50% probability. Hydrogen atoms have been omitted for the sake of clarity. (∼1.92 Å) in propylene-bis(biuret)-copper complex,
Na2[Cu(pr(bi)2)]·3Me2SO21
and in LCuCl [where
L = 2,4-bis(2,6-diisopropylphenylimido)pentane]22 are
similar to the Cu–N bond length observed in 1. HN
N
+Cu(OAc)2
DCM/Ethanol
1
.H2O
LH
O
Cu
O
N
N
+
Scheme 1. Synthesis of complex 1. (∼1.92 Å) in propylene-bis(biuret)-copper complex,
Na2[Cu(pr(bi)2)]·3Me2SO21
and in LCuCl [where
L = 2,4-bis(2,6-diisopropylphenylimido)pentane]22 are
similar to the Cu–N bond length observed in 1. The electrochemistry of 1 is presented in figure 2
where it may be seen that complex 1 shows a well-
behaved reversible single reduction corresponding to
the generation of an anionic radical (scheme 2). Similar reduction process is well-known in related
phenalenyl-based spiro-conjugated boron systems.1,2
This reduction wave appears at a more negative value
(−1.75 V) than the spiro-conjugated boron compounds
(∼−1.0 V) bearing the N,N-phenalenyl ligands.1a This HN
N
+Cu(OAc)2
DCM/Ethanol
1
.H2O
LH
O
Cu
O
N
N
+
Scheme 1. Synthesis of complex 1. (
)
p
py
(
)
pp
p
Na2[Cu(pr(bi)2)]·3Me2SO21
and in LCuCl [where
L = 2,4-bis(2,6-diisopropylphenylimido)pentane]22 are
similar to the Cu–N bond length observed in 1. The electrochemistry of 1 is presented in figure 2
where it may be seen that complex 1 shows a well-
behaved reversible single reduction corresponding to
the generation of an anionic radical (scheme 2). 3.2
X-ray crystal structures of 1 Similar reduction process is well-known in related
phenalenyl-based spiro-conjugated boron systems.1,2
This reduction wave appears at a more negative value
(−1.75 V) than the spiro-conjugated boron compounds
(∼−1.0 V) bearing the N,N-phenalenyl ligands.1a This HN
N
+Cu(OAc)2
DCM/Ethanol
1
.H2O
LH
O
Cu
O
N
N
+
Scheme 1. Synthesis of complex 1. The electrochemistry of 1 is presented in figure 2
where it may be seen that complex 1 shows a well-
behaved reversible single reduction corresponding to
the generation of an anionic radical (scheme 2). Similar reduction process is well-known in related
phenalenyl-based spiro-conjugated boron systems.1,2
This reduction wave appears at a more negative value
(−1.75 V) than the spiro-conjugated boron compounds
(∼−1.0 V) bearing the N,N-phenalenyl ligands.1a This LH LH Scheme 1. Synthesis of complex 1. 142 Arup Mukherjee et al. Table 1. Selected bond lengths [Å] and angles [◦] for complex 1. Bond
Distance
Bond
Angles
Cu(1)–N(2)
1.907(6)
N(2)–Cu(1)–N(1)
95.2(3)
Cu(1)–N(1)
1.912(6)
N(2)–Cu(1)–O(2)
162.7(2)
Cu(1)–O(2)
2.051(5)
N(1)–Cu(1)–O(2)
101.1(2)
Cu(1)–O(1)
2.079(5)
N(2)–Cu(1)–O(1)
100.8(2)
N(1)–C(11)
1.31(10)
N(1)–Cu(1)–O(1)
162.7(2)
N(1)–C(15)
1.475(10)
O(2)–Cu(1)–O(1)
63.9(2)
N(2)–C(1)
1.333(10)
C(11)–N(1)–C(15)
119.3(6)
N(2)–C(14)
1.481(10)
C(11)–N(1)–Cu(1)
125.4(5)
O(1)–C(16)
1.271(10)
C(15)–N(1)–Cu(1)
115.1(5)
O(2)–C(16)
1.272(10)
C(1)–N(2)–Cu(1)
127.1(5)
C(1)–C(2)
1.449(11)
C(14)–N(2)–Cu(1)
114.6(5)
C(1)–C(12)
1.461(11
C(16)–O(1)–Cu(1)
88.1(5) Table 1. Selected bond lengths [Å] and angles [◦] for complex 1. indicates that the reduction is comparatively more dif-
ficult in this system to that of boron-based systems.1a
This may be attributed to the lower oxidation state of
the copper (+2) in neutral complex 1 as compared to
that of cationic spiro-conjugated boron (in +3 oxidation
state) compounds. important intermediate such as 1,2-amino alcohol or α-
hydroxy carboxylic acids. As a result, there have been
considerable efforts towards the development of syn-
thetic methods using Henry reaction.4–8 Recent stud-
ies have revealed that copper can actively catalyse the
Henry reaction.9–15 The new copper complex 1 was tested for its effi-
ciency as catalyst in Henry reaction. The catalytic reac-
tion was carried out at 25◦C for 15 h resulting in a
moderate yield (∼60%) of the corresponding nitroaldol
adduct. Addition of Et3N (10 mol%) greatly enhances
the conversion rate (> 90%) of the catalytic reaction
(table 2). We carried out the catalytic Henry reaction
in a range of solvents such as methanol, ethanol, iso-
propanol, THF, toluene, dichloromethane, and chloro-
form to find out the best solvent. 3.2
X-ray crystal structures of 1 Ethanol was found to
be the best solvent leading to more than 90% isolated
yield (table 3). 3.3
Application to Henry reaction The Henry reaction or the nitroaldol reaction is an
important organic transformation leading to the new C–
C bonds. It allows access to a range of synthetically -2.0
-1.9
-1.8
-1.7
-1.6
-1.5
-2.0
-1.5
-1.0
-0.5
0.0
Current(µA)
Potential (Volts vs.SCE )
E1/2 = -1.75 V
Figure 2. Cyclic voltammetry of complex 1 generating an
anionic radical by one electron reduction process. -2.0
-1.9
-1.8
-1.7
-1.6
-1.5
-2.0
-1.5
-1.0
-0.5
0.0
Current(µA)
Potential (Volts vs.SCE )
E1/2 = -1.75 V
Figure 2. Cyclic voltammetry of complex 1 generating an
anionic radical by one electron reduction process. form to find out the best solvent. Ethanol was found to
be the best solvent leading to more than 90% isolated
yield (table 3). After the most productive reaction condition was
established, we carried out the Henry reaction on
another nitro aldehyde (2-nitro benzaldehyde) using the
+e
-e
O
Cu
O
N
N
O
Cu
O
N
N
+
Scheme 2. Generation of an anionic radical by one elec-
tron reduction of 1. -2.0
-1.9
-1.8
-1.7
-1.6
-1.5
-2.0
-1.5
-1.0
-0.5
0.0
Current(µA)
Potential (Volts vs.SCE )
E1/2 = -1.75 V After the most productive reaction condition was
established, we carried out the Henry reaction on
another nitro aldehyde (2-nitro benzaldehyde) using the Current(µA) +e
-e
O
Cu
O
N
N
O
Cu
O
N
N
+
Scheme 2. Generation of an anionic radical by one elec-
tron reduction of 1. Potential (Volts vs.SCE ) Scheme 2. Generation of an anionic radical by one elec-
tron reduction of 1. Figure 2. Cyclic voltammetry of complex 1 generating an
anionic radical by one electron reduction process. 143 Phenalenyl-based ligand for transition metal chemistry: application in Henry reaction Table 2. Standardization of catalytic Henry reactiona using complex 1 as catalyst. Table 2. Standardization of catalytic Henry reactiona using complex 1 as catalyst. H
O
O2N
CH3NO2
Catalyst (10 mol%)
OH
O2N
NO2
Ethanol, 25 ˚C
Entry
Catalyst
Time (h)
Yield (%)b
1
Complex 1
22
65
2
Complex 1 + Et3N (10 mol%)
15
92
aTh
i
i d
i h 0 66
l
f C H NO CHO
d 3 3
l
f aThe reactions were carried out with 0.66 mmol of C6H4NO2CHO and 3.3 mmol of
CH3NO2 in 2 mL EtOH at 25◦C. 3
2
bIsolated yield after flash chromatographic purification. Table 3. 4.
Conclusion 2. 2. (a) Mandal S K, Samanta S, Itkis M E, Jensen D W, Reed
R W, Oakley R T, Tham F S, Donnadieu B and Haddon
R C 2006 J. Am. Chem. Soc. 128 1982; (b) Pal S K, Itkis
M E, Tham F S, Reed R W, Oakley R T and Haddon R
C 2005 Science 309 281; (c) Itkis M E, Chi X, Cordes A
W and Haddon R C 2002 Science 296 1443 In summary, we have synthesized and characterized the
first transition metal complex of a phenalenyl-based lig-
and to be utilized as catalyst for carrying out Henry
reaction. This result establishes that the phenalenyl-
based ligands can be used for the development of
transition metal chemistry to design catalyst for homo-
geneous organic transformation. The complex was syn-
thesized by direct mixing of the reagents in high
yield. The electrochemistry of this complex clearly
demonstrates that the empty non-bonding orbital of the
phenalenyl ligand can accept an electron to generate the
anionic radical. This result supports that this system has
potential to prepare the mixed organic (free electron in
phenalenyl part) inorganic [free electron in copper(II)]
hybrid magnetic system. Also it could find application
for preparation mixed organic–inorganic tunnel junc-
tion by external spin injection. Complex 1 exhibits high
catalytic efficiency in the C–C bond forming Henry
reactions. This study establishes that the phenalenyl-
based ligand system can not only be used as building
block for the preparation of molecular conductors but
also they can be used as useful ligands for making effi-
cient catalysts. Currently, we have been working on
different metals to synthesize active catalysts bearing
phenalenyl-based ligands for useful organic catalytic
transformations. 3. Haddon R C 1975 Nature 256 394 4. Palomo C, Oiarbide M and Laso A 2007 Eur. J. Org. Chem. 2561 5. Bandini M, Piccinelli F, Tommasi S, Umani-Ronchi A
and Ventrici C 2007 Chem. Commun. 616 6. Trost B M, Yeh V S C, Ito H and Bremeyer N 2002 Org. Lett. 4 2621 7. Kudoh T, Ishikawa T, Shimizu Y and Saito S 2003 Org. Lett. 5 3875 8. Paintner F F, Allmendinger L, Bauschke G and Klemann
P 2005 Org. Lett. 7 1423 9. Christensen C, Juhl K and Jørgensen K A 2001 Chem. Commun. 2222 10. Christensen C, Juhl K, Hazell R G and Jørgensen K A
2002 J. Org. Chem. 67 4875 11. References complex 1 as a catalyst. The results of the catalytic reac-
tions are presented in table 4 exhibiting high isolated
yield. 1. (a) Mandal S K, Itkis M E, Chi X, Samanta S, Lidsky
D, Reed R W, Oakley R T, Tham F S and Haddon R
C 2005 J. Am. Chem. Soc. 127 8185; (b) Chi X, Itkis
M E, Patrick B O, Barclay T M, Reed R W, Oakley R T,
Cordes A W and Haddon R C 1999 J. Am. Chem. Soc. 121 10395 4.
Conclusion Evans D A, Seidel D, Rueping M, Lam H W, Shaw J T
and Downey C W 2003 J. Am. Chem. Soc. 125 12692 y
12. Ginotra S K and Singh V K 2001 Org. Biomol. Chem. 5
3932 13. Arai T, Watanabe M and Yanagisawa A 2007 Org. Lett. 9 3595 14. Bandini M, Benaglia M, Sinisi R, Tommasi S and
Umani-Ronchi A 2007 Org. Lett. 9 2151 15. Mei L, Ming T H, Rong L Q, Jie S, Zhong Y S and Liang
L X 2009 J. Chem. Sci. 121 435 16. Kottke T and Stalke D 1993 J. Appl. Crystallogr. 26
615 17. Bruker SAINT v7.34A. 2005 Bruker AXS Inc. Madison
280 (WI, USA) 3.3
Application to Henry reaction Effect of solvent on Henry reaction using 1 as catalyst.a
H
O
O2N
CH3NO2
Catalyst (10 mol%)
OH
O2N
NO2
Solvent, 25 ˚C
Entry
Catalyst
Solvent
Time (h)
Yield (%)b
1
1+ Et3N
Methanol
15
88
2
1+ Et3N
Ethanol
15
92
3
1+ Et3N
Isopropanol
15
86
4
1+ Et3N
THF
15
75
5
1+ Et3N
Toluene
15
65
6
1+ Et3N
DCM
15
72
7
1+ Et3N
CHC13
15
68
aThe reactions were carried out with 0.66 mmol of C6H4NO2CHO and 3.3 mmol of
CH3NO2 in 2 mL solvent for 15 h at 25◦C in the presence of 10 mol% Et3N. bIsolated yield after flash chromatographic purification. Table 3. Effect of solvent on Henry reaction using 1 as catalyst.a aThe reactions were carried out with 0.66 mmol of C6H4NO2CHO and 3.3 mmol of
CH3NO2 in 2 mL solvent for 15 h at 25◦C in the presence of 10 mol% Et3N. bIsolated yield after flash chromatographic purification. Table 4. Henry reaction using 1 as catalyst on different alde-
hyde substrates.a
CH3NO2
Catalyst (10 mol%)
Ethanol, 25 ˚C
OH
ArCHO
Ar
NO2
Entry
Catalyst
Aldehyde
Yield (%)b
1
Complex 1
4-NO2C6H4CHO
92
2
Complex 1
2-NO2C6H4CHO
90
aThe
reactions
were
carried
out
with
0.66 mmol
of
C6H4NO2CHO and 3.3 mmol of CH3NO2 in 2 mL EtOH for
15 h at 25◦C in the presence of 10 mol% Et3N. bIsolated yield after flash chromatographic purification. Table 4. Henry reaction using 1 as catalyst on different alde-
hyde substrates.a
CH3NO2
Catalyst (10 mol%)
Ethanol, 25 ˚C
OH
ArCHO
Ar
NO2
Entry
Catalyst
Aldehyde
Yield (%)b
1
Complex 1
4-NO2C6H4CHO
92
2
Complex 1
2-NO2C6H4CHO
90
aThe
reactions
were
carried
out
with
0.66 mmol
of
C6H4NO2CHO and 3.3 mmol of CH3NO2 in 2 mL EtOH for
15 h at 25◦C in the presence of 10 mol% Et3N. bIsolated yield after flash chromatographic purification. Table 4. Henry reaction using 1 as catalyst on different alde-
hyde substrates.a Table 4. Henry reaction using 1 as catalyst on different alde-
hyde substrates.a 144 Arup Mukherjee et al. Acknowledgements 18. Sheldrick G M 2008 SADABS 2008/1 Göttingen Financial support from the Department of Science and
Technology, New Delhi, India through SERC Fast-
Track Grant SR/FT/CS-020/2008 and IISER, Kolkata
is acknowledged. AM and TKS are thankful to
IISER, Kolkata and Council of Scientific and Industrial
Research (CSIR), New Delhi for research fellowships. 19. Sheldrick G M 2008 Acta Crystallogr. Sect. A. 64 112 20. Lai G, Wang S and Wang Z 2008 Tetrahedron: Asymme-
try 19 1813 21. Barbier J P, Biyyadh A E, Kappenstein C, Mabiala N
and Hugel R P 1985 Inorg. Chem. 24 3615 22. Holland P L and Tolman W B 1999 J. Am. Chem. Soc. 121 7270
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https://openalex.org/W3197399723
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English
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Improved bacterial leaf blight disease resistance in the major elite Vietnamese rice cultivar TBR225 via editing of the OsSWEET14 promoter
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PloS one
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cc-by
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PLOS ONE PLOS ONE RESEARCH ARTICLE OPEN ACCESS Citation: Duy PN, Lan DT, Pham Thu H, Thi Thu
HP, Nguyen Thanh H, Pham NP, et al. (2021)
Improved bacterial leaf blight disease resistance in
the major elite Vietnamese rice cultivar TBR225 via
editing of the OsSWEET14 promoter. PLoS ONE
16(9): e0255470. https://doi.org/10.1371/journal. pone.0255470 Improved bacterial leaf blight disease
resistance in the major elite Vietnamese rice
cultivar TBR225 via editing of the OsSWEET14
promoter Phuong Nguyen Duy1☯, Dai Tran Lan1,2☯, Hang Pham Thu1, Huong Phung Thi Thu1,
Ha Nguyen Thanh1, Ngoc Phuong Pham1¤, Florence Auguy3, Huong Bui Thi Thu4, Tran
Bao Manh5, Sebastien Cunnac3, Xuan Hoi PhamID1* a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 1 Department of Molecular Pathology, Institute of Agricultural Genetics, Vietnam Academy of Agricultural
Sciences, Hanoi, Vietnam, 2 Faculty of Natural Sciences, Department of Applied Biology and Agriculture,
Quynhon University, Quynhon, Vietnam, 3 PHIM Plant Health Institute, Univ Montpellier, IRD, CIRAD,
INRAE, Institut Agro, Montpellier, France, 4 Vietnam National University of Agriculture, Hanoi, Vietnam,
5 ThaiBinh Seed Corporation, Thaibinh, Vietnam ☯These authors contributed equally to this work. ¤ Current address: Faculty of Biology, Hanoi University of Sciences, Hanoi, Vietnam
* xuanhoi.pham@gmail.com ☯These authors contributed equally to this work. q
y
¤ Current address: Faculty of Biology, Hanoi University of Sciences, Hanoi, Vietnam
* xuanhoi.pham@gmail.com Abstract TBR225 is one of the most popular commercial rice varieties in Northern Vietnam. However,
this variety is highly susceptible to bacterial leaf blight (BLB), a disease caused by Xantho-
monas oryzae pv. oryzae (Xoo) which can lead to important yield losses. OsSWEET14
belongs to the SWEET gene family that encodes sugar transporters. Together with other
Clade III members, it behaves as a susceptibility (S) gene whose induction by Asian Xoo
Transcription-Activator-Like Effectors (TALEs) is absolutely necessary for disease. In this
study, we sought to introduce BLB resistance in the TBR225 elite variety. First, two Vietnam-
ese Xoo strains were shown to up-regulate OsSWEET14 upon TBR225 infection. To inves-
tigate if this induction is connected with disease susceptibility, nine TBR225 mutant lines
with mutations in the AvrXa7, PthXo3 or TalF TALEs DNA target sequences of the OsS-
WEET14 promoter were obtained using the CRISPR/Cas9 editing system. Genotyping
analysis of T0 and T1 individuals showed that mutations were stably inherited. None of the
examined agronomic traits of three transgene-free T2 edited lines were significantly different
from those of wild-type TBR225. Importantly, one of these T2 lines, harboring the largest
homozygous 6-bp deletion, displayed decreased OsSWEET14 expression as well as a sig-
nificantly reduced susceptibility to a Vietnamese Xoo strains and complete resistance to
another one. Our findings indicate that CRISPR/Cas9 editing conferred an improved BLB
resistance to a Vietnamese commercial elite rice variety. Introduction Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major bacterial
disease that causes 10%-20% annual reduction in rice production worldwide [1]. The use of
improved rice varieties resistant to Xoo is probably the most efficient, economical and environ-
mentally-friendly way to control BLB. The virulence of Xoo depends on the transcriptional activation of specific host disease-sus-
ceptibility (S) genes by a subgroup of bacterial type III effectors, called transcription activator-
like effectors (TALEs) [2]. Upon translocation into the plant cell, TALEs bind to specific host
nuclear gene promoter sequences termed Effector-Binding Elements (EBEs) and induce target
gene expression to the benefit of the pathogen. The central repetitive domain of TALEs is
responsible for DNA target sequence binding. DNA binding involves recognition principles
that have been largely deciphered and applied to the computational prediction of TALEs target
DNA sequences [3,4]. This and earlier work has fostered the identification of TALEs transcrip-
tional targets in the rice genome and ultimately, of rice BLB S genes [2]. All Xoo strains recurrently target S genes belonging to the SWEET gene family and coding
for transmembrane sugar exporter proteins [3]. The over accumulation of SWEETs due to
TALE induction is presumed to provide an additional ration of apoplastic carbohydrates for
full bacterial pathogen multiplication and disease expression [5]. Although all five rice clade
III SWEET genes can function as S genes for bacterial blight, only three, namely OsSWEET11,
OsSWEET13 and OsSWEET14, are known to be targeted by several unrelated TALEs in nature
[6–11]. OsSWEET11 is activated by PthXo1 [6], OsSWEET13 is targeted by different variants
of PthXo2 [11,12], while OsSWEET14 is a target of multiple TAL effectors, including AvrXa7,
PthXo3, TalC and TalF [7–9]. Previous studies established that rice resistance to Xoo resulting from "TALE-unresponsive"
alleles can be conferred by natural DNA polymorphisms or targeted editing of EBEs located in
OsSWEET genes promoters of rice germplasm accessions or engineered rice varieties, respec-
tively [6,13–16]. For example, early resistance engineering work has used TALENs to individu-
ally alter the AvrXa7, TalC or TalF EBEs in the OsSWEET14 promoter and successfully
obtained resistance to some Asian Xoo strains [13,15]. Editor: R. M. Sundaram, ICAR-Indian Institute of
Rice Research, INDIA Editor: R. M. Sundaram, ICAR-Indian Institute of
Rice Research, INDIA Received: October 22, 2020
Accepted: July 17, 2021
Published: September 9, 2021 Published: September 9, 2021 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.0255470 Copyright: © 2021 Duy et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files. 1 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0255470
September 9, 2021 PLOS ONE Improved bacterial leaf blight disease resistance in Vietnamese rice cultivar TBR225 Funding: This work was supported by the National
Technology Innovation Program of Vietnam (Grant
No. M.36.DN/18) funded by the Vietnam Ministry
of Science and Technology (https://most.gov.vn/
vn/pages/Trangchu.aspx) and ThaiBinh Seed
Corporation (https://thaibinhseed.com.vn/trang-
chu.aspx?lang=en-US). - All equipments, labs and
nethouses for this work were supported by
Institute of Agricultural Genetics, Vietnam Academy
of Agricultural Sciences - Financial support came
from the Vietnam Ministry of Science and
Technology (https://most.gov.vn/vn/pages/
Trangchu.aspx) and ThaiBinh Seed Corporation
(https://thaibinhseed.com.vn/trang-chu.aspx?lang=
en-US). - The funders had no role in study design,
data collection and analysis, decision to publish the
manuscript. However, Tran Manh Bao who’s
employed by ThaiBinh Seed Corporation has
contributed to the reviewing and editing the
manuscript. His name was added as an author of
the manuscript. - The funder (ThaiBinh Seed
Corporation) supported the research materials (rice
cultivar TBR225) for this study. - The funders
provided support in the form of salaries for authors
(Pham Xuan Hoi, Nguyen Duy Phuong, Pham Thu
Hang and Nguyen Thanh Ha and Tran Manh Bao),
but did not have any additional role in the study
design, data collection and analysis, decision to
publish or preparation of the manuscript. The
specific roles of these authors are articulated in the
‘author contributions’ section. PLOS ONE | https://doi.org/10.1371/journal.pone.0255470
September 9, 2021 Introduction However, strains collected in Asian
countries such as China, Japan, Phillippines, Taiwan, Thailand, India, Nepal or South Korea
can express combinations of up to three major TALEs redundantly targeting clade III OsS-
WEET genes with either PthXo3 or AvrXa7 being occasionally associated with PthXo2 [11,17]. Broad BLB resistance engineering thus required multiplex OsSWEET promoters EBE editing
using the CRISPR/Cas9 system [11,12]. Competing interests: Authors XP, PD, HPT, HNT,
and TB are employee of ThaiBinh Seed
Corporation. This does not alter our adherence to
PLOS ONE policies on sharing data and materials. There are no patents, products in development or
marketed products associated with this research to
declare. The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-
9 nuclease (CRISPR/Cas9) system is a simple and efficient gene-editing tool developed in the
past few years [18,19]. Moreover, the targeted mutations generated by CRISPR/Cas9 can be
stably transmitted to the next generation. Thus, CRISPR/Cas9 has become a routine tool in
plant laboratories around the world to create various mutants for many applications, including
the genetic improvement of crops [20]. BLB is a major rice disease which occurs in many rice cultivating areas of Vietnam [21,22]. Most Vietnamese commercial rice varieties, including TBR225, are susceptible to BLB, result-
ing in annual yield loss of about 15–30% on average [23]. A few studies have identified rice
resistance genes effective against Vietnamese Xoo lineages [22,23]. However, no information is
currently available on the nature of Vietnamese Xoo TALEs and their corresponding S genes. Despite the large number of mapped rice BLB resistance genes [24,25], there is a need for alter-
native breeding approaches that enable the rapid introduction of broad BLB resistance in elite
varieties in order to cope with swift pathogen populations adaptive shifts in the fields [11,26]. PLOS ONE | https://doi.org/10.1371/journal.pone.0255470
September 9, 2021 2 / 16 PLOS ONE Improved bacterial leaf blight disease resistance in Vietnamese rice cultivar TBR225 Here, we report on the identification of OsSWEET14 as a transcriptional target of Vietnam-
ese Xoo. CRISPR/Cas9-mediated mutagenesis of the OsSWEET14 promoter in TBR225, a
major elite variety in rice production areas of North Vietnam, is shown to confer BLB resis-
tance without detectable yield penalty. The current study found this quintessential S gene to be
associated with the virulence of Vietnamese Xoo strains. Gene expression analysis Gene expression analyses were carried out as described previously [29] by RT-PCR method. The rice leaves were infiltrated with the indicated bacterial strains and used for total RNA
extraction 48 h post inoculation using the TRIzol reagent (Invitrogen, USA). One microgram
of RNA was used for each RT-PCR with oligo (dT) primer followed by PCR with OsS-
WEET14-specific primers (forward 50-ACTTGCAAGCAAGAACAGTAGT-30 and reverse 50-AT
GTTGCCTAGGAGACCAAAGG-30). An Eppendorf Mastercycler ep Gradient S was used for 35
PCR cycles. The OsEF1α gene was used as a constitutive control [15] using specific primers
(forward 50-GAAGTCTCATCCTACCTGAAGAAG-30 and reverse 50-GTCAAGAGCCTCAAGCA
AGG-30). gRNA design The OsSWEET14 promoter (GenBank, accession number: AP014967.1) was amplified by PCR
with forward primer 5’-TTGCGGCTCATCAGTTTCTC-3’ and reverse primer 5’-CTAGGAGAC
CAAAGGCGAAG-3’ from genomic DNA of TBR225 rice plants and ligated in pGEM-T Easy
vector (Promega) for sequencing. The gRNA target sequence (Fig 1A) for editing the TBR225
OsSWEET14 promoter was designed based on the sequence of the cloned TBR225 OsS-
WEET14 promoter using a combination of two bioinformatics tools CRISPR-P v2.0 [30] and
CCTop [31]. A gRNA sequence with high on-target and low off-target scores in both predic-
tion tools was chosen for vector construction. Plant and pathogen materials Rice cultivar TBR225 (Oryza sativa L. ssp. indica) were obtained from ThaiBinh Seed Cor. [27]. All edited and wild-type (WT) TBR225 plants were grown in a net-house under the fol-
lowing average conditions: 30˚C for 14 h (light) and 25˚C for 10 h (dark) with 80% humidity. The Xoo VXO_11 and VXO_15 strains used in this study were isolated from diseased leaves
collected in Hanoi-Vietnam in 2013 and 2016, respectively. Bacteria were cultured as described
in Zhou et al. (2015) [28]. Introduction This is an important step for the
future design and implementation of broad-spectrum BLB-resistance in elite rice varieties
using genome editing in Vietnam. Vector construction The Cas9 rice expression vector (pUbi-Cas9) [32] and the sgRNA expression vector (pENTR-
sgRNA) under the control of the OsU6 promoter [33] were used to construct the pCas9/OsS-
WEET14-gRNA expression vector. The complementary oligonucleotides with appropriate
4-bp overhangs were synthesized by Macrogen (Korea). After heat denaturation, the comple-
mentary oligonucleotides (50-gtgtGGTGCTAAGCTCATCAAGCC-3’ and 50-aaacGGCTT
GATGAGCTTAGCACC-3’) were first annealed to each other, phosphorylated, and ligated into
the BsaI-digested vector pENTR-sgRNA. The integrity of the inserted fragment was verified by
sequencing. Subsequently, the sgRNA cassette was cloned into pUbi-Cas9 using the Gateway 3 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0255470
September 9, 2021 PLOS ONE Improved bacterial leaf blight disease resistance in Vietnamese rice cultivar TBR225 Fig 1. CRISPR/Cas9-induced OsSWEET14 promoter modification in TBR225 rice. (A) A region of the OsSWEET14
promoter containing four EBEs (TalC, PthXo3, AvrXa7 and TalF) and putative TATA box from TBR225. The target
site (complementary to the guide RNA) is shown in the box, immediately following the protospacer adjacent motif
(PAM). (B) T-DNA region of the CRISPR/Cas9-mediated genome editing construct carrying OsSWEET14-sgRNA
(indicated by the black box). The expression of Cas9 is driven by the maize ubiquitin promoter (P-Ubi); the expression
of the OsSWEET14-sgRNA is driven by the rice OsU6 promoter (P-OsU6a); the expression of HPT is driven by two
CaMV35S promoters (P-2×35S); T-35S, T-Nos and TTTTTT: Gene terminators; LB and RB: Left and right border,
respectively. (C) Alignment of the OsSWEET14 promoter fragment in the nine T0 transgenic TBR225 rice plants edited
in the AvrXa7, PthXo3 and TalF EBEs. The lines on top of the wild-type sequence represent the binding sites of
AvrXa7, PthXo3 and TalF. The arrow indicates the expected cutting site of the Cas9 complex used in this study. The
labels on the left indicate the name of examined mutant lines; (a1) and (a2) distinguish alleles in the same line. The
numbers on the right indicate the type of mutation and the number of nucleotides involved; (+) and (-) indicate
insertion and deletion, respectively. Fig 1. CRISPR/Cas9-induced OsSWEET14 promoter modification in TBR225 rice. (A) A region of the OsSWEET14
promoter containing four EBEs (TalC, PthXo3, AvrXa7 and TalF) and putative TATA box from TBR225. The target
site (complementary to the guide RNA) is shown in the box, immediately following the protospacer adjacent motif
(PAM). (B) T-DNA region of the CRISPR/Cas9-mediated genome editing construct carrying OsSWEET14-sgRNA
(indicated by the black box). Vector construction The expression of Cas9 is driven by the maize ubiquitin promoter (P-Ubi); the expression
of the OsSWEET14-sgRNA is driven by the rice OsU6 promoter (P-OsU6a); the expression of HPT is driven by two
CaMV35S promoters (P-2×35S); T-35S, T-Nos and TTTTTT: Gene terminators; LB and RB: Left and right border,
respectively. (C) Alignment of the OsSWEET14 promoter fragment in the nine T0 transgenic TBR225 rice plants edited
in the AvrXa7, PthXo3 and TalF EBEs. The lines on top of the wild-type sequence represent the binding sites of
AvrXa7, PthXo3 and TalF. The arrow indicates the expected cutting site of the Cas9 complex used in this study. The
labels on the left indicate the name of examined mutant lines; (a1) and (a2) distinguish alleles in the same line. The
numbers on the right indicate the type of mutation and the number of nucleotides involved; (+) and (-) indicate
insertion and deletion, respectively. https://doi.org/10.1371/journal.pone.0255470.g001 https://doi.org/10.1371/journal.pone.0255470.g001 LR clonase (Life Technologies) (Fig 1B). The resulting construct was confirmed by Sanger
sequencing of the insertion junctions. LR clonase (Life Technologies) (Fig 1B). The resulting construct was confirmed by Sanger
sequencing of the insertion junctions. Agrobacterium-mediated rice transformation The pCas9-OsSWEET14-gRNA was electroporated into Agrobacterium tumefaciens EHA105
and the resulting strain was used to transform rice using the method described by Hiei et al. (1994) [34]. The presence of the transgene in the genome of T0 hygromycin-resistant plants or
segregating T1 individuals was evaluated by PCR using 50-ATGGCCCCAAAGAAGAAG-30 and
50- GCCTCGGCTGTCTCGCCA-30 primers specific for Cas9. T1 individuals were analyzed by
PCR using Cas9, OsSWEET14-gRNA (50- GGATCATGAACCAACG-30 and 50- GAATTCGATAT
CAAGCTT-30) and HPT (5’-AAACTGTGATGGACGACACCGT-3’ and 50- GTGGCGATCCTGC
AAGCTCC -30) specific diagnostic primer pairs together with a positive control pair (50-TTG
CGGCTCATCAGTTTCTC-30 and 50- TGGATCAGATCAAAGGCAAC -30) specific to the OsS-
WEET14 promoter. Bacterial blight inoculation Rice cultivation and disease assays were done according to the methods of Blanvillain-Bau-
fume´ et al. (2017) [15]. Bacteria were cultured in PSA media (10 g/liter peptone, 10 g/liter
sucrose, 1 g/liter glutamic acid, 15 g/liter Bacto Agar) at 28˚C for two days [35] and inoculated
at an optical density (OD600) of 0.5 (infiltrations) or 0.4 (leaf clipping) in water. For lesion
length measurements, at least three inoculated leaves per plant and three plants for each line
were measured 14 days after inoculation (DAI), and scored as follows: high resistance (lesion
length < 8 cm), moderate resistance (lesion length 8–12 cm) and susceptibility (lesion PLOS ONE | https://doi.org/10.1371/journal.pone.0255470
September 9, 2021 4 / 16 PLOS ONE Improved bacterial leaf blight disease resistance in Vietnamese rice cultivar TBR225 length > 12 cm). For gene expression analyses, 4-cm leaf sections infiltrated with bacterial sus-
pensions were collected at 48 h after inoculation for RNA extraction. Experiments included
samples from three pooled biological replicate leaves. The plants inoculated with distilled
water only were used as negative controls. Analysis of OsSWEET14 edited allele sequences To determine the nature of the mutation at the target site, all transgenic T0 or T1 plants were
analyzed by PCR using genomic DNA (50 ng) as a template and OsSWEET14 specific primers
(50-TTGCGGCTCATCAGTTTCTC-30 and 50- TGGATCAGATCAAAGGCAAC -30). The PCR
products were directly sequenced using the Sanger method. The sequencing chromatograms
were decoded using the Degenerate Sequence Decoding method [36] in order to identify the
mutations. Evaluation of major agronomic traits under net-house conditions WT and selected mutant plants were planted under net-house conditions in a randomized pot
design experiment. At maturity, five plants of each line were investigated for the following
agronomic traits: growth duration, plant height, number of tillers per plant, number of grains
per panicle, number of filled grains per panicle and yield (seed mass) per plant. The experi-
ment was repeated three times, so a total of fifteen plants were evaluated for each line. Analysis of potential off-target editing Off-target sequences were predicted with the CCTop tool [31] against the OsSWEET14 pro-
moter sgRNA and the rice Nipponbare genome with default parameters. A total of 18 potential
off-target sequences were identified. Three of them were located in coding regions (S2 Table). These regions were amplified by PCR using the specific primers listed in S2 Table and analyzed
by sequencing. PLOS ONE | https://doi.org/10.1371/journal.pone.0255470
September 9, 2021 Vietnamese Xoo strains induce OsSWEET14 during infection of the
TBR225 rice variety OsSWEET14/Os11N3 was previously identified as a susceptibility gene for Xoo strains relying
on either of the AvrXa7, PthXo3, TalF (formerly Tal5) or TalC TALEs for infection of the rice
cultivars Nipponbare and Kitaake [11]. Because Xoo strains tend to frequently target this gene,
we first sequenced a region of the OsSWEET14 promoter from rice cultivar TBR225 to exam-
ine if it also carries documented target EBEs. Based on the Nipponbare genome sequence in
database (AP014967.1), the region encompassing 1343 bp sequence upstream and 52 bp
sequence downstream of the predicted transcription start site of OsSWEET14 gene from
TRB225 rice cultivar was PCR amplified and sequenced (S1 Fig). The promoter region includ-
ing the putative TATA box (TATAAA) and the AvrXa7, PthXo3, TalF/Tal5 and TalC EBEs
(Fig 1A), located 319 bp to 216 bp upstream of the ATG initiation codon, showed 100% iden-
tity to the Nipponbare sequence. This therefore implied that in principle, the TBR225 OsS-
WEET14 promoter can be recognized by characterized major Xoo TALEs. As shown in Fig 2A, we also challenged TBR225 plants with two Vietnamese Xoo strains
VXO_11 and VXO_15, both originating from the Hanoi province, using leaf clipping assays. We consistently obtained typical extended disease lesions 14 days after inoculation (25.5 cm
and 26.6 cm average lesions length for VXO_11 and VXO_15, respectively in the experiment
of S2 Fig), indicating that the TBR225 variety is susceptible to BLB. PLOS ONE | https://doi.org/10.1371/journal.pone.0255470
September 9, 2021 5 / 16 PLOS ONE Improved bacterial leaf blight disease resistance in Vietnamese rice cultivar TBR225 Fig 2. OsSWEET14 is likely a susceptibility gene for Vietnamese Xoo strains in rice cultivar TBR225. (A) Representative images of
the disease lesions obtained 14 days after leaf clipping inoculation of TBR225 rice leaves with Vietnamese Xoo strains VXO_11 and
VXO_15 or with water (CT). The chevrons above the leaves indicate the maximum visible extent of lesions away from the inoculation
point on the left (B). OsSWEET14 expression pattern obtained by RT-PCR two day post-infiltration of TBR225 rice leaves with
Vietnamese Xoo strains. CT Plants were inoculated with water only. The experiment was repeated three times. https://doi org/10 1371/journal pone 0255470 g002 Fig 2. OsSWEET14 is likely a susceptibility gene for Vietnamese Xoo strains in rice cultivar TBR225. Vietnamese Xoo strains induce OsSWEET14 during infection of the
TBR225 rice variety (A) Representative images of
the disease lesions obtained 14 days after leaf clipping inoculation of TBR225 rice leaves with Vietnamese Xoo strains VXO_11 and
VXO_15 or with water (CT). The chevrons above the leaves indicate the maximum visible extent of lesions away from the inoculation
point on the left (B). OsSWEET14 expression pattern obtained by RT-PCR two day post-infiltration of TBR225 rice leaves with
Vietnamese Xoo strains. CT Plants were inoculated with water only. The experiment was repeated three times. https://doi.org/10.1371/journal.pone.0255470.g002 https://doi.org/10.1371/journal.pone.0255470.g002 To test if OsSWEET14 is a potential direct virulence target of Vietnamese Xoo strains, we
infiltrated TBR225 rice leaves with the two Vietnamese Xoo strains. Forty-eight hours post
infiltration, TBR225 plants inoculated with VXO strains displayed a strong induction of OsS-
WEET14 relative to water controls (Fig 2B). These results suggest that OsSWEET14 is a tran-
scriptional target of VXO strains and that it may act as a susceptibility gene in TBR225. CRISPR/Cas9 design for OsSWEET14 promoter editing Our main objective was to engineer resistance to BLB caused by Vietnamese Xoo strains. To
this end, we subsequently sought to specifically modify the OsSWEET14 promoter in TBR225
rice with CRISPR/Cas9-mediated editing. Previous work revealed that while African Xoo
strains rely on TalC and occasionally, TalF, all Asian Xoo strains use either PthXo3- or Avr-
Xa7-like TALEs to activate OsSWEET14 [11]. Because the talC gene is currently exclusively
found in African strains, we reasoned that it is unlikely that Vietnamese strains carry a talC
copy. Thus, to maximize our chances to perturb all remaining documented EBEs, we selected
a 20-bp nucleotide target site overlapping the PthXo3, AvrXa7 and TalF EBEs and having a
predicted cut site located near the 3’-end of the AvrXa7 EBE (Fig 1A). The recombinant binary
plasmid pCas9/OsSWEET14-gRNA for CRISPR/Cas9 mediated editing of OsSWEET14 was
transformed into the rice variety TBR225 via Agrobacterium-mediated transformation (S1
Table). A total of nine TBR225 transformants were selected from 10 independent PCR-vali-
dated transgenic T0 TBR225 plants to further investigate CRISPR/Cas9-targeted mutagenesis
of the OsSWEET14 promoter. In order to decipher the nature of the editing events in OsS-
WEET14, the promoter sequencing data of transgenic lines were analyzed using the Degener-
ate Sequence Decoding software [36]. All 9 T0 transgenic plants harbored at least an editing
event (Fig 1C): two were heterozygous mutant/wild type, two had homozygous mutations, and
five had bi-allelic mutations. Regarding the type of mutations, 66.7% were nucleotide dele-
tions, 11.1% of the mutations were nucleotide insertions and no substitution was detected
(Table 1). Inheritance of CRISPR/Cas9-induced mutations in the T1 generation To assess the inheritance of the CRISPR/Cas9-induced OsSWEET14 mutations in the next
generation, all T0 mutant transgenic plants (Fig 1C) were allowed to self-pollinate, and T1
transgenic plants were randomly selected in the progeny of T0 plants for sequencing and PLOS ONE | https://doi.org/10.1371/journal.pone.0255470
September 9, 2021 6 / 16 PLOS ONE Improved bacterial leaf blight disease resistance in Vietnamese rice cultivar TBR225 analysis of their edited site (Table 2). All T1 individuals derived from T0 plants previously gen-
otyped as homozygous possessed the same allele as their parent, suggesting stable inheritance
Table 1. Frequencies of mutant genotypes and target mutation types in T0 transgenic plants. Mutant genotype ratiosa (%)
Mutation type ratiosb (%)
Heterozygote
Homozygote
Bi-allelic
Deletion
Insertion
Substitution
22.2 (2/9)
22.2 (2/9)
55.6 (5/9)
66.7 (12/18)
11.1 (2/18)
0 (0/18)
a (Number of on-target mutant genotype/total number of on-target mutant genotypes) x 100%. b (Number of allele mutation type/number of all allele mutation types) x 100%. https://doi.org/10.1371/journal.pone.0255470.t001 Table 1. Frequencies of mutant genotypes and target mutation types in T0 transgenic plants. Mutant genotype ratiosa (%)
Mutation type ratiosb (%)
Heterozygote
Homozygote
Bi-allelic
Deletion
Insertion
Substitution
22.2 (2/9)
22.2 (2/9)
55.6 (5/9)
66.7 (12/18)
11.1 (2/18)
0 (0/18)
a (Number of on-target mutant genotype/total number of on-target mutant genotypes) x 100%. b (Number of allele mutation type/number of all allele mutation types) x 100%. https //doi org/10 1371/journal pone 0255470 t001 Table 1. Frequencies of mutant genotypes and target mutation types in T0 transgenic plants. a (Number of on-target mutant genotype/total number of on-target mutant genotypes) x 100%. b (Number of allele mutation type/number of all allele mutation types) x 100%. analysis of their edited site (Table 2). All T1 individuals derived from T0 plants previously gen-
otyped as homozygous possessed the same allele as their parent, suggesting stable inheritance
of the mutations to the next generation. Similarly, the T1 progeny of each of both bi-allelic and
heterozygous mutation T0 lines showed a segregation ratio which is consistent with Mendelian
segregation (χ2 < χ2
0.05, 2 = 5.99), indicating that the CRISPR/Cas9-induced mutations in T0
plants were transmitted as expected to the next generation. Interestingly, no new mutant allele
was detected in the T1 generation of both heterozygous mutants L-21 and L-27, even though
most of them still carried the transgene. Inheritance of CRISPR/Cas9-induced mutations in the T1 generation Overall, consistent with previous similar studies, our
results indicate that the CRISPR/Cas9-mediated mutations generated here are stably transmit-
ted to the next generation in a Medelian fashion. “+” and “-” indicate respectively, insertion and deletion, of the indicated number of nucleotides.
“w”, wild type. yp
Number of homozygous mutant plants without T-DNA. and “-” indicate respectively, insertion and deletion, of the indicated number of nucleotides.
, wild type. https://doi.org/10.1371/journal.pone.0255470.t002 w , wild type.
Number of homozygous mutant plants without T-DNA. yp
Number of homozygous mutant plants without T-DNA. “+” and “-” indicate respectively, insertion and deletion, of the indicated number of nucleotides.
“w”, wild type. ectively, insertion and deletion, of the indicated number of nucleotides. n and deletion, of the indicated number of nucleotides. pe.
homozygous mutant plants without T-DNA. “+” and “-” indicate respectively, insertion and deletion, of the indicated number of nucleotides. “+” and “-” indicate respectively, insertion and deletion, of the indicated number of nucleotides.
“ ”
ild t Xoo To characterize the BLB-resistance phenotype of the generated rice mutants, three T-DNA-
free, homozygous TBR225 edited lines, namely, L-5.7(-6), L-31.12(-3) and L-15.4(+1) with
OsSWEET14 promoter alleles corresponding respectively to L-5-a1 (6bp deletion), L-31
(3bp deletion) and L-15 (1bp insertion) in Fig 1C, were established. Selected T1 individuals
were propagated to obtain T2 seeds which were used to perform BLB susceptibility assays. Edited T2 and WT TBR225 plants were inoculated by leaf-clipping with the VXO_11 and
VXO_15 strains at the eight-week stage. The inoculated leaves of wild type TBR225 plants
and of edited lines L-15.4(+1) and L-31.12(-3) developed long water-soaked lesions typical
of BLB, ranging from 18.3 cm to 29.0 cm in length. In contrast, the edited line L-5.7(-6),
harboring a longer 6-bp deletion at the target site, displayed high (1.2 cm average lesion
length) and moderate (7.3 cm average lesion length) resistance to VXO_11 and VXO_15
strains, respectively (Fig 3). Means comparisons with a Tukey’s HSD test further indicated
that irrespective of the inoculated strain, the mean lesion lengths measured on the L-15.4
(+1), L-31.12(-3) or wild type lines were not significantly different. In contrast, the mean
lesion lengths recorded on the L-5.7(-6) mutant line were significantly different from those
obtained on the wild type and the two other edited lines challenged with either of the Viet-
namese strains (Fig 3B). Furthermore, our off-target editing analysis on line L-5.7(-6) did
not reveal unintended modifications of other annotated rice loci (S2 Table and S5 Fig), indi-
cating that the 6-bp deletion in the OsSWEET14 promoter is probably responsible for this
phenotype. Consistent with disease assays and as shown in Fig 3C, whereas a semiquantitative RT-PCR
signal for OsSWEET14 expression was detected on the parental variety and the L-15.4(+1) and
L-31.12(-3) edited lines following VXO_11 and VXO_15 infiltration, this amplicon was unde-
tectable in the resistant L-5.7(-6) line. In conclusion, this data shows that the 6-bp deletion in the AvrXa7/PthXo3 EBE reduces
dramatically OsSWEET14 expression following VXO strains inoculation and confers resis-
tance to these strains. In contrast, shorter modifications on the 3’-end of this EBE are insuffi-
cient to perturb OsSWEET14 expression after inoculation and do not confer detectable
protection against the corresponding strains. Xoo Finally, while these results strongly support the
view that OsSWEET14 functions as a unique susceptibility gene in the interaction between
strain VXO_11 and the TBR225 rice variety, the resistance to strain VXO_15 is not as dramatic
and may suggest that other mechanisms partially counteract the effects of the AvrXa7/PthXo3
EBE 6-bp deletion in edited TBR225 plants. Selection of transgene-free mutant TBR225 rice lines To identify T-DNA free T1 rice plants containing a mutation in EBEs of the OsSWEET14 pro-
moter, PCR analysis was carried out using primers specific to Cas9, sgRNA and HPT
sequences (Table 2). A T1 individual was considered devoid of the transgene if the control
amplification of the OsSWEET14 promoter was successful and if none of the PCR reactions
with independent primer pairs designed on the T-DNA produced a detectable diagnostic
band. The results of this PCR screen show that the T-DNA could be segregated out in the prog-
eny of most T0 lines, with 88.9% of the T0 lines generating T-DNA-free progeny. In total, 44 of
221 analyzed edited T1 plants did not generate a specific amplicon from the T-DNA construct
and 15 of them were homozygous mutant harboring the desired OsSWEET14 modifications. Our results demonstrate that transgene-free, homozygous mutant individuals could be
obtained in the segregating progeny of selfed T0 individuals. Table 2. Transmission of CRISPR/Cas9 editing events to the T1 generation. T0 plant
Genotype
Allele(s)
No. of T1 plants tested
Mutation inheritance in the T1 generation
No. of T-DNA-free plants
Alleles segregation
χ2 (1:2:1)
L-4
Bi-allelic
-5/-3
32
10 (-5), 18 (-5/-3), 4 (-3)
2,750
5 (2)
L-5
Bi-allelic
-6/+1
44
9 (-6), 22 (-6/+1), 13 (+1)
0,727
10 (2)
L-7
Bi-allelic
-4/-3
38
14 (-4), 17 (-4/-3), 7 (-3)
3,000
11 (4)
L-15
Homozygote
+1
5
5 (+1)
-
1 (1)
L-21
Heterozygote
-3
26
3 (-3), 13 (-3/wt), 10 (wt)
3,769
7 (1)
L-27
Bi-allelic
-5/-4
7
1 (-5), 3(-5/-4), 3 (-4)
1,286
0
L-29
Heterozygote
-5
33
6 (-5), 19 (-5/wt), 8 (wt)
1,000
2 (0)
L-31
Homozygote
-3
15
15 (-3)
-
5 (5)
L-54
Bi-allelic
-3/-2
21
3 (-3), 12 (-3/-2), 6(-2)
1,286
3 (0) Table 2. Transmission of CRISPR/Cas9 editing events to the T1 generation. PLOS ONE | https://doi.org/10.1371/journal.pone.0255470
September 9, 2021 7 / 16 PLOS ONE Improved bacterial leaf blight disease resistance in Vietnamese rice cultivar TBR225 TBR225 OsSWEET14 promoter edited lines agronomic performances are
undistinguishable from the parental variety To determine if mutations in the OsSWEET14 promoter affect agronomic traits of TRB225
rice plants, three independent homozygous mutant lines were analyzed by measuring their
growth duration, plant height, number of tillers per plant, number of grains per panicle, num-
ber of filled grains per panicle, yield per plant and amylose content under net-house conditions
(see picture of S3 Fig). ANOVA tests and Student’s t tests showed that the mutant lines dis-
played no significant difference to TBR225, in terms of the examined agronomic traits, under
our net-house conditions (Table 3). These results suggest that the tested CRISPR/Cas9-in-
duced mutations in the OsSWEET14 promoter did not negatively impact the main agronomic
traits of TBR225. 8 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0255470
September 9, 2021 PLOS ONE Improved bacterial leaf blight disease resistance in Vietnamese rice cultivar TBR225 Fig 3. BLB resistance assays for homozygous mutant rice lines L-5.7(-6), L-15.4(+1) and L-31.12(-3). (A) Leaves were photographed 14 days post-leaf
clipping inoculation of Xoo strains VXO_11 and VXO_15; arrow heads indicate the end of the lesion. (B) Mean lesion lengths (bars) and standard deviations
(error bars). Values were measured 14 days post-leaf clipping inoculation of two Xoo strains VXO_11 and VXO_15 and were computed from at least three
leaves from each of three plants. Asterisks indicate significant differences relative to wild type plants (Tukey’s HSD test; P < 0.05). The number in the
parentheses following the line name indicates the type of mutation and the number of nucleotides involved. The letters above strain labels indicate
susceptibility score (R—high resistance; M–moderate resistance; S—susceptibility). The experiment was repeated three times. (C) OsSWEET14 expression
pattern obtained by RT-PCR two day post-infiltration of genome edited homozygous mutant rice lines L-31.12(-3), L-15.4(+1) and L-5.7(-6) and parental
TBR225 rice leaves with Vietnamese Xoo strains. This experiment was repeated two times with similar results. S ONE
Improved bacterial leaf blight disease resistance in Vietnamese rice cultivar TBR225 Fig 3. BLB resistance assays for homozygous mutant rice lines L-5.7(-6), L-15.4(+1) and L-31.12(-3). (A) Leaves were photographed 14 days post-leaf
clipping inoculation of Xoo strains VXO_11 and VXO_15; arrow heads indicate the end of the lesion. (B) Mean lesion lengths (bars) and standard deviations
(error bars). Values were measured 14 days post-leaf clipping inoculation of two Xoo strains VXO_11 and VXO_15 and were computed from at least three
leaves from each of three plants. TBR225 OsSWEET14 promoter edited lines agronomic performances are
undistinguishable from the parental variety Asterisks indicate significant differences relative to wild type plants (Tukey’s HSD test; P < 0.05). The number in the
parentheses following the line name indicates the type of mutation and the number of nucleotides involved. The letters above strain labels indicate
susceptibility score (R—high resistance; M–moderate resistance; S—susceptibility). The experiment was repeated three times. (C) OsSWEET14 expression
pattern obtained by RT-PCR two day post-infiltration of genome edited homozygous mutant rice lines L-31.12(-3), L-15.4(+1) and L-5.7(-6) and parental
TBR225 rice leaves with Vietnamese Xoo strains. This experiment was repeated two times with similar results. https://doi.org/10.1371/journal.pone.0255470.g003 PLOS ONE PLOS ONE Table 3. Agronomic traits evaluation of homozygous T2 mutant lines. Lines
Growth duration (day) Plant height (cm) No. of tillers per plant No. of grains per panicle No. of filled grains per panicle Amylose content (%)
WT
108.4 ± 1.1a
86.6 ± 3.2a
5 ± 0.7a
144.4 ± 4.9a
125 ± 4.5a
13.2 ± 0.38a
L-5.7(-6)
108 ± 1.2a
86.4 ± 4.3a
5.2 ± 0.4a
144.2 ± 4.4a
123.4 ± 5.5a
13.7 ± 0.35a
L-15.4(+1)
107.8 ± 0.8a
86.4 ± 5.0a
4.8 ± 0.4a
147.8 ± 5.1a
121.8 ± 3.0a
13.5 ± 0.41a
L-31.12
(-3)
108 ± 1.2a
88.4 ± 4.3a
5.4 ± 0.5a
144.6 ± 5.3a
124.2 ± 7.4a
13.8 ± 0.21a
Five plants per line were measured. Experiments were repeated three time. Means followed by the same letter do not differ significantly (P < 0.05). https://doi.org/10.1371/journal.pone.0255470.t003 Table 3. Agronomic traits evaluation of homozygous T2 mutant lines. Five plants per line were measured. Experiments were repeated three time. Means followed by the same letter do not differ significantly (P < 0.05). https://doi.org/10.1371/journal.pone.0255470.t003 improvement of agriculturally relevant elite rice cultivars for pertinent traits using the
CRISPR/Cas9 technology (see for example [38–42]) is gradually increasing but is still limited. improvement of agriculturally relevant elite rice cultivars for pertinent traits using the
CRISPR/Cas9 technology (see for example [38–42]) is gradually increasing but is still limited. TBR225 [27], a major commercial rice variety cultivated in large areas of Northern Viet-
nam, has the advantages of early maturity, high and stable yield, as well as cooking quality. However, it is very susceptible to BLB. Here, the CRISPR/Cas9-mediated editing method was
applied in order to rapidly improve the BLB resistance of TBR225 by modifying the AvrXa7,
PthXo3 and TalF EBEs on the promoter of OsSWEET14. Of the three generated homozygous
mutant lines tested for resistance, the one carrying the largest deletion at the target site (6 bp)
showed a significantly improved resistance to infection with two Xoo strains VXO_11 and
VXO_15. Therefore, using the major commercial rice variety TBR225 as an example, we illus-
trate the advantages of CRISPR/Cas9 tool for rice breeding. In the present study, the frequency of individuals with CRISPR/Cas9-induced mutations in
T0 transgenic plants was 90%, which is similar to previous observation [33]. We obtained only
two heterozygous mutant/wild type lines versus seven homozygous or bi-allelic mutant lines. PLOS ONE | https://doi.org/10.1371/journal.pone.0255470
September 9, 2021 Discussion Recently, the CRISPR/Cas9 system has emerged as a powerful tool for gene editing in many
organisms including plants. Because of its specificity and efficiency, this system has been
widely used to improve important agronomic traits of major crops such as rape, tomato, soy-
bean, rice, wheat and maize [37]. Excluding easy-to-transform reference accessions such as
Nipponbare and Kitaake that are widely used in the laboratory, the number of reports on the PLOS ONE | https://doi.org/10.1371/journal.pone.0255470
September 9, 2021 PLOS ONE | https://doi.org/10.1371/journal.pone.0255470
September 9, 2021 9 / 16 Improved bacterial leaf blight disease resistance in Vietnamese rice cultivar TBR225 PLOS ONE [11] who studied 30 combinations of EBE
mutations in the OsSWEET11, OsSWEET13 and OsSWEET14 promoters of the IR64 or Ciher-
ang-Sub1 varieties and detected only a single line with abnormal agronomic traits. Some individual Xoo strains have evolved a set of distinct TALE effectors that collectively
target several members of the clade III SWEET family. The presence of these redundant
TALEs thereby trumps single “loss-of-tale-responsiveness” resistance alleles [11,12,17,47]. For
example, Kitaake lines carrying TALEN-induced mutation in the SWEET14 promoter [13,15]
exhibit resistance to strains which depend exclusively on matching AvrXa7/PthXo3 for clade
III SWEET family induction. Likewise, the natural xa13 allele [48] or CRISPR/Cas9-induced
mutation in the SWEET11 promoter [11] exhibit resistance to strains such as PXO99 which
depend exclusively on PthXo1, for virulence. However, the BLB resistance of the Kitaake lines
harboring mutations in both AvrXa7/PthXo3 (OsSWEET14) and PthXo1 (OsSWEET11) EBEs
was defeated by Xoo strains expressing simultaneously the AvrXa7/PthXo3 and PthXo2B
TALEs [11]. Recently, the stacking of EBE-edited alleles in several OsSWEET promoters have
overcome this limitation and was shown to achieve a broad spectrum of resistance to strains
from most BLB-prone countries in Asia [11,12]. All of the three T2 lines tested for BLB resistance were affected for the AvrXa7/PthXo3 EBE
and conserved an otherwise wild type TalF EBE (Fig 1C). The homozygous mutant TBR225
line L-5.7(-6) carrying a 6-bp deletion in the AvrXa7/PthXo3 EBE exhibited a significantly
enhanced resistance to two Vietnamese Xoo strains compared to WT TBR225. The L-15.4(+1)
and L-31.12(-3) lines that harbored more subtle alterations in the 3’-end of this EBE (a 1-bp
insertion and a 3-bp deletion, respectively) in contrast remained susceptible to VXO strains. Our OsSWEET14 expression analysis after Vietnamese Xoo strains inoculation (Fig 1C) sug-
gests that these editing events did not alter the EBE sequence sufficiently to compromise pro-
moter recognition by an AvrXa7/PthXo3-like Vietnamese TALE. With less than 2 cm average
lesion length, the resistance of line L-5.7(-6) (6-bp deletion) to the VXO_11 strain is rather
extreme (versus average lesion length of 20.1 cm on wild type plants). Moreover, in this line,
OsSWEET14 expression following bacterial inoculation is strongly reduced relative the paren-
tal line and the two other edited lines, which suggest that in this case, recognition by an
AvrXa7/PthXo3-like Vietnamese TALE is abrogated. PLOS ONE This high frequency of mutated alleles is another proof that the CRISPR/Cas9 system is indeed
an efficient tool for gene editing in plant. We also observed the stable transmission of edited
alleles to subsequent generations. This is a common phenomenon that has been repeatedly
documented for rice plants carrying CRISPR/Cas9-induced mutations [38,40,43,44]. In this
study, we obtained only two types of induced mutations in T0 plants: insertion (11.1%) and
deletion (66.7%), but no substitution were observed. In some earlier studies, new mutations
were continuously obtained in the T1 offspring of heterozygous T0 mutants because the Cas9
complex remains active on edited targets until the seed or PAM regions cease to be functional
[35,37,43]. In contrast, here, all the T1 plants generated from both heterozygous lines L-21 and
L-29, regardless of whether they had a CRISPR/Cas9 T-DNA transgene integrated in their
genome, did not show any new mutation. We could also readily obtain transgene-free plants
from most of the T1 segregation populations without any laborious crossing or backcrossing
steps, which illustrates an advantage of the CRISPR/Cas9 technology compared to conven-
tional breeding. Clade III SWEET family proteins are involved in a number of biological processes such as
seed and pollen development or pathogen susceptibility [45]. Their inactivation has previously
been shown to cause pleiotropic and/or detrimental effects. For example, both ossweet11 single
and ossweet11-ossweet15 double Kitaake rice mutants showed defects in endosperm develop-
ment and filling [46]. In addition, RNA-mediated silencing of either Os11N3/OsSWEET14 [7]
or Os8N3/OsSWEET11 [6] in BLB resistant Kitaake lines causes negative effects on seed pro-
duction. In contrast, here, we show that T-DNA-free TBR225 plants harboring homozygous
mutations generated with the CRISPR/Cas9 system in the AvrXa7/PthXo3 EBE of the OsS-
WEET14 promoter exhibited enhanced Xoo resistance but did not show any significant PLOS ONE | https://doi.org/10.1371/journal.pone.0255470
September 9, 2021 10 / 16 PLOS ONE Improved bacterial leaf blight disease resistance in Vietnamese rice cultivar TBR225 difference in all examined agronomic traits compared to wild-type plants under net-house
growth conditions. It is conceivable that limited modifications in promoter regions do not
affect the normal expression of SWEET genes in contrast to KO or silenced lines. Our findings
are consistent with the previous work of Oliva et al. PLOS ONE | https://doi.org/10.1371/journal.pone.0255470
September 9, 2021 PLOS ONE Consistent with OsSWEET14 expression
analysis and as shown in S4 Fig, the Talvez [49] target prediction scores for AvrXa7 and
PthXo3 on the OsSWEET14 promoter L-5-a1 allele sequence of line L-5.7(-6) are markedly
lower than on the wild type promoter sequence. This is not the case however for the edited
alleles carried by lines L-15.4(+1) and L-31.12(-3) (respectively L-15 and L-31 in S4 Fig) whose
Talvez scores are identical or slightly lower than those of the wild type promoter sequence. The magnitude of the effect of the 6-bp deletion allele on susceptibility to VXO_11 is com-
parable to the dramatic effect of previously characterized alterations of the same EBEs in the
Kitaake background against the PXO86 strain that possesses a single TALE, AvrXa7, targeting
OsSWEET14 for clade III OsSWEET gene induction [15]. By analogy, this suggests that OsS-
WEET14 is also the only clade III OsSWEETs target of VXO_11 in the TBR225 background
but, in order to confirm this hypothesis an examination of other clade III OsSWEET genes
expression patterns in response to this strain would be required. The situation with the
VXO_15 strain is not as straightforward to interpret and will require further investigations. Although the 6-bp deletion in the AvrXa7/PthXo3 EBE did provide an increased resistance to
the edited plants, the VXO_15 strain caused intermediate disease severity (7.3 cm average
lesion length on Fig 3). This incomplete resistance is unlikely to result from the partial but still PLOS ONE | https://doi.org/10.1371/journal.pone.0255470
September 9, 2021 11 / 16 PLOS ONE Improved bacterial leaf blight disease resistance in Vietnamese rice cultivar TBR225 productive recognition of subsequences of the altered EBE by a VXO_15 AvrXa7/PthXo3-like
TALE because OsSWEET14 expression is similarly decreased in response to either this strain
or VXO_11 (Fig 3C). Alternatively, contrary to all Asian Xoo examined so far, but similar to
African Xoo [11,15], VXO_15 may have the intrinsic potential to cause disease in the absence
of clade III OsSWEET gene induction, a phenomenon that seems to be dependent on the
edited rice variety genetic background [42]. More likely, analogous to other Asian strains,
VXO_15 may encode alternative TALEs, such as PthXo2B or PthXo1 that compensate the loss
of OsSWEET14 induction by targeting other clade III OsSWEET genes. S1 Fig. Nucleotide sequence of the OsSEET14 promoter in TBR225.
(TIF) S2 Fig. Virulence of Vietnamese Xoo strains VXO_11 and VXO_15 on TBR225 rice. Grey
points correspond to individual lesion length measurements while the black points indicate
the calculated average value. The line range represents standard deviation. (TIF) PLOS ONE In this regard, deci-
phering clade III OsSWEET genes expression patterns in combination with long read genome
sequencing will ultimately help describe TALEs variability in Vietnamese Xoo strains and its
functional impact on OsSWEET genes induction. In conclusion, we showed that editing specific EBEs of Xoo TALEs via CRISPR/Cas9 tool is
an efficient method for improving BLB resistance of elite rice varieties such as TBR225 without
detectable yield penalties. This also uncovered the potential diversity of TALEs in Vietnamese
Xoo population, which will thus require future investigations to address the TALE repertoires
of Vietnamese Xoo strains in order to generate broad-spectrum BLB-resistant rice varieties in
Vietnam. S3 Fig. Picture of an individual plant from the homozygous mutant rice lines L-5.7(-6).
(TIF) S4 Fig. Talvez scoring of AvrXa7, PthXo3 and TalF target EBES in the edited OsSWEET14
promoter allele sequences. Score values are represented both by the length of the horizontal
bar and a fill color scale. Higher Talvez prediction scores reflect a better match between a pre-
dicted EBE and the sequence of RVD of the query TALE. (TIF) S5 Fig. Amplicon sequencing of predicted off-target sites for the OsSWEET14 promoter-
sgRNA in annotated exons of the TBR225 edited line L-5.7(-6). Potential unintended target
sequences including the PAM are highlighted in boxes. They are all identical to the expected
wild type Nipponbare sequences. (TIF) S1 Table. Key figures on the TBR225 transformation procedure for OsSWEET14 promoter
editing.
(DOCX) S1 Table. Key figures on the TBR225 transformation procedure for OsSWEET14 promoter
editing. (DOCX) S2 Table. Output of the CCTop tool used with the OsSWEET14 promoter sgRNA for off-
target prediction on the rice Nipponbare genome. (DOCX) S1 Raw images. Original photograph used in Fig 2 for the RT-PCR gels panel. (DOCX) 12 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0255470
September 9, 2021 PLOS ONE Improved bacterial leaf blight disease resistance in Vietnamese rice cultivar TBR225 Author Contributions Conceptualization: Phuong Nguyen Duy, Sebastien Cunnac, Xuan Hoi Pham. Conceptualization: Phuong Nguyen Duy, Sebastien Cunnac, Xuan Hoi Pham. Formal analysis: Phuong Nguyen Duy, Dai Tran Lan, Sebastien Cunnac, Xuan Hoi Pham. Funding acquisition: Phuong Nguyen Duy, Tran Bao Manh, Xuan Hoi Pham. Investigation: Phuong Nguyen Duy, Dai Tran Lan, Hang Pham Thu, Huong Phung Thi Thu,
Ha Nguyen Thanh, Ngoc Phuong Pham, Florence Auguy, Sebastien Cunnac. Methodology: Phuong Nguyen Duy, Dai Tran Lan, Hang Pham Thu, Ha Nguyen Thanh,
Sebastien Cunnac, Xuan Hoi Pham. Project administration: Xuan Hoi Pham. Project administration: Xuan Hoi Pham. Resources: Hang Pham Thu, Ha Nguyen Thanh, Florence Auguy. Resources: Hang Pham Thu, Ha Nguyen Thanh, Florence Auguy. Supervision: Sebastien Cunnac, Xuan Hoi Pham. Validation: Sebastien Cunnac, Xuan Hoi Pham. Writing – original draft: Phuong Nguyen Duy, Dai Tran Lan. Writing – review & editing: Huong Bui Thi Thu, Tran Bao Manh, Sebastien Cunnac, Xuan
Hoi Pham. Acknowledgments We are grateful to Msc. Pham Thi Van, Dr. Cao Le Quyen and Dr. Nguyen Van Cuu from the
Institute of Agricultural Genetics for rice transformation experiments, Msc. Nguyen Thi Thu
Ha from the Institute of Agricultural Genetics for managing the Xoo strains collection and
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Relationship Between Epicardial Fat volume on Cardiac CT and Atherosclerosis Severity in Three-Vessel Coronary Artery Disease: A Single-Center Cross-Sectional Study
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Research Square (Research Square)
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Sun et al. BMC Cardiovascular Disorders (2022) 22:76
https://doi.org/10.1186/s12872-022-02527-7 Sun et al. BMC Cardiovascular Disorders (2022) 22:76
https://doi.org/10.1186/s12872-022-02527-7 Open Access Relationship between epicardial fat volume
on cardiac CT and atherosclerosis severity
in three‑vessel coronary artery disease:
a single‑center cross‑sectional study Yu Sun1,2,3, Xiao‑gang Li2,3, Kai Xu4, Jie Hou1,2,3, Hong‑rui You2, Rong‑rong Zhang2, Miao Qi1,2,3, Li‑bo Zhang2,3,
Li‑sheng Xu1, Stephen E. Greenwald5 and Ben‑qiang Yang2,3* Abstract Background: The ideal treatment strategy for stable three-vessel coronary artery disease (CAD) patients are difficult
to determine and for patients undergoing conservative treatment, imaging evidence of coronary atherosclerotic
severity progression remains limited. Epicardial fat volume (EFV) on coronary CT angiography (CCTA) has been con‑
sidered to be associated with coronary atherosclerosis. Therefore, this study aims to evaluate the relationship between
EFV level and coronary atherosclerosis severity in three-vessel CAD. Methods: This retrospective study enrolled 252 consecutive patients with three-vessel CAD and 252 normal control
group participants who underwent CCTA between January 2018 and December 2019. A semi-automatic method
was developed for EFV quantification on CCTA images, standardized by body surface area. Coronary atherosclerosis
severity was evaluated and scored by the number of coronary arteries with ≥ 50% stenosis on coronary angiography. Patients were subdivided into groups on the basis of lesion severity: mild (score = 3 vessels, n = 85), moderate (3.5
vessels ≤ score < 4 vessels, n = 82), and severe (4 vessels ≤ score ≤ 7 vessels, n = 85). The independent sample t-test,
analysis of variance, and logistic regression analysis were used to evaluate the associations between EFV level and
severity of coronary atherosclerosis. Results: Compared with normal controls, three-vessel CAD patients had significantly higher EFV level (65 ± 22 mL/
m2 vs. 48 ± 19 mL/m2; P < 0.001). In patients with three-vessel CAD, there was a progressive decline in EFV level as the
score of coronary atherosclerosis severity increased, especially in those patients with a body mass index (BMI) ≥ 25 kg/
m2 (75 ± 21 mL/m2 vs. 72 ± 22 mL/m2 vs. 62 ± 17 mL/m2; P < 0.05). Multivariable regression analysis showed that
both BMI (OR 3.40, 95% CI 2.00–5.78, P < 0.001) and the score of coronary atherosclerosis severity (OR 0.49, 95% CI
0.26–0.93, P < 0.05) were independently related to the change of EFV level. Conclusion: Three-vessel CAD patients do have higher EFV level than the normal controls. While, there may be an
inverse relationship between EFV level and the severity of coronary atherosclerosis in patients with three-vessel CAD. *Correspondence: bqyang888@sina.com
2 Department of Radiology, General Hospital of Northern Theater
Command, 83 Wenhua RoadLiaoning Province, Shenyang 110016,
People’s Republic of China
Full list of author information is available at the end of the article Background three-vessel CAD were initially included. Patients with
previous percutaneous coronary intervention (n = 147)
or coronary artery bypass grafting (n = 297), pericardial
effusion (n = 38), coronary anomalies (n = 187), malig-
nant chest tumor (n = 12), or systemic autoimmune
disease (n = 3) were excluded. A further 418 patients
without invasive coronary angiography (ICA) data were
excluded. Patients with ICA-confirmed one- (n = 39) and
two-vessel (n = 215) CAD were also excluded. Finally, 252
consecutive patients with ICA-confirmed three-vessel
CAD were enrolled (Fig. 1). The time interval between
CCTA imaging and ICA operating of all patients was
(37 ± 15) days. Three-vessel CAD was defined as ≥ 50%
luminal diameter stenosis present in three main epicar-
dial coronary arteries on the ICA data, including the
left anterior descending artery, left circumflex, and right
coronary artery, with or without the left main artery
involvement. Patients suffering from three-vessel coronary artery dis-
ease (CAD) often have long-standing and complex coro-
nary atherosclerosis and are at an increased risk of adverse
cardiovascular events [1]. Many randomized clinical tri-
als results have revealed no significant differences in sur-
vival benefit between the two most widely used treatment,
namely percutaneous coronary intervention and coronary
artery bypass grafting, for the therapy of multivessel CAD
patients [2–4]. Recently, another multicenter randomized
trial reported that, in stable CAD, there was no evidence
that an initial invasive strategy reduced the risk of adverse
cardiovascular events in comparison to medical therapy
alone [5]. Therefore, it is difficult to make the best choice
of treatment strategy for stable three-vessel CAD patients. For patients who have undergone initial conservative treat-
ment, evaluation of coronary atherosclerotic severity and
assessment of its progression are in great demand, as an
important aid in deciding further treatment, such as adjust-
ment of drug dose or the ideal timing of invasive strategies. For the normal control group, 252 age-, gender- and
body mass index (BMI)-matched subjects were selected. They underwent CCTA because of chest pain from Janu-
ary 2018 to December 2019 and all had negative coronary
atherosclerosis and no known cardiovascular disease. Epicardial adipose tissue is a type of visceral fat that is
located between the surface of the myocardium and the
visceral pericardial layer. Background By secreting a large variety of bio-
active molecules, it can modulate vascular inflammation
via paracrine signaling mechanisms which, in turn con-
tribute to the development of coronary atherosclerosis and
the destabilization of existing atherosclerotic lesions [6]. Many clinical studies have reported that the epicardial fat
volume (EFV) using coronary CT angiography (CCTA) is
associated with the presence and severity of coronary ath-
erosclerosis and with the characteristics of atherosclerotic
plaques [7–9]. Measuring the volume of epicardial fat has
been considered as a potential imaging biomarker of dis-
ease progression and response to treatment [10]. However,
clinical evidence of the relationship between EFV level
and coronary atherosclerosis severity in three-vessel CAD
patients remains limited, except for that arising from sub-
group analysis in previously reported literature [11]. © 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
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licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom‑
mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Sun et al. BMC Cardiovascular Disorders (2022) 22:76 Sun et al. BMC Cardiovascular Disorders Page 2 of 10 Keywords: Epicardial adipose tissue, Epicardial fat volume, Computed tomography angiography, Three-vessel
coronary artery disease CCTA data acquisition CCTA was performed using a 256-slice CT scanner (Bril-
liance CT, Philips Medical Systems, Cleveland, USA). Detailed scanning parameters were listed as follows: tube
voltage, 120 kVp; detector collimation, 128 × 0.625 mm;
pitch, 0.16; rotation time, 0.27 s; slice thickness, 0.9 mm;
section increment, 0.45 mm. Tube current was set using
the ECG-based tube current modulation technique. About 60–70 mL of iodine contrast agent was injected
at a rate of 4.5–5.5 mL/s via a high-pressure injector,
followed by a 20–30 mL flush of saline at the same rate. Threrfore, in the present study, we sought to investigate
the relationship between EFV level assessed with CCTA
and coronary atherosclerosis severity in three-vessel CAD. Cardiovascular risk factors and clinical data Demographic characteristics and clinical data (summa-
rized in Table 1) were collected for all patients, includ-
ing BMI, smoking history, hypertension, dyslipidemia,
diabetes mellitus and family history of CAD. In addition,
measures of left ventricular (LV) function assessed with
transthoracic echocardiography were obtained from the
hospital records. These included LV end-diastolic diame-
ter, LV ejection fraction (LVEF), and the ratio of diastolic
mitral inflow velocity E peak and A peak (E/A). Coronary
collateral circulation status and SYNTAX score assessed
with ICA data were also collected. Study population In this single-center retrospective cross-sectional study,
between 1 January 2018 and 31 December 2019, 1608
consecutive patients who underwent CCTA scan-
ning in our hospital and had CCTA-based diagnosis of Sun et al. BMC Cardiovascular Disorders Sun et al. BMC Cardiovascular Disorders (2022) 22:76 Page 3 of 10 Fig. 1 Study flowchart. CT computed tomography, CAD coronary artery disease, PCI percutaneous coronary intervention, CABD coronary artery
bypass grafting, ICA invasive coronary angiography the heart. Epicardial fat was defined as all voxels with
attenuation values between − 190 HU and − 30HU. Thus all voxels having attenuation values within this
range and lying within the pericardium were thresh-
olded accordingly and used for EFV level quantifica-
tion (Fig. 2). The value of EFV was normalized to body
surface area (BSA) where the BSA was calculated with
the Mosteller equation [13]. Epicardial fat attenuation
index (FAI) was defined as the average attenuation of
adipose tissue within the pre-specified attenuation
window of − 190 HU to − 30 HU. A prospective ECG-gated CCTA was triggered using a
bolus tracking technique with a trigger threshold of 100
Hounsfield units (HU) in the descending aorta. The mean
estimated effective radiation dose of the CCTA scan was
(4.6 ± 1.2) mSv. EFV level and epicardial FAI measurement EFV level quantification was quantified on axial CCTA
images at 75% of the R-R interval. A semi-automatic
method was developed for segmenting the pericar-
dium and measuring the amount of EFV. Briefly, the
pericardial contour was automatically delineated by
a U-net framework, details of which are described in
a previous report from our group [12]. The segmen-
tation results were further checked and modified by
two experienced cardiac imaging physicians (J.H. and
M.Q.) blinded to both the study plan and the clini-
cal data. Modifications, including manual small scale
enlargment or shrinking of the imaging annotation
range, were made to ensure that the segmentation
results of the pericardial boundary matched exactly
with its anatomical structure. The upper boundary of
the pericardium was taken as the bifurcation of the
pulmonary trunk and the lower limit was the apex of For reproducibility assessment, images from 30
patients with three-vessel CAD were randomly
selected. The pericardial contour was automatically
delineated using the U-net framework mentioned
above. Two radiologists (J.H. and M.Q.) reviewed and
modified the automatic pericardial segmentation to
ensure a good match with the pericardial anatomy. After four weeks, one of the radiologists (J.H.) reviewed
and modified the pericardial segmentation output
again. The value of EFV and epicardial FAI derived
from the semi-automatic segmentation results of the
pericardium was employed for reproducibility assess-
ment using intraclass correlation coefficient analysis. Sun et al. BMC Cardiovascular Disorders (2022) 22:76 Sun et al. BMC Cardiovascular Disorders Page 4 of 10 Table 1 Basic characteristics and clinical data of study population
CAD coronary artery disease, EFV epicardial fat volume, BSA body surface area, FAI fat attenuation index, BMI body mass index, LV left ventricular, EF ejection fraction,
CCC coronary collateral circulation
*Patients were divided into two subgroups according to median EFV level (118 mL)
# P values refer to the comparison between three-vessel CAD patients and normal controls. Statistical analysis Coronary atherosclerosis severity was quantified by the
number of coronary arteries having ≥ 50% stenosis on
ICA [11, 14]. Stenoses in the left anterior descending,
left circumflex, and right coronary artery were each
scored as a single vessel lesion and stenoses in the left
main coronary artery were more strongly weighted
by being scored as two vessel lesions. Stenoses in the
remaining secondary branches of the coronary arter-
ies (such as the diagonal artery, obtuse marginal artery,
posterolateral artery, and posterior descending artery)
with ≥ 50% stenosis were each scored as 0.5 of a lesion. The three-vessel CAD patients were subdivided into
three groups according to coronary atherosclerosis
severity: mild (score = 3 vessels, n = 85), moderate (3.5
vessels ≤ score < 4 vessels, n = 82), and severe (4 ves-
sels ≤ score ≤ 7 vessels, n = 85). Patients with more than
three vessel lesions were divided into two subgroups
based on the median score (4 lesions) of coronary ath-
erosclerosis severity. Continuous variables were expressed as mean ± standard
deviation or medians and interquartile range. Categori-
cal variables were expressed as number and percentage. The Kolmogorov–Smirnov test was employed to test
for the normal distribution of continuous data. Com-
parisons of continuous variables were conducted using
the independent t-test and one-way analysis of variance
(ANOVA), with the least significant difference (LSD)
method for multiple comparisons. Comparisons of cat-
egorical variables were performed with the Chi-square
test. The relation of EFV level to mean epicardial FAI,
LV function, and BMI were assessed with Pearson’s cor-
relation analysis. Multivariable logistic regression analy-
sis was used for co-variables that had a significant effect
in the univariable logistic regression analysis, aiming to
identify the independent factors associated with a change
of EFV level. Interobserver and intraobserver agree-
ment for EFV level and epicardial FAI quantification
were assessed by evaluating the intraclass correlation Coronary atherosclerosis severity was quantified by the
number of coronary arteries having ≥ 50% stenosis on
ICA [11, 14]. Stenoses in the left anterior descending,
left circumflex, and right coronary artery were each
scored as a single vessel lesion and stenoses in the left
main coronary artery were more strongly weighted
by being scored as two vessel lesions. EFV level and epicardial FAI measurement The remaining P values refer to comparisons between the low and high
EFV level subgroups of the CAD patients
Variables
Normal control
(n = 252)
Three-vessel CAD patients*
P-value
All patients (n = 252)
Low EFV level
(< 118 mL, n = 125)
High EFV level
(≥ 118 mL, n = 127)
EFV (mL)
89 ± 32
120 ± 42
86 ± 21
154 ± 29
< 0.001#
EFV/BSA (mL/m2)
48 ± 19
65 ± 22
49 ± 12
82 ± 16
< 0.001#
Epicardial FAI (HU)
− 84 ± 9
− 81 ± 5
− 78 ± 4
− 84 ± 4
< 0.001#
Cardiovascular risk factors
Age (years)
61 ± 8
61 ± 8
61 ± 9
62 ± 7
0.163
Male sex, n (%)
193 (77)
193 (77)
93 (74)
100 (79)
0.416
BMI (kg/m2)
25 ± 3
25 ± 3
24 ± 3
26 ± 3
< 0.001
Smoking, n (%)
37 (19)
125 (50)
60 (48)
65 (51)
0.614
Hypertension, n (%)
0 (0)
179 (71)
85 (68)
94 (74)
0.293
Dyslipidemia, n (%)
0 (0)
45 (19)
21 (17)
24 (19)
0.664
Diabetes, n (%)
0 (0)
104 (41)
48 (38)
56 (44)
0.359
CAD family history, n (%)
0 (0)
29 (12)
17 (14)
12 (9)
0.302
LV functional parameters
LV diameter (mm)
48 ± 3.8
48 ± 4.3
48 ± 4.5
49 ± 4.0
0.023
LVEF (%)
61 ± 5.0
60 ± 6.0
60 ± 6.4
60 ± 5.6
0.911
E/A
1.6 ± 0.3
1.3 ± 0.4
1.2 ± 0.4
1.3 ± 0.4
0.056
Invasive coronary angiography
CCC status, n (%)
NA
78 (31)
40 (32)
38 (30)
0.721
SYNTAX score
NA
29 (23, 36)
30 (23, 36)
29 (23, 35)
0.793 Table 1 Basic characteristics and clinical data of study population d into two subgroups according to median EFV level (118 mL) isease, EFV epicardial fat volume, BSA body surface area, FAI fat attenuation index, BMI body mass index, LV left ventricular, EF ejection fra
al circulation Statistical analysis Stenoses in the
remaining secondary branches of the coronary arter-
ies (such as the diagonal artery, obtuse marginal artery,
posterolateral artery, and posterior descending artery)
with ≥ 50% stenosis were each scored as 0.5 of a lesion.h The three-vessel CAD patients were subdivided into
three groups according to coronary atherosclerosis
severity: mild (score = 3 vessels, n = 85), moderate (3.5
vessels ≤ score < 4 vessels, n = 82), and severe (4 ves-
sels ≤ score ≤ 7 vessels, n = 85). Patients with more than
three vessel lesions were divided into two subgroups
based on the median score (4 lesions) of coronary ath-
erosclerosis severity. Sun et al. BMC Cardiovascular Disorders (2022) 22:76 Sun et al. BMC Cardiovascular Disorders Page 5 of 10 Fig. 2 Semi-automatic quantification of EFV level from CCTA images. Axial CCTA images at 75% of the R-R interval were processed for pericardial
segmentation using the U-Net framework. The initial results were checked and modified by two radiologists and the epicardial fat was identified
as those voxels lying between upper and lower threshold values of − 30 and − 190 HU. EFV epicardial fat volume, CCTA coronary computed
tomography angiography Fig. 2 Semi-automatic quantification of EFV level from CCTA images. Axial CCTA images at 75% of the R-R interval were processed for pericardial
segmentation using the U-Net framework. The initial results were checked and modified by two radiologists and the epicardial fat was identified
as those voxels lying between upper and lower threshold values of − 30 and − 190 HU. EFV epicardial fat volume, CCTA coronary computed
tomography angiography significantly higher EFV in overweight patients than
in those with normal body weight (EFV: 134 ± 41 vs
105 ± 39 mL, t = 5.786, P < 0.001; EFV/BSA: 70 ± 21
vs 60 ± 21 mL/m2, t = 3.603, P < 0.001). Hypertensive
patients had a significantly higher EFV (123 ± 42 vs
110 ± 42 mL, t = 2.229, P = 0.027; EFV/BSA: 67 ± 21 vs
60 ± 22 mL/m2, t = 2.322, P = 0.021). Study population characteristics A total of 252 consecutive three-vessel CAD patients
and 252 control group participants were included in this
retrospective study. Detailed baseline demographic and
clinical data are listed in Table 1. Compared with lesion-
free controls, three-vessel CAD patients had significantly
higher EFV level (EFV: 120 ± 42 vs 89 ± 32 mL, EFV/
BSA: 65 ± 22 vs 48 ± 19 mL/m2; P < 0.001) and higher
mean epicardial FAI (− 81 ± 5 vs − 84 ± 9 HU, P < 0.001). According to the median EFV level (118 cm3), three-
vessel CAD patients were categorized into low (< 118 mL,
n = 125) and high EFV level subgroups (≥ 118 mL,
n = 127). Compared with the low EFV subgroup, patients
with high EFV had significantly higher BMI (26 ± 3 vs
24 ± 3 kg/m2, P < 0.001) and larger LV diameter (49 ± 4.0
vs 48 ± 4.5 mm, P = 0.023) (Table 1). Pearson correlation analysis showed that, for all CAD
subjects, EFV was positively and significantly correlated
with BMI (r = 0.407, P < 0.001) and negatively, with mean
epicardial FAI (r = − 0.709, P < 0.001). Statistical analysis In comparison to
patients who had normal LV diastolic function (E/A > 1,
n = 203), those with LV diastolic dysfunction (E/A < 1,
n = 49) had a lower EF and lower EFV relative to BSA,
although only the latter was statistically significant (EFV:
110 ± 43 vs 122 ± 42 mL, t = − 1.868, P = 0.063; EFV/
BSA: 60 ± 22 vs 67 ± 21 mL/m2, t = − 1.988, P = 0.048). However, there were no significant differences of EFV
level between the two subgroups in the variables related
to gender, smoking, dyslipidemia, diabetes mellitus, or
family history of CAD (P > 0.05). coefficient (ICC). Statistical analysis was performed with
SPSS software (SPSS statistics, version 26.0, IBM Corp.). Two-sided testing was used and P < 0.05 was considered
to be statistically significant. Relation of EFV, FAI, and LV function to the severity
of coronary atherosclerosis Relation of EFV level to risk factors and LV function
Subgroup analysis of EFV level, separated into
two groups by median BMI (25 kg/m2), revealed a Relation of EFV level to risk factors and LV function Subgroup analysis of EFV level, separated into
two groups by median BMI (25 kg/m2), revealed a Table 2 and Fig. 3a, b show that increasing steno-
sis severity was significantly associated with reduced Sun et al. BMC Cardiovascular Disorders (2022) 22:76 Sun et al. Relation of EFV level to risk factors and LV function BMC Cardiovascular Disorders Page 6 of 10 Table 2 Comparison of EFV level, FAI, and LV function in three-
vessel CAD
EFV epicardial fat volume, FAI fat attenuation index, LV left ventricular, CAD
coronary artery disease, BSA body surface area, EF ejection fraction, BMI body
mass index
*Patients were subdivided into groups according to their coronary
atherosclerosis severity: mild (score = 3 vessels), moderate (3.5 ≤ score < 4
vessels), and severe (4 ≤ score ≤ 7 vessels)
# Patients were categorized by median BMI (25 kg/m2)
Variables
Score of coronary atherosclerosis
severity*
P-value
Mild
Moderate
Severe
All patients (n = 252)
85 (34)
82 (32)
85 (34)
EFV (mL)
126 ± 45
124 ± 45
110 ± 34
0.045
EFV/BSA (mL/m2)
69 ± 23
67 ± 23
60 ± 17
0.036
Epicardial FAI (HU)
− 81 ± 5.5
− 77 ± 4.5
− 73 ± 4.3
< 0.001
LV diameter (mm)
48 ± 4.3
48 ± 4.5
48 ± 4.0
0.967
LVEF (%)
60 ± 6.3
60 ± 5.6
60 ± 6.2
0.952
E/A
1.3 ± 0.4
1.3 ± 0.3
1.3 ± 0.4
0.931
Patients stratified by
BMI#
BMI < 25 kg/m2
(n = 124)
41 (33)
37 (30)
46 (37)
EFV (mL)
107 ± 43
107 ± 42
103 ± 31
0.86
EFV/BSA (mL/m2)
62 ± 24
62 ± 24
58 ± 17
0.857
Epicardial FAI (HU)
− 79 ± 6.6
− 77 ± 4.7
− 72 ± 3.9
< 0.001
LV diameter (mm)
48 ± 4.3
47 ± 4.3
48 ± 4.0
0.448
LVEF (%)
60 ± 5.8
61 ± 6.1
60 ± 6.2
0.788
E/A
1.2 ± 0.5
1.3 ± 0.3
1.2 ± 0.3
0.945
BMI ≥ 25 kg/m2
(n = 128)
44 (34)
45 (35)
39 (31)
EFV (mL)
144 ± 40
139 ± 43
118 ± 35
0.01
EFV/BSA (mL/m2)
75 ± 21
72 ± 22
62 ± 17
0.013
Epicardial FAI (HU)
− 82 ± 4.0
− 78 ± 4.4
− 74 ± 4.5
< 0.001
LV diameter (mm)
49 ± 4.2
49 ± 4.4
49 ± 4.2
0.802
LVEF (%)
60 ± 6.7
60 ± 5.2
59 ± 6.2
0.725
E/A
1.3 ± 0.4
1.3 ± 0.3
1.3 ± 0.4
0.825 level from the mild, moderate to severe coronary ath-
erosclerosis subgroups in the higher BMI group (EFV:
144 ± 40 vs 139 ± 43 vs 118 ± 35 mL, P = 0.01; EFV/
BSA: 75 ± 21 vs 72 ± 22 vs 62 ± 17 mL/m2, P = 0.013)
(Table 2; Fig. Reproducibility assessmentfi EFV epicardial fat volume, FAI fat attenuation index, LV left ventricular, CAD
coronary artery disease, BSA body surface area, EF ejection fraction, BMI body
mass index Intraclass correlation coefficient analysis revealed excel-
lent interobserver and intraobserver agreement for EFV
level and epicardial FAI measurement. Interobserver
analysis showed 0.97, 0.95 agreement for EFV level
and epicardial FAI, respectively. Intraobserver analysis
showed 0.99, 0.97 agreement for EFV level and epicardial
FAI, respectively. # Patients were categorized by median BMI (25 kg/m2) EFV. There was a statistically significant inverse rela-
tionship between EFV and severity of coronary ath-
erosclerosis (126 ± 45 vs 124 ± 45 vs 110 ± 34 mL,
F = 3.818, P = 0.023). Post hoc tests using the LSD
method showed that the differences between the mild
and severe coronary atherosclerosis subgroups were
statistically significant (t = 16.212, P = 0.012), as well as
for the comparison between the moderate and severe
subgroups (t = 14.301, P = 0.028). When normalized
to BSA, the relationship was maintained and remained
significant although the effect was weaker (69 ± 23 vs
67 ± 23 vs 60 ± 17 mL/m2, P = 0.036). EFV. There was a statistically significant inverse rela-
tionship between EFV and severity of coronary ath-
erosclerosis (126 ± 45 vs 124 ± 45 vs 110 ± 34 mL,
F = 3.818, P = 0.023). Post hoc tests using the LSD
method showed that the differences between the mild
and severe coronary atherosclerosis subgroups were
statistically significant (t = 16.212, P = 0.012), as well as
for the comparison between the moderate and severe
subgroups (t = 14.301, P = 0.028). When normalized
to BSA, the relationship was maintained and remained
significant although the effect was weaker (69 ± 23 vs
67 ± 23 vs 60 ± 17 mL/m2, P = 0.036). Analysis of factors related to the change of EFV level Analysis of factors related to the change of EFV level
Univariate and multivariable logistic regression analysis
showed that BMI (OR 3.40, 95% CI 2.00–5.78, P < 0.001)
and coronary atherosclerosis severity (OR 0.49, 95% CI
0.26–0.93, P = 0.03) were independently associated with
the change of EFV level in all patients (Table 3). Relation of EFV level to risk factors and LV function 3c, d). Post hoc tests using the LSD
method showed that, in comparison to the severe ath-
erosclerosis subgroup, both the mild (EFV: t = 25.678,
P = 0.004; EFV/BSA: t = 12.670, P = 0.005) and the
moderate atherosclerosis subgroups (EFV: t = 20.480,
P = 0.020; EFV/BSA: t = 10.164, P = 0.023) had signifi-
cantly higher EFVs. However, no significant relation-
ship between EFV level and atheroslcerotic severity
was seen in patients with BMIs below the median value
Additionally, there was a significant positive associa-
tion between the severity of coronary atherosclerosis
and the mean attenuation due to epicardial fat (FAI)
(− 81 ± 5.5 vs − 77 ± 4.5 vs − 73 ± 4.3 HU, P < 0.001)
for the mild, moderate and severe atherosclerosis lev-
els, respectively. However, there were no significant
differences between the mild, moderate, and severe
atherosclerosis subgroups in the variables related to LV
function, namely LV diameter, LVEF, or E/A (P > 0.05). Discussionh The results show that three-vessel CAD patients had
significantly higher EFV levels than those of the normal
controls. Furthermore, stratifying the severity of athero-
sclerosis into mild, moderate and severe levels showed
that there was an inverse relationship between EFV level
assessed with CCTA and coronary atherosclerosis sever-
ity in three-vessel CAD. Many clinical studies have demonstrated that EFV
level on CCTA is related to the presence and severity of
coronary atherosclerosis. For instance, in the Framing-
ham Heart Study, measurements in more than 3000 indi-
viduals showed that increased EFV was associated with
the presence of CAD [8]. EFV level was also related to When grouped according to the median BMI (25 kg/
m2), there was, similarly, a progressive decline in EFV Sun et al. BMC Cardiovascular Disorders (2022) 22:76 Sun et al. BMC Cardiovascular Disorders Page 7 of 10 Fig. 3 Relation of EFV level to coronary atherosclerosis severity in three-vessel CAD. Three-vessel CAD patients with more severe coronary
atherosclerosis have significantly lower EFV level (a, b), especially in those patients with higher BMI (≥ 25 kg/m2) (c, d). EFV, epicardial fat volume;
CAD, coronary artery disease; BMI, body mass index. Fig. 3 Relation of EFV level to coronary atherosclerosis severity in three-vessel CAD. Three-vessel CAD patients with more severe coronary
atherosclerosis have significantly lower EFV level (a, b), especially in those patients with higher BMI (≥ 25 kg/m2) (c, d). EFV, epicardial fat volume
CAD, coronary artery disease; BMI, body mass index. atherosclerosis, chronic hypoxia and an unfavorable
hemodynamic environment would affect the regula-
tory component of the epicardial fat proteasome, which
would accelerate the apoptosis of epicardial adipocytes. This hypothesis is supported by evidence that several
genes involved in cellular function are downregulated
in the epicardial adipocytes of advanced CAD [15]. Fur-
thermore, recent evidence has revealed a bidirectional
signaling between epicardial adipose tissue and vascular
inflammatory cells [16, 17]. Epicardial adipocytes could,
therefore, modulate structural changes and inflamma-
tion in the vascular wall via paracrine mechanisms and
vascular wall-derived inflammatory factors could block
the differentiation of epicardial adipocytes. In three-
vessel CAD patients, the remodelling of adipocytes due
to vessel inflammation was more obvious than that in
patients with mild or moderate CAD. Therefore, the end
result could be large proportion of small and immature
epicardial perivascular pre-adipocytes around vessels,
thus leading to a reduction of EFV. Discussionh These observa-
tions are also consistent with previous studies which
have reported increased epicardial FAI in patients with
advanced CAD or acute coronary syndrome [20, 21]. Additionally, we observed a significantly higher EFV in
hypertensive subjects and those in the higher BMI group,
again in line with previous studies [22, 23]. We also found
that in patients with LV diastolic dysfunction there was a
tendency towards lower EFV, although this was not sta-
tistically significant. A possible explanation is that epicar-
dial fat is associated with both visible epicardial coronary
atherosclerosis and coronary microvascular dysfunction,
both of which would lead to impaired myocardial perfu-
sion and contractile function [24].h There are several limitations to the study. First, it was
a retrospective single-center study and the sample size
was relatively small. Second, even though we adopted
a commonly used method for classifying the severity
of coronary atherosclerosis [11, 14], it did not account
for the stability of the atherosclerotic plaque. Third, it
has been reported that statin therapy is associated with
a reduction in epicardial fat accumulation, especially
when given in high doses (atorvastatin 80 mg/day) [25]. However, in this study, all the CAD patients were given
moderate doses (atorvastatin 10–20 mg/day), this being
based on expert recommendation for Chinese subjects
[26]. Whether a significant reduction of EFV in three-
vessel CAD patients with severe coronary atherosclerosis
is associated with moderate statin therapy needs further
clinical studies to confirm. to changes in the composition of epicardial adipocytes
which could increase the degree to which they attenu-
ate incident x-rays. If this were to happen, fewer voxels
would fall within the thresholded range and, this would
lead to an apparent reduction in EFV. This effect is likely
to be seen in CAD patients who have had long-standing
and complex coronary atherosclerosis, which is associ-
ated with irreversible changes in epicardial perivascular
adipocyte composition, such as inflammatory infiltra-
tion, microvascular remodeling and extracellular fibro-
sis [18, 19]. Furthermore, according to the bidirectional
interaction hypothesis mentioned above [16, 17], epicar-
dial fat preadipocytes tend to be of smaller size and con-
tain less intracellular lipid than that of mature epicardial
adipocytes. The loss of balance between lipid and water
content in epicardial perivascular preadipocytes would
result in a rise of epicardial FAI. Discussionh The second explana-
tion for reduced EFV in those with severe CAD is related coronary atherosclerosis severity, coronary artery cal-
cium score, and the characteristics of unstable athero-
sclerotic plaques [8–10]. Here, we found that three-vessel
CAD patients had significantly higher EFVs than those of
the normal controls, which is consistent with the previ-
ous studies [8–10]. Interestingly, we observed a progressive decline in EFV
level with increasing coronary atherosclerotic severity in
all three-vessel CAD patients, although it was statisti-
cally significant only those with BMI ≥ 25 kg/m2. Gorter
et al. [11] also reported that patients with more severe
coronary atherosclerosis tended to have lower EFVs
although their study sample contained only 128 subjects. We believe there are two plausible explanations for this
inverse association between disease severity and EFV. One is that fewer numbers of epicardial adipocytes and
an increased number of small and immature epicardial
perivascular preadipocytes in patients with severe coro-
nary atherosclerosis would imply a reduced absolute
EFV level. In the three-vessel CAD patients with chronic
coronary vascular inflammation and severe coronary Sun et al. BMC Cardiovascular Disorders (2022) 22:76 Sun et al. BMC Cardiovascular Disorders Page 8 of 10 Page 8 of 10 Table 3 Factors associated with EFV level in three-vessel CAD
EFV epicardial fat volume, CAD coronary artery disease, OR odds ratio, BMI body
mass index, LV left ventricular, EF ejection fraction, CCC coronary collateral
circulation
* Patients were divided into lower (< 25 kg/m2) and higher (≥ 25 kg/m2) BMI
groups
# Patients were divided into groups according to their coronary atherosclerosis
severity: mild (score = 3 vessels), moderate (3.5 ≤ score < 4 vessels), and severe
(4 ≤ score ≤ 7 vessels)
Variables
Univariable analysis
Multivariable analysis
(univariable P < 0.05)
OR (95% CI)
P value
OR (95% CI)
P value
Age
1.02 (0.99; 1.06)
0.163
Gender
0.70 (0.38; 1.19)
0.168
Smoking
1.21 (0.74; 1.98)
0.452
Hypertension
1.45 (0.81; 2.60)
0.211
Dyslipidemia
1.40 (0.75; 2.63)
0.294
Diabetes
1.26 (0.76; 2.07)
0.366
BMI*
3.60 (2.14; 6.05)
< 0.001
3.40 (2.00; 5.78)
< 0.001
CAD family history 0.66 (0.30; 1.45)
0.304
LV diameter
1.07 (1.00; 1.14)
0.025
1.06 (0.99; 1.13)
0.104
LVEF
1.00 (0.96; 1.05)
0.91
E/A
1.94 (0.98; 3.84)
0.058
Score of coronary
atherosclerosis
severity#
0.49 (0.27; 0.90)
0.022
0.49 (0.26; 0.93)
0.03
CCC status
0.91 (0.53; 1.55)
0.72
SYNTAX score
0.99 (0.97; 1.02)
0.57 had significantly higher epicardial FAI. Discussionh The change of epicar-
dial fat composition in three-vessel CAD patients with
more severe coronary atherosclerosis leads to an increase
of epicardial FAI, which, in turn, leads to a significant
reduction of EFV level when measuring CCTA images
using threshold-based methods. This hypothesis is con-
sistent with the results reported here in which three-
vessel CAD patients with severe coronary atherosclerosis Conclusions Patients with three-vessel CAD have significantly higher
EFV level than age-, gender- and BMI-matched normal
controls. Furthermore, our study results show that there
is an potential inverse relationship between EFV level
and coronary atherosclerosis severity in three-vessel
CAD. Therefore, the value of clinical applications of the
current study may be that a fall in EFV level on CCTA
images during follow-up of patients with three-vessel
CAD is a potential imaging biomarker of disease progres-
sion, which will contribute to the clinical management of
three-vessel CAD. However, future larger-scale studies
are required for the validation of our findings. Abbreviations
BMI: Body mass index; BSA: Body surface area; CAD: Coronary artery disease;
CCTA: Coronary computed tomography angiography; EF: Ejection fraction;
EFV: Epicardial fat volume; FAI: Fat attenuation index; HU: Hounsfield units; ICA:
Invasive coronary angiography; LV: Left ventricular. Abbreviations Abbreviations
BMI: Body mass index; BSA: Body surface area; CAD: Coronary artery disease;
CCTA: Coronary computed tomography angiography; EF: Ejection fraction;
EFV: Epicardial fat volume; FAI: Fat attenuation index; HU: Hounsfield units; ICA:
Invasive coronary angiography; LV: Left ventricular. Declarations 11. Gorter PM, Vos AMD, Graaf YVD, Stella PR, Doevendans PA, Meijs MFL,
et al. Relation of epicardial and pericoronary fat to coronary athero‑
sclerosis and coronary artery calcium in patients undergoing coronary
angiography. Am J Cardiol. 2008;102:380–5. Acknowledgements
Not applicable. Acknowledgements
Not applicable. Page 9 of 10 Sun et al. BMC Cardiovascular Disorders (2022) 22:76 Sun et al. BMC Cardiovascular Disorders (2022) 22:76 Sun et al. BMC Cardiovascular Disorders Availability of data and materials 10. Mancio J, Azevedo D, Saraiva F, Azevedo AI, Pires-Morais G, Leite-Moreira
A, et al. Epicardial adipose tissue volume assessed by computed tomog‑
raphy and coronary artery disease: a systematic review and meta-analysis. Eur Heart J Cardiovasc Imaging. 2018;19:490–7. The datasets used and/or analysed during the current study are available from
the corresponding author on reasonable request. Funding
Thi
d This study is supported by the National Natural Science Foundation of China
(61773110), the Key Research and Development Program of Liaoning Prov‑
ince, China (2018225024). The funders play an important role in study design,
data analysis, and preparation of the manuscript. 9. Yu WJ, Liu B, Zhang FF, Wang JF, Shao XL, Yang XY, et al. Association of
epicardial fat volume with increased risk of obstructive coronary artery
disease in Chinese patients with suspected coronary artery disease. J Am
Heart Assoc. 2021;10:e018080. Author details 1 College of Medicine and Biological Information Engineering, Northeastern
University, Shenyang, People’s Republic of China. 2 Department of Radiology,
General Hospital of Northern Theater Command, 83 Wenhua RoadLiaoning
Province, Shenyang 110016, People’s Republic of China. 3 Key Laboratory
of Cardiovascular Imaging and Research of Liaoning Province, Shenyang,
People’s Republic of China. 4 Department of Cardiology, General Hospital
of Northern Theater Command, Shenyang, People’s Republic of China. 5 Blizard
Institute, Barts and The London School of Medicine and Dentistry, Queen Mary
University of London, London, UK. 15. Camarena V, Sant D, Mohseni M, Salerno T, Zaleski ML, Wang G, et al. Novel atherogenic pathways from the differential transcriptome
analysis of diabetic epicardial adipose tissue. Nutr Metab Cardiovasc Dis. 2017;27:739–50. 16. Margaritis M, Antonopoulos AS, Digby J, Lee R, Reilly S, Coutinho P, et al. Interaction between vascular wall and perivascular adipose tissue reveal
novel roles for adiponectin in the regulation of endothelial nitric oxide
synthase function in human vessels. Circulation. 2013;127:2209–21. synthase function in human vessels. Circulation. 2013;127:2209–2 17. Antonopoulos AS, Sanna F, Sabharwal N, Thomas S, Oikonomou EK,
Herdman L, et al. Detecting human coronary inflammation by imaging
perivascular fat. Sci Transl Med. 2017;9:eaal2658. Received: 8 December 2021 Accepted: 24 February 2022 Received: 8 December 2021 Accepted: 24 February 2022 18. Oikonomou EK, Antoniades C. The role of adipose tissue in cardiovascular
health and disease. Nat Rev Cardiol. 2019;16:83–99. 19. Mancio J, Oikonomou EK, Antoniades C. Perivascular adipose tissue and
coronary atherosclerosis. Heart. 2018;104:1654–62. 20. Liu ZH, Wang SJ, Wang YQ, Zhou NB, Shu J, Stamm C, et al. Association
of epicardial adipose tissue attenuation with coronary atherosclerosis
in patients with a high risk of coronary artery disease. Atherosclerosis. 2019;284:230–6. Consent for publication
Not applicable. 14. Scanlon PJ, Faxon DP, Audet AM, Carabello B, Dehmer GJ, Eagle KA, et al. ACC/AHA guidelines for coronary angiography. A report of the American
College of Cardiology/American Heart Association Task Force on practice
guidelines (Committee on Coronary Angiography). Developed in col‑
laboration with the Society for Cardiac Angiography and Interventions. J
Am Coll Cardiol. 1999;33:1756–824. Competing interests The authors declare that they have no competing interests. Authors’ contributions 5. Maron DJ, Hochman JS, Reynolds HR, Bangalore S, O’Brien SM, Boden WE,
et al. Initial invasive or conservative strategy for stable coronary disease. N
Engl J Med. 2020;382:1395–407. YS collected and analyzed the study population data, and was a major
contributor in writing the manuscript. XGL contributed to the development
of deep learning model used in this study and the interpretation of data. KX contributed to the design of this study and the analysis of data. JH, and
MQ contributed to the collection and interpretation of data. HRY, RRZ, LBZ,
LSX, and SEG contributed to the development of deep learning model. BQY
contributed to the conception of the work and analysis of data, and was a
major contributor in revising the manuscript. All authors read and approved
the final manuscript. 6. Mazurek T, Opolski G. Pericoronary adipose tissue: a novel therapeu‑
tic target in obesity-related coronary atherosclerosis. J Am Coll Nutr. 2015;34:244–54. 7. Lu MT, Park J, Ghemigian K, Mayrhofer T, Puchner SB, Liu T, et al. Epicardial
and paracardial adipose tissue volume and attenuation—association
with high-risk coronary plaque on computed tomographic angiography
in the ROMICAT II trial. Atherosclerosis. 2016;251:47–54. in the ROMICAT II trial. Atherosclerosis. 2016;251:47–54. 8. Thanassoulis G, Massaro JM, Hoffmann U, Mahabadi AA, Vasan RS,
O’Donnell CJ, et al. Prevalence, distribution, and risk factor correlates of
high pericardial and intrathoracic fat depots in the Framingham heart
study. Circ Cardiovasc Imaging. 2010;3:559–66. Ethics approval and consent to participate The study protocol was approved by the ethics committee of the General
Hospital of Northern Theater Command (Y [2021] 012) and the study com‑
plied with the Declaration of Helsinki. Informed consent was waived by the
committee because of the retrospective nature of this study. 12. Li XG, Sun Y, Xu LS, Greenwald SE, Zhang LB, Zhang RR, et al. Auto‑
matic quantification of epicardial adipose tissue volume. Med Phys. 2021;48:4279–90. 13. Mosteller RD. Simplified calculation of body-surface area. N Engl J Med. 1987;317:1098. Consent for publication
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https://openalex.org/W3012964330
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https://uu.diva-portal.org/smash/get/diva2:1443715/FULLTEXT01
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English
| null |
Two-Step Size-Exclusion Nanofiltration of Prothrombin Complex Concentrate Using Nanocellulose-Based Filter Paper
|
Biomedicines
| 2,020
|
cc-by
| 13,038
|
Received: 29 February 2020; Accepted: 21 March 2020; Published: 26 March 2020 Abstract: Coagulation Factor IX-rich protrhombin complex concentrate (FIX-PCC) is a therapeutic
biologic product that consists of a mixture of several human plasma-derived proteins, useful for treating
hemophilia B. Due to its complex composition, FIX-PCC is very challenging to bioprocess through
virus removing nanofilters in order to ensure its biosafety. This article describes a two-step filtration
process of FIX-PCC using a nanocellulose-based filter paper with tailored porosity. The filters were
characterized with scanning electron microscopy (SEM), cryoporometry with differential scanning
calorimetry, and nitrogen gas sorption. Furthermore, in order to probe the filter’s cut-offsize rejection
threshold, removal of small- and large-size model viruses, i.e., ΦX174 (28 nm) and PR772 (70 nm), was
evaluated. The feed, pre-filtrate, and permeate solutions were characterized with mass-spectrometric
proteomic analysis, dynamic light scattering (DLS), sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), and analytical size-exclusion high-performance liquid chromatography
(SEHPLC). By sequential filtration through 11 µm pre-filter and 33 µm virus removal filter paper,
it was possible to achieve high product throughput and high virus removal capacity. The presented
approach could potentially be applied for bioprocessing other protein-based drugs. Keywords: Mille-feuille filter; Cladophora cellulose; hemophilia B; protein aggregates; virus
removal filtration biomedicines biomedicines Levon Manukyan 1
, Athanasios Mantas 1
, Mikhail Razumikhin 2, Andrey Katalevsky 3,
Eugen Golubev 4 and Albert Mihranyan 1,* Levon Manukyan 1
, Athanasios Mantas 1
, Mikhail Razumikhin 2, Andrey Katalevsky 3
Eugen Golubev 4 and Albert Mihranyan 1,* 1
Nanotechnology and Functional Materials, Department of Materials Science and Engineering,
Uppsala University, Box 534, 751 21 Uppsala, Sweden; levon.manukyan@angstrom.uu.se (L.M.);
athanasios.mantas@angstrom.uu.se (A.M.) 2
Nacimbio JC, 10, 2nd Volkonsky lane, 127473 Moscow, Russia; razumikhin.m@gmail.com
3
Biopharmgarant LLC, Stancionnaya 45, 600901 Vladimir, Russia; andreyalexandr2003@yandex.ru
4
National Research Center for Hematology, Novyi Zykovskiy proezd 4, 125167 Moscow, Russia;
ev-genius@bk.ru 2
Nacimbio JC, 10, 2nd Volkonsky lane, 127473 Moscow, Russia; razumikhin.m@gmail.com
3
Biopharmgarant LLC, Stancionnaya 45, 600901 Vladimir, Russia; andreyalexandr2003@yandex.ru
4
National Research Center for Hematology, Novyi Zykovskiy proezd 4, 125167 Moscow, Russia;
ev-genius@bk.ru g
*
Correspondence: albert.mihranyan@angstrom.uu.se; Tel.: +46-18-471-7940 g
*
Correspondence: albert.mihranyan@angstrom.uu.se; Tel.: +46-18-471-7940 biomedicines biomedicines 1. Introduction p
p
gg
g
It is known that the levels of different impurities are highly dependent on the type of
chromatographic separation that was used during plasma treatment [3,18,20]. The most extensively
described impurities in vitamin K-dependent clotting factors include inter-α-trypsin-inhibitor (ITI),
complement 4b binding protein (C4BP), and vitronectin (VN). C4BP is a large glycoprotein of
570 kDa [21] in the shape of an octopus that consists of seven α-chains connected to a single β-chain
by disulfide bonds [22–24]. The α-chains are responsible for binding C4b, while the β-chain has
high-affinity for VN (protein S), forming large complexes [22,25]. The molecular conformation of C4BP
is highly dependent on the surrounding medium composition [11,19]. In the charged state the α-chains
repel each other, thereby occupying much larger volume than the same molecule in the uncharged state. In its open structure form, C4BP would have a diameter of approximately 66 nm since each α-chain
arm is 33 nm long [11,22]. Varying salt concentrations have been shown to affect the compactness of
C4BP molecule and thereby the flux properties of the virus removal filter [19]. Another large Mw
impurity present often in FIX products is ITI. ITI (225 kDa) is a large complex that consists of one light
and several heavy chains (H1-H3) covalently linked by a chondroitin sulfate chain. The heavy chains
of ITI proteins function as hyaluronic acid (HA) binding proteins, whereas the light chain, also called
bikunin, functions as a serine protease inhibitor upon activation [26]. The third extensively described
impurity is VN [27]. The monomeric form of VN has a cryptic hydrophobic pocket, which upon
exposure and conformational changes exhibits heparin- and C5b-7 binding activity [28]. Normally,
only 2% of VN in plasma shows heparin-binding activity but its fraction increases manifold during
coagulation [29]. VN also presents a free thiol group capable of disulfide bonding [30]. When unfolded
VN is highly prone to polymerization and may form aggregates with Mw up to 1000 kDa [28]. Studies
on nanofiltration of FIX products where VN aggregates were detectable confirmed its role as a filter
foulant [27]. Extensive coverage of various impurities at different intermediate stages during FIX
manufacturing is discussed elsewhere [15,18]. So far successful virus removal filtration of FIX products has been described in the literature only
for a limited number of commercial filters. 1. Introduction Replacement therapy using plasma-derived Factor IX (FIX) products is a life-saving treatment for
patients with hemophilia B. Both recombinant and plasma-derived FIX show high efficacy in clinical
trials [1]. Production of FIX normally involves multiple steps. High purity FIX is obtained from
prothrombin complex concentrate (PCC), which is a mixture of vitamin K-dependent clotting factors,
e.g., factor II (prothrombin), V, VII, IX, and X, and clotting inhibitors, e.g., protein C, Z, and S [2]. PCC
preparation is a highly complex mixture of proteins and may contain up to 50% of proteins other than
FIX [3]. Both highly purified FIX and PCC can be used for hemophilia B treatment [4]. Also, PCC
preparation may be useful for prevention of bleeding due to overdose of oral anticoagulants and liver
dysfunctions [2,3]. Biomedicines 2020, 8, 69; doi:10.3390/biomedicines8040069 www.mdpi.com/journal/biomedicines www.mdpi.com/journal/biomedicines 2 of 18 Biomedicines 2020, 8, 69 As it is with all plasma-derived products, the viral safety of FIX-rich PCC is a critical issue [5]. According to current regulations, at least two orthogonal virus clearance steps must be implemented
to ensure viral safety of the final product [6]. The steps to mitigate virus contamination of FIX and
PCC products include donor screening for known blood-borne viruses, i.e., HIV 1–2, HBV, HCV, HAV,
and parvo B19; virus inactivation, such as solvent/detergent, mixed chemical inactivation (tri-n-butyl
phosphate) and detergent (nonionic, polysorbate, and polyethylene oxide) treatment; and incubating
intermediate product in controlled temperature (usually 6 h at 25 ◦C) [3,7,8]. Dry heat treatment
may also be used after lyophilization, e.g., 100 ◦C for 1 h or 80 ◦C for 72 h. In the past, steam
treatment at 60 ◦C (190 mbar) for 10 h or 80 ◦C (375 mbar) for 1 h was reported [3,7,8]. Presently,
virus removal nanofiltration has become widely used as a robust and reliable method for ensuring
viral safety. Nanofiltration is attractive because it is capable of physically removing all types of
viruses from protein solution as opposed to virus inactivation. Several authors have described the
application of virus removal nanofiltration for FIX industrial products using Planova 15/20/35N [9–15],
Viresolve NFP [11,16–19], and Ultipor DV50 filters [2]. The conclusion of these studies is that filtration
of plasma-derived FIX-rich products is challenging due to the presence of large molecular weight
impurities and protein aggregates. 2.4. Filtration Setup Pre-filtration and filtration steps were performed in a 47 mm diameter Advantech KST 47 filter
holder. Prior to filtration, the pre- and filter papers were wetted in order to extrude the air by
running 20 mL of PBS. The pre-filtration steps with 6 and 11 µm pre-filters were performed at 1 bar
transmembrane pressure, and the filtrations with 33 µm filters were carried out at 1 or 3 bar. The
permeate solutions were collected and for filtrations of the larger volume, permeate was collected in
one or three fractions and saved. 2.1. Materials Cladophora cellulose was provided by FMC Biopolymer (batch 3095-10; Newark, DE, USA). FIX-rich PCC was provided by National Center for Hematology, Moscow, Russia, as lyophilized
powder. Coliphages ΦX174 (ATCC 13706-B1™) and PR772 (BAA-769-B1), and the host bacteria
Escherichia coli (Migula) Castellani and Chalmers C (ATCC 13706) and K12 J53-1(R15) [HER 1221]
(BAA-769) strains were obtained from ATCC (Manassas, VA, USA). Agar (214530) was obtained
from BD (Franklin Lakes, NJ, USA). Tryptone (LP0042B) and yeast extract (Oxoid) (LP0021) were
obtained from Thermo Fisher Scientific. Phosphate-buffered saline (P4417), 2-mercaptoethanol (M3148),
sodium chloride (S5886), sodium phosphate dibasic (71640) and 2-mercaptoethanol (M3148) were
purchased from Sigma-Aldrich (Saint Louis, MO, USA). Any kD™Mini-PROTEAN® TGX Stain-Free™
protein gels (4568125), tris/glycine/SDS running buffer (1610732), 4x Laemmli Sample Buffer (1610747),
and Precision Plus Protein™unstained protein standards (1610363) were purchased from Bio-Rad
(Hercules, CA, USA). 2.2. Filter Preparation Filters of different thickness were prepared from Cladophora cellulose dispersion (0.1 wt.%) made
by microfluidization with 200 µm (twice) and 100 µm hole sized chambers at 1800 bar using LM20
Microfluidizer (Microfluidics, Westwood, MA, USA). Furthermore, the wet cake was made by draining
the dispersion over a membrane (Durapore, 0.65 µm hydrophilic PVDF “DVPP”, Merck Millipore,
Burlington, MA, USA) in a funnel, driven by vacuum. Obtained cellulose cakes were dried at 140 ◦C to
produce pre-filters and 80 ◦C for filter papers using hot-press (Carver Model 4122CE, Carver, Wabash,
IN, USA). 1. Introduction A novel type of virus removal filter paper was developed
at Uppsala University, which is produced by adapting traditional paper making technology and
consists of 100% cellulose nanofibers [31,32]. The pore size and flux properties of the filter paper can
be controlled, which opens new opportunities to model fundamental aspects of bioprocessing [32–34]. The filter paper was previously validated in numerous studies to remove several large and small-size
model viruses, including retroviruses (xMuLV, 100 nm) [35], parvoviruses (MVM, 20 nm) [32,36],
and model phages (ΦX174, 28 nm) [37–39]. Recently, it was shown that this nanocellulose-based virus
removal filter paper is useful for bioprocessing human plasma-derived IgG [40]. 3 of 18 Biomedicines 2020, 8, 69 In this article, for the first time the filtration of FIX-rich PCC using a nanocellulose-based virus
removal filter paper is described. Furthermore, a two-step size-exclusion nanofiltration process is
developed to remove foulants and ensure efficient virus removal filtration of FIX-rich PCC using
nanocellulose-based virus removal filter paper. FIX-rich PCC was used as a model for a highly
complex plasma-derived product to simulate industrial bioprocesses where impurities may greatly
affect product yield and biosafety. 2.3. Dissolution of Factor IX-rich PCC Lyophilized FIX-rich PCC samples were reconstituted by dissolving in phosphate-buffered saline
(PBS). No visible particles could be seen after reconstitution, and the solution was clear and transparent. Upon dissolution, the conductivity and pH values were 15.4 mS cm−1 and 7.4, respectively. 2.7. Dynamic Light Scattering Particle size distribution was obtained from dynamic light scattering (DLS) using a Zetasizer
Nano ZS (Malvern, UK) instrument. All experiments were performed in triplicates. 2.8. Polyacrylamide Gel Electrophoresis 2.9. Analytical SEHPLC Samples were analyzed by size-exclusion high-pressure liquid chromatography using Hitachi
Chromaster HPLC-UV system with bioZen 1.8 µm SEC-3 (Phenomenex, Torrance, CA, USA) analytical
column. Chromatography was performed with 100 mM sodium phosphate, pH 6.8 mobile phase at
0.3 mL min−1 flow rate for 20 min. 2.5. Scanning Electron Microscopy (SEM) For top-view images samples were fixed onto aluminum stubs with double-adhesive carbon tape,
and for cross-section images the samples were mounted onto aluminum sample holders with screw. Imaging was performed using Zeiss Merlin FEG-SEM instrument (Jena, Germany). To reduce charging 4 of 18 Biomedicines 2020, 8, 69 effects samples were sputtered with Au/Pd with a sputter coater (Polaron, Ashford, UK) was used. The sputtering settings were 4 × 102 mbar and 35 mA, and the sputtering time was 30 s. 2.6. Cryoporometry by Differential Scanning Calorimetry 2.6. Cryoporometry by Differential Scanning Calorimetry 2.8. Polyacrylamide Gel Electrophoresis Protein separation was performed by reducing polyacrylamide gel electrophoresis (SDS-PAGE). Samples were diluted (1:20 v/v) with PBS and Laemmli buffer, and boiled for 10 min. Electrophoretic
separation was carried out at 270 V with Mini-PROTEAN Tetra Vertical Electrophoresis Cell (Bio-Rad,
Hercules, CA, USA). Protein bands were detected with Gel Doc™EZ System (Bio-Rad, Hercules, CA,
USA), and quantified using Image Lab 6.0 analysis software (Bio-Rad). 2.6. Cryoporometry by Differential Scanning Calorimetry Filter paper samples (1.5–2 mg) were soaked into deionized water overnight at room temperature. Water was decanted, and the samples were placed in aluminum crucibles with a lid. Samples were
cooled down to 248.15 K (−25 ◦C) at a rate of 10 K min−1 followed by heating to 277.15 K (4 ◦C) at a
rate of 0.7 K min−1. Measurements were performed in five replicates. The pore size was calculated according to Landry [41]: The pore size was calculated according to Landry [41]: ∆T = −19.082
rp −1.12 −0.1207
(1) (1) where rp is the radius of pore (nm) and ∆T is the difference between the peak maximum for melting
of pore-confined water and peak value for melting of bulk water, experimentally determined at
0.6 ± 0.01 ◦C. where rp is the radius of pore (nm) and ∆T is the difference between the peak maximum for melting
of pore-confined water and peak value for melting of bulk water, experimentally determined at
0.6 ± 0.01 ◦C. 2.10. LCMS Equal amounts (20 µg) of protein samples were taken out for digestion. After reduction and
alkylation, the proteins were on-filter digested by trypsin using 3 kDa centrifugal filters (Millipore
Tullagreen, Ireland) according to a standard operating procedure. Obtained peptides were dried
using a speedvac system. Pellets were resolved in 60 µL of 0.1% formic acid and further diluted
four times prior to nano-LCMS/MS. Tandem mass spectrometry was performed by applying HCD
in the QEx-Orbitrap mass spectrometer (Thermo Finnigan, San Jose, CA, USA), equipped with a
reversed-phase C18-column by 35 min long gradient. Database searches were performed using the Sequest algorithm, embedded in Proteome Discoverer
1.4 (Thermo Fisher Scientific, Waltham, MA, USA) against Homo Sapience proteome extracted from
Uniprot, Release June 2019 with 95% confidence level per protein. 2.11. Bacteriophage Filtration and Titration 3.1. One-Step 33 µm Filtration of FIX-Rich PCC
3.1. One-Step 33 μm Filtration of FIX-Rich PCC 3.1. One-Step 33 µm Filtration of FIX-Rich PCC
3.1. One-Step 33 μm Filtration of FIX-Rich PCC When the FIX-rich PCC at 20 L m−2 volumetric load was filtered through the 33 µm mille-feuille
filter paper, a rapid flux decline was observed, e.g., from about 80 L m−2 h−1 to about 10 L m−2 h−1 at
3 bar overhead pressure. DLS analysis of the feed and permeate samples revealed that the feed sample
showed widely distributed fraction of protein impurities above 70 nm, which were not detectable after
filtration as shown in Figure 1. Notably, these large-size impurities could not be detected in the volume
distribution profiles of the feed sample but only in the intensity distribution plots, which suggests that
the original amount of the aggregates is small. In the permeate sample, no particle fractions above
40 nm were detected by DLS. When the FIX-rich PCC at 20 L m−2 volumetric load was filtered through the 33 μm mille-feuille
filter paper, a rapid flux decline was observed, e.g., from about 80 L m−2 h−1 to about 10 L m−2 h−1 at 3
bar overhead pressure. DLS analysis of the feed and permeate samples revealed that the feed sample
showed widely distributed fraction of protein impurities above 70 nm, which were not detectable
after filtration as shown in Figure 1. Notably, these large-size impurities could not be detected in the
volume distribution profiles of the feed sample but only in the intensity distribution plots, which
suggests that the original amount of the aggregates is small. In the permeate sample, no particle
fractions above 40 nm were detected by DLS. Figure 1. DLS profiles of feed and permeate samples after 1-step filtration through 33 μm filter paper
at 1 and 3 bar. Figure 1. DLS profiles of feed and permeate samples after 1-step filtration through 33 µm filter paper
at 1 and 3 bar. Figure 1. DLS profiles of feed and permeate samples after 1-step filtration through 33 μm filter paper
at 1 and 3 bar. Figure 1. DLS profiles of feed and permeate samples after 1-step filtration through 33 µm filter paper
at 1 and 3 bar. To investigate if significant changes were recorded in the protein molecular weight distribution
in the permeate sample, SDS-PAGE analysis was performed, as shown in Figure 2. 2.11. Bacteriophage Filtration and Titration Coliphages PR772 and ΦX174 were spiked to the pre-filtered solutions to obtain final titer about 106
plaque forming units (PFU) mL−1 before filtration was performed. Bacteriophage titer was determined
by PFU assay by double agar overlay method. Briefly, ten-fold serially diluted bacteriophage samples 5 of 18 Biomedicines 2020, 8, 69 were mixed with host E. coli strains and melted soft agar, and poured on the surface of prepared hard
agar plate, followed by incubation at 37 ◦C for 5 h. Biomedicines 2020, 8, x FOR PEER REVIEW
5 of 18 were mixed with host E. coli strains and melted soft agar, and poured on the surface of prepared hard
agar plate, followed by incubation at 37 ◦C for 5 h. Biomedicines 2020, 8, x FOR PEER REVIEW
5 of 18 p
y
Bacteriophage titer was calculated using Equation (1):
B
t
i
h
tit
l
l t d
i
ti
1 Bacteriophage titer was calculated using Equation (1): log10
PFU mL−1
= log10
N
V·d
(2)
ulated using equation 1:
𝑜𝑔10(𝑃𝐹𝑈 𝑚𝑙−1) = 𝑙𝑜𝑔10 ( 𝑁
V ∙d )
(2) (2)
(2) where N is the number of plaques, V is the volume (typically 0.1 mL) of added virus and d is the
dilution factor. where N is the number of plaques, V is the volume (typically 0.1 mL) of added virus and d is the
dilution factor where N is the number of plaques, V is the volume (typically 0.1 mL) of added virus and d is the
dilution factor. where N is the number of plaques, V is the volume (typically 0.1 mL) of added virus and d is the
dilution factor The virus retention was expressed as log10 reduction value (LRV):
dilution factor. The virus retention was expressed as log10 reduction value (LRV): The virus retention was expressed as log10 reduction value (LRV):
dilution factor. The virus retention was expressed as log10 reduction value (LRV): LRV = log10
PFUfeed
PFUpermeate
(3)
𝐿𝑅𝑉= 𝑙𝑜𝑔10
𝑃𝐹𝑈𝑓𝑒𝑒𝑑
𝑃𝐹𝑈𝑝𝑒𝑟𝑚𝑒𝑎𝑡𝑒
(3). (3)
3). 3.1. One-Step 33 µm Filtration of FIX-Rich PCC
3.1. One-Step 33 μm Filtration of FIX-Rich PCC Additional
proteomics analysis of the detected bands was not performed as it was outside of the scope of the
present work. It is seen in Figure 2 that all major fractions in the permeate sample were reduced
compared to the feed. The observed decrease in total protein fraction is concordant to that reported
earlier for PCC product filtered through Planova 15N filter [14]. In all, it appears that the large
molecular weight protein fractions are the main reason for the observed fouling. To investigate if significant changes were recorded in the protein molecular weight distribution in
the permeate sample, SDS-PAGE analysis was performed, as shown in Figure 2. Additional proteomics
analysis of the detected bands was not performed as it was outside of the scope of the present work. It is seen in Figure 2 that all major fractions in the permeate sample were reduced compared to the
feed. The observed decrease in total protein fraction is concordant to that reported earlier for PCC
product filtered through Planova 15N filter [14]. In all, it appears that the large molecular weight
protein fractions are the main reason for the observed fouling. 6 of 18
6 of 18 Biomedicines 2020, 8, 69
Biomedicines 2020, 8, x FO Figure 2. SDS-PAGE analysis of permeate fractions after 1 step filtration through 33 μm filter paper
kDa
250
150
100
75
50
37
25
20
15
10
Feed
Feed
Permeate
Band 1
Band 2
Band 3
Band 4
Band 5
Band 6
Permeate
1 bar
3 bar
Figure 2. SDS-PAGE analysis of permeate fractions after 1 step filtration through 33 µm filter paper at 1
and 3 bar. Figure 2. SDS-PAGE analysis of permeate fractions after 1 step filtration through 33 μm filter paper
at 1 and 3 bar. kDa
250
150
100
75
50
37
25
20
15
10
Feed
Feed
Perme
Band 1
Band 2
Band 3
Band 4
Band 5
Band 6
Perme
1 bar
3 bar Figure 2. SDS-PAGE analysis of permeate fractions after 1 step filtration through 33 μm filter paper
Figure 2. SDS-PAGE analysis of permeate fractions after 1 step filtration through 33 µm filter paper at 1
and 3 bar. Figure 2. SDS-PAGE analysis of permeate fractions after 1 step filtration through 33 μm filter paper
at 1 and 3 bar. at 1 and 3 bar. 3.1. One-Step 33 µm Filtration of FIX-Rich PCC
3.1. One-Step 33 μm Filtration of FIX-Rich PCC Development and Validation of Two-Step Size-Exclusion Bioprocess for FIX-Rich PCC Nanofiltration
Development and Validation of Two-Step Size-Exclusion Bioprocess for FIX-Rich PCC Nanofiltration 3.2. Development and Validation of Two-Step Size-Exclusion Bioprocess for FIX-Rich PCC Nanofiltration
The nanocellulose-based filter paper platform provides the possibilities to relatively easily tailo
the pore size distribution of the filter paper to a specific cut off value This could be achieved fo
The nanocellulose-based filter paper platform provides the possibilities to relatively easily tailor
the pore-size distribution of the filter paper to a specific cut-offvalue. This could be achieved for
instance by varying the thickness of the filter paper. The nanocellulose-based filter paper platform provides the possibilities to relatively easily tailor
the pore-size distribution of the filter paper to a specific cut-off value. This could be achieved for
instance by varying the thickness of the filter paper. the pore-size distribution of the filter paper to a specific cut-off value. This could be achieved for
instance by varying the thickness of the filter paper. Figure 3 shows the SEM images of the filters with varying thickness, including their top-view
and cross-section It is seen from the images of the cross-sections of the filters that they indeed feature
Figure 3 shows the SEM images of the filters with varying thickness, including their top-view
and cross-section. It is seen from the images of the cross-sections of the filters that they indeed feature
varying thicknesses. y
y
g
p p
Figure 3 shows the SEM images of the filters with varying thickness, including their top-view
and cross-section. It is seen from the images of the cross-sections of the filters that they indeed feature
varying thicknesses. varying thicknesses. varying thicknesses. Figure 3. SEM images of top-view and cross-sections of 6, 11, and 33 μm filter papers. Figure 3. SEM images of top-view and cross-sections of 6, 11, and 33 µm filter papers. Figure 3. SEM images of top-view and cross-sections of 6, 11, and 33 µm filter papers. 7 of 18 Biomedicines 2020, 8, 69 To derive information about the pore size of the filter cryoporometry analysis was performed. Figure 4 shows the typical CP-DSC curves of the studied samples and the boxplots of the derived
pore width modes. Cryoporometry analysis has the benefit that it probes the pores in the wet state,
and it is a relatively quick and highly automated and reliable method. 3.1. One-Step 33 µm Filtration of FIX-Rich PCC
3.1. One-Step 33 μm Filtration of FIX-Rich PCC In this method, the samples are
first frozen to −40 ◦C and then slowly thawed. As the ice crystals start to melt, there is a detectable
endotherm peak. When the water is located inside mesopores (i.e., 2–50 nm pore width), there will be
a melting point depression as opposed to bulk water, present outside pores or in macropores (above
50 nm). In our experiments, bulk water melts at around 0.6 ◦C. The larger the melting point depression,
the smaller are the pores. As seen from Figure 4 there is a trend of decreasing pore width mode with
increasing thickness. Biomedicines 2020, 8, x FOR PEER REVIEW
7 of 18
To derive information about the pore size of the filter cryoporometry analysis was performed. Figure 4 shows the typical CP-DSC curves of the studied samples and the boxplots of the derived
pore width modes. Cryoporometry analysis has the benefit that it probes the pores in the wet state,
and it is a relatively quick and highly automated and reliable method. In this method, the samples
are first frozen to −40 °C and then slowly thawed. As the ice crystals start to melt, there is a detectable
endotherm peak. When the water is located inside mesopores (i.e., 2–50 nm pore width), there will
be a melting point depression as opposed to bulk water, present outside pores or in macropores
(above 50 nm). In our experiments, bulk water melts at around 0.6 °C. The larger the melting point
depression, the smaller are the pores. As seen from Figure 4 there is a trend of decreasing pore width mode with increasing thickness. Figure 4. Typical cryoporometry DSC curves (A) and boxplot of pore width modes (B) for 6, 11, and
33 μm filter papers (n = 7). * p < 0.05, *** p < 0.01
Figure 4. Typical cryoporometry DSC curves (A) and boxplot of pore width modes (B) for 6, 11, and
33 µm filter papers (n = 7). * p < 0.05, *** p < 0.01. Figure 4. Typical cryoporometry DSC curves (A) and boxplot of pore width modes (B) for 6, 11, and
33 μm filter papers (n = 7). * p < 0.05, *** p < 0.01
Figure 4. Typical cryoporometry DSC curves (A) and boxplot of pore width modes (B) for 6, 11, and
33 µm filter papers (n = 7). * p < 0.05, *** p < 0.01. 3.1. One-Step 33 µm Filtration of FIX-Rich PCC
3.1. One-Step 33 μm Filtration of FIX-Rich PCC To assess the particle rejection cut-off for each filter, model probes with 2 different particle sizes
were used in the form of bacteriophages, i.e., PR772 (70 nm) and ΦX174 (28 nm) phages, see Table 1
and 2, respectively. These probes provide a highly sensitive tool for assessing the size-dependent
rejection capability of the filters with varying thickness, i.e., 6, 11, and 33 μm. The 33 μm mille-feuille
filter paper shows the lowest hydraulic flux, i.e., 38 L m−2 h−1 bar−1, and the highest virus removal
capacity for both small- and large-size viruses, i.e., LRV ≥5.7. The 6 μm filter in the series exhibits the
fastest flux, i.e., 405 L m−2 h−1 bar−1, but poor virus removal capacity, i.e., LRV <1 and <2 for 28 nm and
70 nm model phages. The flux and virus removal properties of 11 µm filter are intermediate to the
other two filters, wherein the 11 µm filter paper shows high clearance towards 70 nm virus, i.e., >5.7,
and moderate clearance toward 28 nm one, i.e., LRV 3.5–4.5, and hydraulic flux of 125 L m−2 h−1 bar−1. Interestingly, the small-size virus removal capacity of 11 µm filter decreased with increasing load
volume, whereas that of 33 µm filter remained unaffected under the experimental conditions. The
latter could probably be due to redistribution of flow through the larger pores when the smaller pores
become clogged in 11 µm filter paper. To assess the particle rejection cut-offfor each filter, model probes with 2 different particle sizes were
used in the form of bacteriophages, i.e., PR772 (70 nm) and ΦX174 (28 nm) phages, see Tables 1 and 2,
respectively. These probes provide a highly sensitive tool for assessing the size-dependent rejection
capability of the filters with varying thickness, i.e., 6, 11, and 33 µm. The 33 µm mille-feuille filter
paper shows the lowest hydraulic flux, i.e., 38 L m−2 h−1 bar−1, and the highest virus removal capacity
for both small- and large-size viruses, i.e., LRV ≥5.7. The 6 µm filter in the series exhibits the fastest
flux, i.e., 405 L m−2 h−1 bar−1, but poor virus removal capacity, i.e., LRV <1 and <2 for 28 nm and
70 nm model phages. 3.1. One-Step 33 µm Filtration of FIX-Rich PCC
3.1. One-Step 33 μm Filtration of FIX-Rich PCC The flux and virus removal properties of 11 µm filter are intermediate to the
other two filters, wherein the 11 µm filter paper shows high clearance towards 70 nm virus, i.e., >5.7,
and moderate clearance toward 28 nm one, i.e., LRV 3.5–4.5, and hydraulic flux of 125 L m−2 h−1
bar−1. Interestingly, the small-size virus removal capacity of 11 µm filter decreased with increasing
load volume, whereas that of 33 µm filter remained unaffected under the experimental conditions. The latter could probably be due to redistribution of flow through the larger pores when the smaller
pores become clogged in 11 µm filter paper. 8 of 18 Biomedicines 2020, 8, 69 Table 1. LRVs for 70 nm (PR772) bacteriophages filtered through 6, 11, and 33 µm filter papers. The results represent the virus clearance data of virus-spiked PBS. Green color code denotes high virus
clearance LRV > 5; Yellow denotes moderate virus clearance (2 < LRV < 5); and pink denotes low virus
clearance (LRV < 1). Thickness (µm)
Load Volume
7.5 L m−2
15 L m−2
23 L m−2
6
1.6 ± 0.2
1.3 ± 0.1
1.1 ± 0.3
11
>5.5 ± 0.2
>5.5 ± 0.2
>5.5 ± 0.2
33
>5.7 ± 0.2
>5.7 ± 0.2
>5.7 ± 0.2
Table 2. LRVs for 28 nm (ΦX174) bacteriophages filtered through 6, 11, and 33 µm filter papers. The results represent the virus clearance data of virus-spiked PBS. Green color code denotes high virus
clearance LRV > 5; Yellow denotes moderate virus clearance (2 < LRV < 5); and pink denotes low virus
edicines 2020, 8, x FOR PEER REVIEW
8
Table 2. LRVs for 28 nm (ΦΧ174) bacteriophages filtered through 6, 11, and 33 μm filter papers. The
results represent the virus clearance data of virus-spiked PBS. Green color code denotes high virus
clearance LRV>5; Yellow denotes moderate virus clearance (2<LRV<5); and pink denotes low virus
l
(LRV 2) Table 2. LRVs for 28 nm (ΦX174) bacteriophages filtered through 6, 11, and 33 µm filter papers. The results represent the virus clearance data of virus-spiked PBS. Green color code denotes high virus
clearance LRV > 5; Yellow denotes moderate virus clearance (2 < LRV < 5); and pink denotes low virus
clearance (LRV < 2). Table 2. LRVs for 28 nm (ΦΧ174) bacteriophages filtered through 6, 11, and 33 μm filter papers. 3.1. One-Step 33 µm Filtration of FIX-Rich PCC
3.1. One-Step 33 μm Filtration of FIX-Rich PCC The
results represent the virus clearance data of virus-spiked PBS. Green color code denotes high virus
clearance LRV>5; Yellow denotes moderate virus clearance (2<LRV<5); and pink denotes low virus
clearance (LRV <2). Table 2. LRVs for 28 nm (ΦX174) bacteriophages filtered through 6, 11, and 33 µm filter papers. The results represent the virus clearance data of virus-spiked PBS. Green color code denotes high virus
clearance LRV > 5; Yellow denotes moderate virus clearance (2 < LRV < 5); and pink denotes low virus
clearance (LRV < 2). Table 2. LRVs for 28 nm (ΦΧ174) bacteriophages filtered through 6, 11, and 33 μm filter papers. The
results represent the virus clearance data of virus-spiked PBS. Green color code denotes high virus
clearance LRV>5; Yellow denotes moderate virus clearance (2<LRV<5); and pink denotes low virus
clearance (LRV <2). Thickness (µm)
Load Volume
7.5 L m−2
15 L m−2
23 L m−2
6
0.7 ± 0.3
0.8 ± 0.3
0.8 ± 0.2
11
4.5 ± 0.5
3.7 ± 0.4
3.5 ± 0.7
33
>5.7 ± 0.4
>5.7 ± 0.4
5.7 ± 0.4
3 3 Two-Step 6 µm/33 µm Filtration of FIX-Rich PCC
Thickness
(μm)
Load Volume
7.5 L m−2
15 L m−2
23 L m−2
6
0.7 ± 0.3
0.8 ± 0.3
0.8 ± 0.2
11
4.5 ± 0.5
3.7 ± 0.4
3.5 ± 0.7
33
>5.7 ± 0.4
>5.7 ± 0.4
5.7 ± 0.4
3 3 Two-Step 6 μm/33 μm Filtration of FIX-Rich PCC 3.3. Two-Step 6 µm/33 µm Filtration of FIX-Rich PCC
3.3. Two-Step 6 μm/33 μm Filtration of FIX-Rich PCC Figure 5 shows the permeate flux through the 6 µm/33 µm filtration sequence at 1 bar. Rapid flux
decline was observed for the permeate after initial plateau. Please note that the flux of pre-filtrate was
so fast that it was not recorded. Figure 5 shows the permeate flux through the 6 μm/33 μm filtration sequence at 1 bar. Rapid
flux decline was observed for the permeate after initial plateau. Please note that the flux of pre-filtrate
was so fast that it was not recorded. Figure 5. Observed permeate flux for two-step 6 μm/33 μm μm filtration at 1 bar. Figure 5. Observed permeate flux for two-step 6 µm/33 µm µm filtration at 1 bar. Figure 5. Observed permeate flux for two-step 6 μm/33 μm μm filtration at 1 bar. Figure 5. 3.1. One-Step 33 µm Filtration of FIX-Rich PCC
3.1. One-Step 33 μm Filtration of FIX-Rich PCC Figure 7 shows the results of SDS-PAGE analysis of the collected samples. It is seen from
graph that all bands showed decreasing intensity. Even after 6 μm pre-filtration, some decline i
band intensity was observed. The bands for lower Mw fractions, i.e., bands 4–6, were reduced
greater extent after pre-filtration than those of the larg Figure 7. SDS-PAGE analysis of pre-filtrate and permeate fractions after 2-step filtration throu
Figure 7. SDS-PAGE analysis of pre-filtrate and permeate fractions after 2-step filtration through
6 µm/33 µm filter paper at 1 bar. Figure 7. SDS-PAGE analysis of pre-filtrate and permeate fractions after 2-step filtration throu
Figure 7. SDS-PAGE analysis of pre-filtrate and permeate fractions after 2-step filtration through
6 µm/33 µm filter paper at 1 bar. μm/33 μm filter paper at 1 bar. Overall, the results from the filtration with 6 μm/33 μm sequence suggest that the large
impurities were not removed during the pre-filtration step and, subsequently, caused filter fo
d h
b l
d
i ld d
i
h
d
Overall, the results from the filtration with 6 µm/33 µm sequence suggest that the large Mw
impurities were not removed during the pre-filtration step and, subsequently, caused filter fouling and
thereby low product yield during the second step. 3.1. One-Step 33 µm Filtration of FIX-Rich PCC
3.1. One-Step 33 μm Filtration of FIX-Rich PCC Observed permeate flux for two-step 6 µm/33 µm µm filtration at 1 bar. Figure 6 shows the DLS results for pre-filtrate and permeate samples for the 6 μm/33 μm
filtration sequence at 1 bar. It was observed the fraction of large colloids was not removed by 6 μm
pre-filtration. However, no aggregates were observed in the permeate sample after filtration through
f l
Figure 6 shows the DLS results for pre-filtrate and permeate samples for the 6 µm/33 µm filtration
sequence at 1 bar. It was observed the fraction of large colloids was not removed by 6 µm pre-filtration. However, no aggregates were observed in the permeate sample after filtration through 33 µm filter. 9 of 18
y
μ
hrough Biomedicines 2020, 8, 69
pre-filtration. Howe
33
filt 3 μm filter. Figure 6. DLS analysis 11 µm filter with further filtration with 33 µm filter at 1 bar TMP DLS profiles of
feed, pre-filtrate, and permeate samples after 2-step filtration through 11 µm/33 µm filter paper at 1 bar. Biomedicines 2020, 8, x FOR PEER REVIEW
Figure 6. DLS analysis 11 µm filter with further filtration with 33 µm filter at 1 bar TMP DLS prof
of feed, pre-filtrate, and permeate samples after 2-step filtration through 11 μm/33 μm filter pape OR PEER REVIEW Figure 6. DLS analysis 11 µm filter with further filtration with 33 µm filter at 1 bar TMP DLS profiles of
feed, pre-filtrate, and permeate samples after 2-step filtration through 11 µm/33 µm filter paper at 1 bar. Figure 6. DLS analysis 11 µm filter with further filtration with 33 µm filter at 1 bar TMP DLS pr
of feed, pre-filtrate, and permeate samples after 2-step filtration through 11 μm/33 μm filter pap Figure 7 shows the results of SDS-PAGE analysis of the collected samples. It is seen from the
graph that all bands showed decreasing intensity. Even after 6 µm pre-filtration, some decline in the
band intensity was observed. The bands for lower Mw fractions, i.e., bands 4–6, were reduced to a
greater extent after pre-filtration than those of the larger Mw, i.e., bands 1–3. In the permeate sample
all band intensities were further decreased. LCMS analysis suggested that key coagulation factors IX,
X, V as well as prothrombin were not removed following the two-step 6 µm/33 µm filtration sequence,
as shown in Appendix Tables A1–A3. 1 bar. and thereby low product yield during the seco
3.4. Two-Step 11 µm/33 µm Filtration of FIX-Rich PCC 3.4. Two-Step 11 μm/33 μm Filtration of FIX-Rich PCC
In another set of experiments, the pre-filtration was performed using 11-μm filter paper follo
by filtration with 33 μm filter at 1 bar. Figure 8 shows the flux data of permeate for 11 μm/33
In another set of experiments, the pre-filtration was performed using 11-µm filter paper followed
by filtration with 33 µm filter at 1 bar. Figure 8 shows the flux data of permeate for 11 µm/33 µm
filtration sequence. Increasing the thickness of the pre-filter from 6 to 11 µm significantly affected the 10 of 18 Biomedicines 2020, 8, 69 results. The flux values for pre-filtration indicated rapid fouling as observed above for 33 µm filtration. However, in the second step of 11 µm/33 µm filtration sequence, i.e., through 33 µm filters, stable flux
was observed for the entire processed volume, Figure 7. The results contrast starkly those observed for
6 µm/33 µm filtration sequence as shown in Figure 4. Biomedicines 2020, 8, x FOR PEER REVIEW
10 of 18
Biomedicines 2020, 8, x FOR PEER REVIEW
10 of 18 Figure 8. Observed permeate flux for two-step 11 μm/33 μm μm filtration at 1 bar. Figure 8. Observed permeate flux for two-step 11 µm/33 µm µm filtration at 1 bar. Figure 8. Observed permeate flux for two-step 11 μm/33 μm μm filtration at 1 bar. Figure 8. Observed permeate flux for two-step 11 μm/33 μm μm filtration at 1 bar. Figure 8. Observed permeate flux for two-step 11 µm/33 µm µm filtration at 1 bar. Figure 8. Observed permeate flux for two-step 11 μm/33 μm μm filtration at 1 bar. Figure 9 shows the results of DLS analysis of the pre-filtrate and permeate samples. It is seen
that the fraction of large-size impurities, which was clearly visible in the feed solution, was absent
both in the pre-filtrate and permeate fractions of 11 μm/33 μm filtration sequence. The latter suggests
that pre-filtration with 11 μm filter paper efficiently removes the large-size impurities, unlike pre-
filtration with 6 μm filter paper. Additional SEHPLC analysis was performed on these samples as
shown in Figure 8. It is seen in the graph that the peak retention times and relative intensities are
similar in all three samples except for the early peak at 0.5 min in the feed sample. and thereby low product yield during the seco
3.4. Two-Step 11 µm/33 µm Filtration of FIX-Rich PCC This peak, which
corresponds to the largest protein fraction was not detectable in pre-filtrate and permeate samples. Figure 9 shows the results of DLS analysis of the pre-filtrate and permeate samples. It is seen that
the fraction of large-size impurities, which was clearly visible in the feed solution, was absent both in
the pre-filtrate and permeate fractions of 11 µm/33 µm filtration sequence. The latter suggests that
pre-filtration with 11 µm filter paper efficiently removes the large-size impurities, unlike pre-filtration
with 6 µm filter paper. Additional SEHPLC analysis was performed on these samples as shown in
Figure 8. It is seen in the graph that the peak retention times and relative intensities are similar in all
three samples except for the early peak at 0.5 min in the feed sample. This peak, which corresponds to
the largest protein fraction was not detectable in pre-filtrate and permeate samples. Figure 9 shows the results of DLS analysis of the pre-filtrate and permeate samples. It is seen
that the fraction of large-size impurities, which was clearly visible in the feed solution, was absent
both in the pre-filtrate and permeate fractions of 11 μm/33 μm filtration sequence. The latter suggests
that pre-filtration with 11 μm filter paper efficiently removes the large-size impurities, unlike pre-
filtration with 6 μm filter paper. Additional SEHPLC analysis was performed on these samples as
shown in Figure 8. It is seen in the graph that the peak retention times and relative intensities are
similar in all three samples except for the early peak at 0.5 min in the feed sample. This peak, which
corresponds to the largest protein fraction was not detectable in pre-filtrate and permeate samples. Figure 9. DLS (A) and SEHPLC (B) profiles of feed, pre-filtrate, and permeate samples after two-step
Figure 9. DLS (A) and SEHPLC (B) profiles of feed, pre-filtrate, and permeate samples after two-step
Figure 9. DLS (A) and SEHPLC (B) profiles of feed, pre-filtrate, and permeate samples after two-step
filtration through 11 µm/33 µm filter paper at 1 bar. Figure 9. DLS (A) and SEHPLC (B) profiles of feed, pre-filtrate, and permeate samples after two-step
Figure 9. DLS (A) and SEHPLC (B) profiles of feed, pre-filtrate, and permeate samples after two-step
Figure 9. DLS (A) and SEHPLC (B) profiles of feed, pre-filtrate, and permeate samples after two-step
filtration through 11 µm/33 µm filter paper at 1 bar. and thereby low product yield during the seco
3.4. Two-Step 11 µm/33 µm Filtration of FIX-Rich PCC SDS-PAGE analysis of pre-filtrate and permeate fractions after 2-step filtration through
11 µm/33 µm filter paper at 1 bar. medicines 2020, 8, x FOR PEER REVIEW
11 of
Figure 10. SDS-PAGE analysis of pre-filtrate and permeate fractions after 2-step filtration through 11
/33
filt
t 1 b Figure 10. SDS-PAGE analysis of pre-filtrate and permeate fractions after 2-step filtration through 11
μm/33 μm filter paper at 1 bar. Figure 10. SDS-PAGE analysis of pre-filtrate and permeate fractions after 2-step filtration through
11 µm/33 µm filter paper at 1 bar. Figure 10. SDS-PAGE analysis of pre-filtrate and permeate fractions after 2-step filtration through 11 Based on the above results, it was concluded that pre-filtration with 11 µm pre-filter removes
the aggregates, which in turn greatly enhances the yield of the 33 µm filtration. To confirm the high
virus removal capacity of 33 µm filter, the filter paper was loaded with much larger volume than that
tested earlier, i.e., 175 L m−2. Figure 11 shows the result of the large load filtration. Following the
filtration, no abrupt filter fouling was detected for the entire processed volume, although some flux
decline could be observed (Figure 11a). Under the experimental conditions, it is estimated that Vmax
of the process will be roughly around 500 L m−2, which is a drastic improvement from 20 L m−2 when
filtering in a single-step process through 33 µm filter paper. Furthermore, the filter paper showed
high model small-size virus removal capacity, wherein LRV was ≥ 5 in all collected fractions (Figure
11b). In particular, no detectable PFUs were observed at all up to 90 L m−2 load volume. In the last
fractions only residual breakthrough (1–2 PFUs per agar plate, corresponding to 0.7 PFU mL−1) was
detected. Thus, it was confirmed that the two-step 11 µm/33 µm filtration provides enhanced
throughput and good capacity to remove small-size virus without abrupt fouling even when
challenged with a relatively large load. Based on the above results, it was concluded that pre-filtration with 11 µm pre-filter removes
the aggregates, which in turn greatly enhances the yield of the 33 µm filtration. To confirm the high
virus removal capacity of 33 µm filter, the filter paper was loaded with much larger volume than that
tested earlier, i.e., 175 L m−2. Figure 11 shows the result of the large load filtration. and thereby low product yield during the seco
3.4. Two-Step 11 µm/33 µm Filtration of FIX-Rich PCC Following the
filtration, no abrupt filter fouling was detected for the entire processed volume, although some flux
decline could be observed (Figure 11A). Under the experimental conditions, it is estimated that Vmax
of the process will be roughly around 500 L m−2, which is a drastic improvement from 20 L m−2 when
filtering in a single-step process through 33 µm filter paper. Furthermore, the filter paper showed high
model small-size virus removal capacity, wherein LRV was ≥5 in all collected fractions (Figure 11B). In particular, no detectable PFUs were observed at all up to 90 L m−2 load volume. In the last fractions
only residual breakthrough (1–2 PFUs per agar plate, corresponding to 0.7 PFU mL−1) was detected. Thus, it was confirmed that the two-step 11 µm/33 µm filtration provides enhanced throughput and
good capacity to remove small-size virus without abrupt fouling even when challenged with a relatively
large load. μ
/
μ
p p
Based on the above results, it was concluded that pre-filtration with 11 µm pre-filter removes
the aggregates, which in turn greatly enhances the yield of the 33 µm filtration. To confirm the high
virus removal capacity of 33 µm filter, the filter paper was loaded with much larger volume than that
tested earlier, i.e., 175 L m−2. Figure 11 shows the result of the large load filtration. Following the
filtration, no abrupt filter fouling was detected for the entire processed volume, although some flux
decline could be observed (Figure 11a). Under the experimental conditions, it is estimated that Vmax
of the process will be roughly around 500 L m−2, which is a drastic improvement from 20 L m−2 when
filtering in a single-step process through 33 µm filter paper. Furthermore, the filter paper showed
high model small-size virus removal capacity, wherein LRV was ≥ 5 in all collected fractions (Figure
11b). In particular, no detectable PFUs were observed at all up to 90 L m−2 load volume. In the last
fractions only residual br Figure 11. Observed flux for FIX-rich PCC permeate for 2-step 11 μm/33 μm filtration with 175 L m-2
load volume at 3 bar (A) and LRV for ΦX174 phage (B). Figure 11. Observed flux for FIX-rich PCC permeate for 2-step 11 μm/33 μm filtration with 175 L m-2
load volume at 3 bar (A) and LRV for ΦX174 phage (B)
Figure 11. and thereby low product yield during the seco
3.4. Two-Step 11 µm/33 µm Filtration of FIX-Rich PCC filtration through 11 μm/33 μm filter paper at 1 bar. The results of the SDS-PAGE analysis for 11 μm /33 μm filtration sequence are summarized in
Figure 10. It is seen from the graph that the band intensities were reduced in the pre-filtrate and
permeate samples as compared to the feed. It should be noted that in general the band intensities
were reduced to a greater extent after pre-filtration with 11 μm filter than with 6 μm filter. The
decrease of band intensity levels in the permeate sample passed through the 33 μm filter after 11 μm
filtration through 11 μm/33 μm filter paper at 1 bar. The results of the SDS-PAGE analysis for 11 μm /33 μm filtration sequence are summarized in
Figure 10. It is seen from the graph that the band intensities were reduced in the pre-filtrate and
permeate samples as compared to the feed. It should be noted that in general the band intensities
were reduced to a greater extent after pre-filtration with 11 μm filter than with 6 μm filter. The
decrease of band intensity levels in the permeate sample passed through the 33 μm filter after 11 μm
The results of the SDS-PAGE analysis for 11 µm /33 µm filtration sequence are summarized in
Figure 10. It is seen from the graph that the band intensities were reduced in the pre-filtrate and
permeate samples as compared to the feed. It should be noted that in general the band intensities were
reduced to a greater extent after pre-filtration with 11 µm filter than with 6 µm filter. The decrease of
band intensity levels in the permeate sample passed through the 33 µm filter after 11 µm filtration was
much less drastic than that for 6 µm/33 µm filtration sequence. In particular, no significant changes 11 of 18 Biomedicines 2020, 8, 69 were observed for bands 1, 2, and 4. For bands 3, 5, and 6 some intensity reduction was further detected
in the permeate sample. LCMS analysis suggested that key coagulation factors IX, X, V as well as
prothrombin were not removed following the two-step 11 µm/33 µm filtration sequence (for details see
Appendix Tables A1, A4 and A5). Biomedicines 2020, 8, x FOR PEER REVIEW
11 of 18 Figure 10. SDS-PAGE analysis of pre-filtrate and permeate fractions after 2-step filtration through 11
μm/33 μm filter paper at 1 bar. Figure 10. 4. Discussion In this article, the filtration of a highly challenging hematologic product was investigated. Considering that FIX-rich PCC inherently consists of many bioactive components and some impurities,
the virus removal filtration of this product is difficult without fouling. The virus removal filtration of
PCC was previously reported using Ultipor DV50 filters, which are dedicated for removal of large-size
viruses but do not ensure viral safety against parvoviruses [2]. Filtration of PCC through small-size
virus removal filters, e.g., Planova 15N, resulted in nearly 39% total protein loss and reduced FIX
and FII activity, which was ascribed to presence of large-size complexes between clotting factors and
high molecular weight impurities [14]. It was further reported in the same study that filtration of
highly purified FIX through Planova 15N not only did not result in the decrease of FIX activity but also
improved its purity [14]. Biomedicines 2020, 8, x FOR PEER REVIEW
12 of 18
of large-size viruses but do not ensure viral safety against parvoviruses [2]. Filtration of PCC through
small-size virus removal filters, e.g., Planova 15N, resulted in nearly 39% total protein loss and
reduced FIX and FII activity, which was ascribed to presence of large-size complexes between clotting
factors and high molecular weight impurities [14]. It was further reported in the same study that
f l
f h
hl
f d
I
h
h
l
l
d d
l
h d
f
I In this work, in order to achieve high virus removal capacity combined with reduced fouling,
a tailored two-step process of filtration with nanocellulose-based filter paper was developed. In particular, sacrificial pre-filters with a thickness of 6 and 11 µm were tested. The increased
thickness of the filters resulted in tighter pore structure as detected by cryoporometry. The observed
effect is explained as follows and illustrated in Figure 12. The mille-feuille filter paper consists of a
stratified 3-dimensional network of cellulose nanofibers, producing a mesh-like stricture. The layered
structure is illustrated in the side-view panel of Figure 12. Considering that the nanofibers are randomly
distributed in each layer, the pores, which percolate throughout the entire depth of the filter, become
tighter with increasing number of layers. The latter is reflected, e.g., in improved virus clearance
properties with increased thickness or enhanced aggregate removal properties. 4. Discussion Based on the results of
PFU titrations of 27 and 70 nm phage particles, it was concluded that the tested filters show varying
particle size rejection threshold as the thickness of the filter is increased. Thus, the observed effect is
due to the combination of the receding pore size and depth effects (increased tortuosity). The latter
enables using pre-filters with tailored cut-offto remove protein aggregates, which eventually results in
improved flux through the dedicated virus removal filter. filtration of highly purified FIX through Planova 15N not only did not result in the decrease of FIX
activity but also improved its purity [14]. In this work, in order to achieve high virus removal capacity combined with reduced fouling, a
tailored two-step process of filtration with nanocellulose-based filter paper was developed. In
particular, sacrificial pre-filters with a thickness of 6 and 11 μm were tested. The increased thickness
of the filters resulted in tighter pore structure as detected by cryoporometry. The observed effect is
explained as follows and illustrated in Figure 12. The mille-feuille filter paper consists of a stratified
3-dimensional network of cellulose nanofibers, producing a mesh-like stricture. The layered structure
is illustrated in the side-view panel of Figure 12. Considering that the nanofibers are randomly
distributed in each layer, the pores, which percolate throughout the entire depth of the filter, become
tighter with increasing number of layers. The latter is reflected, e.g., in improved virus clearance
properties with increased thickness or enhanced aggregate removal properties. Based on the results
of PFU titrations of 27 and 70 nm phage particles, it was concluded that the tested filters show varying
particle size rejection threshold as the thickness of the filter is increased. Thus, the observed effect is
due to the combination of the receding pore size and depth effects (increased tortuosity). The latter
enables using pre-filters with tailored cut-off to remove protein aggregates, which eventually results
in improved flux through the dedicated virus removal filter. Figure 12. Illustration of the mechanism of virus removal with increased thickness of nanocellulose-
based filter paper. Yellow symbols represent large-size model virus and red symbols represent small-
size viruses. Increased thickness of the filter results in tighter pores and enhanced virus clearance. and thereby low product yield during the seco
3.4. Two-Step 11 µm/33 µm Filtration of FIX-Rich PCC Observed flux for FIX-rich PCC permeate for 2-step 11 µm/33 µm filtration with 175 L m−2
load volume at 3 bar (A) and LRV for ΦX174 phage (B). Figure 11. Observed flux for FIX-rich PCC permeate for 2-step 11 μm/33 μm filtration with 175 L m-2
load volume at 3 bar (A) and LRV for ΦX174 phage (B). Figure 11. Observed flux for FIX-rich PCC permeate for 2-step 11 μm/33 μm filtration with 175 L m-2
l
d
l
b
A
d
f
h
Figure 11. Observed flux for FIX-rich PCC permeate for 2-step 11 µm/33 µm filtration with 175 L m−2
load volume at 3 bar (A) and LRV for ΦX174 phage (B). Biomedicines 2020, 8, 69 12 of 18 12 of 18 4. Discussion Overall, the two-step approach presented here is based on the size-exclusion principles and is
the efo e
obu t It
ould thu
fu the
be ada ted i
the
a ufa tu i
of othe
otei
ba ed
Figure 12. Illustration of the mechanism of virus removal with increased thickness of
nanocellulose-based filter paper. Yellow symbols represent large-size model virus and red symbols
represent small-size viruses. Increased thickness of the filter results in tighter pores and enhanced
virus clearance. Figure 12. Illustration of the mechanism of virus removal with increased thickness of nanocellulose-
based filter paper. Yellow symbols represent large-size model virus and red symbols represent small-
size viruses. Increased thickness of the filter results in tighter pores and enhanced virus clearance. Overall, the two-step approach presented here is based on the size-exclusion principles and is
Figure 12. Illustration of the mechanism of virus removal with increased thickness of
nanocellulose-based filter paper. Yellow symbols represent large-size model virus and red symbols
represent small-size viruses. Increased thickness of the filter results in tighter pores and enhanced
virus clearance. 13 of 18 Biomedicines 2020, 8, 69 Overall, the two-step approach presented here is based on the size-exclusion principles and
is therefore robust. It could thus further be adapted in the manufacturing of other protein-based
pharmaceutics, too, including recombinant proteins wherein impurities in the form of host cell proteins
may greatly affect the final yield of the biologics during virus removal nanofiltration. Author Contributions: For research articles with several authors, a short paragraph specifying their individual
contributions must be provided.
The following statements should be used “Conceptualization, A.M.
(Albert Mihranyan) and M.R.; methodology, A.M. (Albert Mihranyan), L.M., A.M. (Athanasios Mantas); supply of
material, E.G., A.K.; formal analysis, L.M.; investigation, L.M, A.M. (Athanasios Mantas); resources, A.M. (Albert
Mihranyan); data curation, L.M. and A.M. (Athanasios Mantas); writing—original draft preparation, L.M., A.M.
(Albert Mihranyan); writing—review and editing, A.M. (Albert Mihranyan) L.M., R.M., A.K.; visualization, L.M.;
supervision, A.M. (Albert Mihranyan); project administration, A.M. (Albert Mihranyan); funding acquisition,
A.M. (Albert Mihranyan). All authors have read and agreed to the published version of the manuscript. Funding: The project was funded by Knut and Alice Wallenberg Foundation (Bridge grant 2018.01141), Swedish
Research Council [Vetenskapsrådet No. 2016-05715], and EU EIT Health Innovation by Idea program [VIREPAP
19104]. EIT Health is supported by the European Institute of Innovation and Technology (EIT), a body of the
European Union that receives support from the European Union’ s Horizon 2020 Research and innovation program. 5. Conclusions A two-step process was developed to both enhance filtration capacity (25-fold) and achieve high
clearance of small-size viruses (LRV >5) using appropriate pre-filter paper. Large-size aggregates were
the main foulants in the feed solution, and by tailoring the properties of the pre-filters the foulants
were efficiently removed. In particular, 11 µm/33 µm filtration was found most suitable. The presented
approach could potentially be applied for bioprocessing other protein-based drugs, both derived from
plasma and produced by recombinant approaches. The article further provides new insights regarding
the mechanism of virus removal in the nanocellulose-based filter paper, highlighting the combined
effect of size exclusion and tortuosity of pore network. Author Contributions: For research articles with several authors, a short paragraph specifying their individual
contributions must be provided. The following statements should be used “Conceptualization, A.M. (Albert Mihranyan) and M.R.; methodology, A.M. (Albert Mihranyan), L.M., A.M. (Athanasios Mantas); supply of
material, E.G., A.K.; formal analysis, L.M.; investigation, L.M, A.M. (Athanasios Mantas); resources, A.M. (Albert
Mihranyan); data curation, L.M. and A.M. (Athanasios Mantas); writing—original draft preparation, L.M., A.M. (Albert Mihranyan); writing—review and editing, A.M. (Albert Mihranyan) L.M., R.M., A.K.; visualization, L.M.;
supervision, A.M. (Albert Mihranyan); project administration, A.M. (Albert Mihranyan); funding acquisition,
A.M. (Albert Mihranyan). All authors have read and agreed to the published version of the manuscript. Funding: The project was funded by Knut and Alice Wallenberg Foundation (Bridge grant 2018.01141), Swedish
Research Council [Vetenskapsrådet No. 2016-05715], and EU EIT Health Innovation by Idea program [VIREPAP
19104]. EIT Health is supported by the European Institute of Innovation and Technology (EIT), a body of the
European Union that receives support from the European Union’ s Horizon 2020 Research and innovation program. Acknowledgments: For LCMS analysis, the services of the Mass Spectrometry Based Proteomics Facility in
Uppsala were used. The authors thank Ganna Shevchenko, Alexander Falk, and Jonas Bergquist for assistance in
performing LCMS analysis. Conflicts of Interest: The corresponding author (A.M. (Albert Mihranyan)) is the inventor behind the IP pertaining
to virus removal filter paper. Acknowledgments: For LCMS analysis, the services of the Mass Spectrometry Based Proteomics Facility in
Uppsala were used. The authors thank Ganna Shevchenko, Alexander Falk, and Jonas Bergquist for assistance in
performing LCMS analysis. Appendix A Table A1. LCMS analysis of FIX-PCC feed solution. Table A1. LCMS analysis of FIX-PCC feed solution. Accession
Description
Score
Coverage
MW [kDa]
calc. pI
P0C0L5
Complement C4-B
1920.89
76.38
192.6
7.27
P0C0L4
Complement C4-A
1898.90
76.03
192.7
7.08
P00734
Prothrombin
1767.89
75.56
70.0
5.90
P19823
Inter-alpha-trypsin inhibitor heavy chain H2
917.12
53.59
106.4
6.86
P19827
Inter-alpha-trypsin inhibitor heavy chain H1
685.72
55.76
101.3
6.79
Q06033
Inter-alpha-trypsin inhibitor heavy chain H3
257.18
40.22
99.8
5.74
P02760
Protein AMBP
199.11
27.56
39.0
6.25
P00740
Coagulation factor IX
134.15
47.29
51.7
5.47
P00742
Coagulation factor X
59.86
38.52
54.7
5.94
P02768
Serum albumin
48.49
28.41
69.3
6.28
P49747
Cartilage oligomeric matrix protein
41.93
14.27
82.8
4.60
P67936
Tropomyosin alpha-4 chain
32.67
39.11
28.5
4.69
P01857
Immunoglobulin heavy constant gamma 1
32.57
35.15
36.1
8.19
P01834
Immunoglobulin kappa constant
30.33
39.25
11.8
6.52
P07225
Vitamin K-dependent protein S
26.15
12.72
75.1
5.67
P0DOY3
Immunoglobulin lambda constant 3
21.63
78.30
11.3
7.24 14 of 18 Biomedicines 2020, 8, 69 Table A1. Cont. Table A1. Cont. Accession
Description
Score
Coverage
MW [kDa]
calc. pI
P51884
Lumican
21.32
19.53
38.4
6.61
B9A064
Immunoglobulin lambda-like polypeptide 5
19.49
41.12
23.0
8.84
P04004
Vitronectin
19.13
14.02
54.3
5.80
P01861
Immunoglobulin heavy constant gamma 4
19.09
20.18
35.9
7.36
Q08380
Galectin-3-binding protein
18.16
9.74
65.3
5.27
P07359
Platelet glycoprotein Ib alpha chain
17.06
8.28
71.5
6.29
P35443
Thrombospondin-4
16.88
6.66
105.8
4.68
P01876
Immunoglobulin heavy constant alpha 1
15.74
24.93
37.6
6.51
P01859
Immunoglobulin heavy constant gamma 2
13.53
21.78
35.9
7.59
P01871
Immunoglobulin heavy constant mu
8.22
4.64
49.4
6.77
Q14515
SPARC-like protein 1
7.09
6.93
75.2
4.81
P04070
Vitamin K-dependent protein C
5.36
7.16
52.0
6.28
O95810
Caveolae-associated protein 2
4.07
4.71
47.1
5.21
P16070
CD44 antigen
3.37
2.83
81.5
5.33
Q99436
Proteasome subunit beta type-7
2.23
8.30
29.9
7.68
P12259
Coagulation factor V
1.95
0.81
251.5
6.05
P07237
Protein disulfide-isomerase
1.69
3.35
57.1
4.87
P10909
Clusterin
1.53
4.01
52.5
6.27 Table A2. LCMS analysis of FIX-PCC pre-filtrate following filtration with 6 µm filter. Table A2. LCMS analysis of FIX-PCC pre-filtrate following filtration with 6 µm filter. Accession
Description
Score
Coverage
MW [kDa]
calc. Appendix A pI
P00734
Prothrombin
2759.75
73.31
70.0
5.90
P0C0L5
Complement C4-B
2411.85
78.61
192.6
7.27
P0C0L4
Complement C4-A
2379.43
75.80
192.7
7.08
P19823
Inter-alpha-trypsin inhibitor heavy chain H2
1480.56
56.77
106.4
6.86
P19827
Inter-alpha-trypsin inhibitor heavy chain H1
1167.61
54.45
101.3
6.79
Q06033
Inter-alpha-trypsin inhibitor heavy chain H3
352.03
43.60
99.8
5.74
P02760
Protein AMBP
263.80
27.56
39.0
6.25
P00740
Coagulation factor IX
216.46
54.66
51.7
5.47
P00742
Coagulation factor X
90.46
37.09
54.7
5.94
P02768
Serum albumin
62.34
36.45
69.3
6.28
P01857
Immunoglobulin heavy constant gamma 1
47.83
39.09
36.1
8.19
P04004
Vitronectin
45.60
23.64
54.3
5.80
P49747
Cartilage oligomeric matrix protein
43.78
19.82
82.8
4.60
P51884
Lumican OS=Homo sapiens
43.31
42.90
38.4
6.61
P01834
Immunoglobulin kappa constant
40.16
49.53
11.8
6.52
P67936
Tropomyosin alpha-4 chain
39.83
40.32
28.5
4.69
P07359
Platelet glycoprotein Ib alpha chain
31.44
12.12
71.5
6.29
P0DOY2
Immunoglobulin lambda constant 2
29.10
75.47
11.3
7.24
Q08380
Galectin-3-binding protein
28.60
18.80
65.3
5.27
P01861
Immunoglobulin heavy constant gamma 4
28.41
22.02
35.9
7.36
P01876
Immunoglobulin heavy constant alpha 1
23.24
25.78
37.6
6.51
B9A064
Immunoglobulin lambda-like polypeptide 5
22.01
38.79
23.0
8.84
P07225
Vitamin K-dependent protein S
20.55
14.35
75.1
5.67
P01859
Immunoglobulin heavy constant gamma 2
19.80
24.23
35.9
7.59
Q14515
SPARC-like protein 1
19.72
9.19
75.2
4.81
P35443
Thrombospondin-4
14.73
4.79
105.8
4.68
P12259
Coagulation factor V
8.97
2.34
251.5
6.05
P13591
Neural cell adhesion molecule 1
8.03
4.55
94.5
4.87
P12814
Alpha-actinin-1
7.56
3.48
103.0
5.41
P0CG38
POTE ankyrin domain family member I
7.35
3.07
121.2
6.21
P01871
Immunoglobulin heavy constant mu
7.06
8.61
49.4
6.77
P04070
Vitamin K-dependent protein C
5.93
10.63
52.0
6.28
P16070
CD44 antigen
5.80
2.70
81.5
5.33
P61981
14-3-3 protein gamma
5.53
10.93
28.3
4.89
P07900
Heat shock protein HSP 90-alpha
3.08
3.42
84.6
5.02
P01023
Alpha-2-macroglobulin
2.56
1.76
163.2
6.46
Q14185
Dedicator of cytokinesis protein 1
1.99
1.66
215.2
7.56
Q562R1
Beta-actin-like protein 2
1.78
7.71
42.0
5.59
T bl A4 LCMS
l
i
f FIX PCC
filt
t f ll
i
filt
ti
ith 11
filt A3. LCMS analysis of FIX-PCC permeate following two-step filtration with 6 µm/33 µm filters. Table A4. LCMS analysis of FIX-PCC pre-filtrate following filtration with 11 µm filter. Table A4. LCMS analysis of FIX-PCC pre-filtrate following filtration with 11 µm filter. Accession
Description
Score
Coverage
MW [kDa]
calc. Appendix A pI
P00734
Prothrombin
2584.88
71.86
70.0
5.90
P0C0L5
Complement C4-B
2441.20
77.69
192.6
7.27
P0C0L4
Complement C4-A
2393.34
74.89
192.7
7.08
P19827
Inter-alpha-trypsin inhibitor heavy chain H1
1154.64
54.34
101.3
6.79
P19823
Inter-alpha-trypsin inhibitor heavy chain H2
1136.27
55.50
106.4
6.86
P02760
Protein AMBP
337.33
25.57
39.0
6.25
Q06033
Inter-alpha-trypsin inhibitor heavy chain H3
332.27
43.15
99.8
5.74
P00740
Coagulation factor IX
151.88
54.66
51.7
5.47
P00742
Coagulation factor X
81.34
41.19
54.7
5.94
P02768
Serum albumin
60.16
37.44
69.3
6.28
P01834
Immunoglobulin kappa constant OS=Homo
sapiens
48.66
81.31
11.8
6.52
P51884
Lumican
42.56
35.50
38.4
6.61
P49747
Cartilage oligomeric matrix protein
41.14
16.12
82.8
4.60
P01857
Immunoglobulin heavy constant gamma 1
40.75
41.21
36.1
8.19
P67936
Tropomyosin alpha-4 chain
31.98
36.29
28.5
4.69
P01861
Immunoglobulin heavy constant gamma 4
30.25
28.13
35.9
7.36
P01860
Immunoglobulin heavy constant gamma 3
28.46
27.06
41.3
7.90
P04004
Vitronectin OS=Homo sapiens
25.83
17.99
54.3
5.80
P0DOY2
Immunoglobulin lambda constant 2
23.29
67.92
11.3
7.24
P07225
Vitamin K-dependent protein S
21.00
11.83
75.1
5.67
P07359
Platelet glycoprotein Ib alpha chain
20.03
8.28
71.5
6.29
Q08380
Galectin-3-binding protein
17.21
12.82
65.3
5.27
P01876
Immunoglobulin heavy constant alpha 1
15.47
19.55
37.6
6.51
P35443
Thrombospondin-4
14.04
4.79
105.8
4.68
P01859
Immunoglobulin heavy constant gamma 2
12.71
27.91
35.9
7.59
P01024
Complement C3
7.05
1.56
187.0
6.40
P12259
Coagulation factor V
6.08
1.57
251.5
6.05
Q14515
SPARC-like protein 1
5.71
6.93
75.2
4.81
P16070
CD44 antigen
5.36
2.70
81.5
5.33
P13591
Neural cell adhesion molecule 1
5.24
2.56
94.5
4.87
P04070
Vitamin K-dependent protein C
5.14
5.86
52.0
6.28
P07900
Heat shock protein HSP 90-alpha
4.64
4.92
84.6
5.02
P61981
14-3-3 protein gamma
3.79
7.29
28.3
4.89
P63104
14-3-3 protein zeta/delta
3.48
6.94
27.7
4.79
Q99436
Proteasome subunit beta type-7
2.11
8.30
29.9
7.68
P25786
Proteasome subunit alpha type-1
1.66
7.98
29.5
6.61 15 of 18 Biomedicines 2020, 8, 69 Table A3. LCMS analysis of FIX-PCC permeate following two-step filtration with 6 µm/33 µm filters. Accession
Description
Score
Coverage
MW [kDa]
calc. Appendix A pI
P00734
Prothrombin
2458.17
67.20
70.0
5.90
P0C0L5
Complement C4-B
2389.47
80.68
192.6
7.27
P0C0L4
Complement C4-A
2352.05
79.59
192.7
7.08
P19823
Inter-alpha-trypsin inhibitor heavy chain H2
1268.03
56.87
106.4
6.86
P19827
Inter-alpha-trypsin inhibitor heavy chain H1
1122.57
59.50
101.3
6.79
Q06033
Inter-alpha-trypsin inhibitor heavy chain H3
352.56
40.00
99.8
5.74
P02760
Protein AMBP
285.56
27.56
39.0
6.25
P00740
Coagulation factor IX
182.33
57.27
51.7
5.47
P00742
Coagulation factor X
80.33
41.19
54.7
5.94
P01834
Immunoglobulin kappa constant
54.45
54.21
11.8
6.52
P49747
Cartilage oligomeric matrix protein
53.77
24.31
82.8
4.60
P01857
Immunoglobulin heavy constant gamma 1
45.54
43.33
36.1
8.19
P02768
Serum albumin
44.26
25.78
69.3
6.28
P07359
Platelet glycoprotein Ib alpha chain
33.83
16.56
71.5
6.29
P01860
Immunoglobulin heavy constant gamma 3
33.53
31.83
41.3
7.90
P0DOY3
Immunoglobulin lambda constant 3
33.11
70.75
11.3
7.24 16 of 18 Biomedicines 2020, 8, 69 Table A4. Cont. Table A4. Cont. Accession
Description
Score
Coverage
MW [kDa]
calc. pI
P01861
Immunoglobulin heavy constant gamma 4
30.81
23.85
35.9
7.36
P04004
Vitronectin
27.21
23.64
54.3
5.80
P51884
Lumican
26.62
35.50
38.4
6.61
P01876
Immunoglobulin heavy constant alpha 1
25.67
29.18
37.6
6.51
Q08380
Galectin-3-binding protein
25.15
17.78
65.3
5.27
P67936
Tropomyosin alpha-4 chain
25.00
44.35
28.5
4.69
P01859
Immunoglobulin heavy constant gamma 2
17.12
25.77
35.9
7.59
P07225
Vitamin K-dependent protein S
16.75
9.62
75.1
5.67
P35443
Thrombospondin-4
16.66
5.52
105.8
4.68
P12259
Coagulation factor V
10.18
1.98
251.5
6.05
P04070
Vitamin K-dependent protein C
7.36
5.86
52.0
6.28
P16070
CD44 antigen
5.64
2.70
81.5
5.33
P13591
Neural cell adhesion molecule 1
5.50
1.75
94.5
4.87
P22105
Tenascin-X
4.42
2.33
458.1
5.17
Q99436
Proteasome subunit beta type-7
4.03
8.30
29.9
7.68
P12814
Alpha-actinin-1
3.68
2.35
103.0
5.41
P27348
14-3-3 protein theta
3.53
6.53
27.7
4.78
P07900
Heat shock protein HSP 90-alpha
1.73
3.14
84.6
5.02 Table A5. LCMS analysis of FIX-PCC permeate following two-step filtration with 11 µm/33 µm filters. Table A5. LCMS analysis of FIX-PCC permeate following two-step filtration with 11 µm/33 µm filters. Accession
Description
Score
Coverage
MW [kDa]
calc. References 1. EMA. Guideline on Clinical Investigation of Recombinant and Human Plasma-derived Factor IX Products. EMA/CHMP/BPWP/144552/2009; 2011. Available online: https://www.ema.europa.eu/en/documents/
scientific-guideline/guideline-clinical-investigation-recombinant-human-plasma-derived-factor-ix-
products_en.pdf (accessed on 25 March 2020). 2. Josi´c, D.; Hoffer, L.; Buchacher, A.; Schwinn, H.; Frenzel, W.; Biesert, L.; Klöcking, H.-P.; Hellstern, P. Manufacturing of a Prothrombin Complex Concentrate Aiming at Low Thrombogenicity. Thromb. Res. 2000,
100, 433–441. [CrossRef] 2. Josi´c, D.; Hoffer, L.; Buchacher, A.; Schwinn, H.; Frenzel, W.; Biesert, L.; Klöcking, H.-P.; Hellstern, P. Manufacturing of a Prothrombin Complex Concentrate Aiming at Low Thrombogenicity. Thromb. Res. 2000,
100, 433–441. [CrossRef] 3. Josi´c, D.; Hoffer, L.; Buchacher, A. Preparation of vitamin K-dependent proteins, such as clotting factors II,
VII, IX and X and clotting inhibitor Protein, C. J. Chromatogr. B. 2003, 790, 183–197. [CrossRef] 4. Burnouf, T.; Michalski, C.; Goudemand, M.; Huart, J.J. Properties of a Highly Purified Human Plasma Factor
IX:c Therapeutic Concentrate Prepared by Conventional Chromatography. Vox. Sang. 1989, 57, 225–232. [CrossRef] [PubMed] 5. Teitel, J.M. Safety of coagulation factor concentrates. Haemophilia 1998, 4, 393–401. [PubMed] 6. EMA. Guideline on Plasma-derived Medicinal Products. EMA/CHMP/BWP/706271/2010; 2011. Available
online: https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-plasma-derived-medicinal-
products_en.pdf (accessed on 25 March 2020). 7. Klamroth, R.; Gröner, A.; Simon, T.L. Pathogen inactivation and removal methods for plasma-derived
clotting factor concentrates. Transfusion 2014, 54, 1406–1417. [CrossRef] [PubMed] 8. Grancha, S.; Herring, S.; Paéz, A.; Ristol, P.; Jorquera, J.I. Factor IX; John Wiley & Sons, Inc.: Hoboken, NJ,
USA, 2012; pp. 81–92. [CrossRef] 9. Koenderman, A.H.L.; ter Hart, H.G.J.; Prins-de Nijs, I.M.M.; Bloem, J.; Stoffers, S.; Kempers, A.; Derksen, G.J.;
Al, B.; Dekker, L.; Over, J. Virus safety of plasma products using 20 nm instead of 15 nm filtration as virus
removing step. Biologicals 2012, 40, 473–481. [CrossRef] 10. Roberts, P.L.; Feldman, P.; Crombie, D.; Walker, C.; Lowery, K. Virus removal from factor IX by filtration:
Validation of the integrity test and effect of manufacturing process conditions. Biologicals 2010, 38, 303–310. [CrossRef] 11. Winkler, C.J.; Jorba, N.; Shitanishi, K.T.; Herring, S.W. Protein sieving characteristics of sub-20-nm pore size
filters at varying ionic strength during nanofiltration of Coagulation Factor IX. Biologicals 2013, 41, 176–183. [CrossRef] 12. Caballero, S.; Diez, J.M.; Belda, F.J.; Otegui, M.; Herring, S.; Roth, N.J.; Lee, D.; Gajardo, R.; Jorquera, J.I. Robustness of nanofiltration for increasing the viral safety margin of biological products. Biologicals 2014, 42,
79–85. [CrossRef] 13. Appendix A pI
P0C0L5
Complement C4-B
2266.18
76.26
192.6
7.27
P0C0L4
Complement C4-A
2219.29
76.26
192.7
7.08
P00734
Prothrombin
2011.89
71.54
70.0
5.90
P19823
Inter-alpha-trypsin inhibitor heavy chain H2
1452.50
55.81
106.4
6.86
P19827
Inter-alpha-trypsin inhibitor heavy chain H1
1205.49
54.23
101.3
6.79
Q06033
Inter-alpha-trypsin inhibitor heavy chain H3
256.28
46.97
99.8
5.74
P02760
Protein AMBP
213.99
25.57
39.0
6.25
P00740
Coagulation factor IX
114.46
45.55
51.7
5.47
P00742
Coagulation factor X
74.31
36.07
54.7
5.94
P02768
Serum albumin
46.51
27.91
69.3
6.28
P01857
Immunoglobulin heavy constant gamma 1
45.77
43.33
36.1
8.19
P49747
Cartilage oligomeric matrix protein
34.16
20.08
82.8
4.60
P01834
Immunoglobulin kappa constant
32.23
49.53
11.8
6.52
P67936
Tropomyosin alpha-4 chain
27.70
37.90
28.5
4.69
P01860
Immunoglobulin heavy constant gamma 3
27.37
25.99
41.3
7.90
P51884
Lumican
25.59
39.94
38.4
6.61
P01861
Immunoglobulin heavy constant gamma 4
24.73
26.61
35.9
7.36
P01859
Immunoglobulin heavy constant gamma 2
23.14
32.82
35.9
7.59
P04004
Vitronectin
22.83
23.43
54.3
5.80
P07359
Platelet glycoprotein Ib alpha chain
21.45
9.82
71.5
6.29
P07225
Vitamin K-dependent protein S
20.38
10.65
75.1
5.67
P0DOY2
Immunoglobulin lambda constant 2
17.99
37.74
11.3
7.24
Q08380
Galectin-3-binding protein
17.44
15.73
65.3
5.27
P01876
Immunoglobulin heavy constant alpha 1
17.06
25.78
37.6
6.51
P35443
Thrombospondin-4
9.76
4.79
105.8
4.68
P12259
Coagulation factor V
8.36
2.11
251.5
6.05
P04070
Vitamin K-dependent protein C
7.60
8.89
52.0
6.28
P61981
14-3-3 protein gamma
5.93
10.53
28.3
4.89
P16070
CD44 antigen
4.96
2.70
81.5
5.33
Q99436
Proteasome subunit beta type-7
4.05
8.30
29.9
7.68
Q14515
SPARC-like protein 1
4.05
3.77
75.2
4.81
P07900
Heat shock protein HSP 90-alpha
2.46
2.60
84.6
5.02
P01023
Alpha-2-macroglobulin
1.97
1.49
163.2
6.46
P22105
Tenascin-X OS=Homo sapiens
1.66
0.66
458.1
5.17
P13591
Neural cell adhesion molecule 1
0.00
1.75
94.5
4.87
Q5UIP0
Telomere-associated protein RIF1]
0.00
1.62
274.3
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https://openalex.org/W2804317271
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https://hal.umontpellier.fr/hal-01850345/document
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English
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Naturally occurring variations in the nod-independent model legume Aeschynomene evenia and relatives: a resource for nodulation genetics
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BMC plant biology
| 2,018
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cc-by
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To cite this version: Clémence Chaintreuil, Xavier Perrier, Guillaume Martin, Joël Fardoux, Gwilym Lewis, et al.. Natu-
rally occurring variations in the nod-independent model legume Aeschynomene evenia and relatives:
a resource for nodulation genetics. BMC Plant Biology, 2018, 18 (1), pp.54 (1-15). 10.1186/s12870-
018-1260-2. hal-01850345 Naturally occurring variations in the nod-independent
model legume Aeschynomene evenia and relatives: a
resource for nodulation genetics Clémence Chaintreuil, Xavier Perrier, Guillaume Martin, Joël Fardoux,
Gwilym Lewis, Laurent Brottier, Ronan Rivallan, Mario Gomez-Pacheco,
Mickaël Bourge, Léo Lamy, et al. Distributed under a Creative Commons Attribution 4.0 International License Abstract Background: Among semi-aquatic species of the legume genus Aeschynomene, some have the unique property of being
root and stem-nodulated by photosynthetic Bradyrhizobium lacking the nodABC genes necessary for the production of Nod
factors. These species provide an excellent biological system with which to explore the evolution of nodulation in legumes. Among them, Aeschynomene evenia has emerged as a model legume to undertake the genetic dissection of the so-called
Nod-independent symbiosis. In addition to the genetic analysis of nodulation on a reference line, natural variation in a
germplasm collection could also be surveyed to uncover genetic determinants of nodulation. To this aim, we investigated
the patterns of genetic diversity in a collection of 226 Nod-independent Aeschynomene accessions. Results: A combination of phylogenetic analyses, comprising ITS and low-copy nuclear genes, along with cytogenetic
experiments and artificial hybridizations revealed the richness of the Nod-independent Aeschynomene group with the
identification of 13 diploid and 6 polyploid well-differentiated taxa. A set of 54 SSRs was used to further delineate taxon
boundaries and to identify different genotypes. Patterns of microsatellite diversity also illuminated the genetic basis of the
Aeschynomene taxa that were all found to be predominantly autogamous and with a predicted simple disomic inheritance,
two attributes favorable for genetics. In addition, taxa displaying a pronounced genetic diversity, notably A. evenia, A. indica
and A. sensitiva, were characterized by a clear geographically-based genetic structure and variations in root and stem
nodulation Conclusion: A well-characterized germplasm collection now exists as a major genetic resource to thoroughly explore the
natural variation of nodulation in response to different bradyrhizobial strains. Symbiotic polymorphisms are expected to be
found notably in the induction of nodulation, in nitrogen fixation and also in stem nodulation. Subsequent genetic analysis
and locus mapping will pave the way for the identification of the underlying genes through forward or reverse genetics. Such discoveries will significantly contribute to our understanding of the molecular mechanisms underpinning how some
Aeschynomene species can be efficiently nodulated in a Nod-independent fashion. Keywords: Aeschynomene, Diversity, Genotype, Legume, Nodulation, Ploidy, Species, Symbiosis Naturally occurring variations in the nod-
independent model legume Aeschynomene
evenia and relatives: a resource for
nodulation genetics Clémence Chaintreuil1,8, Xavier Perrier2,3, Guillaume Martin2,3, Joël Fardoux1,8, Gwilym P. Lewis4, Laurent Brottier1,8,
Ronan Rivallan2,3, Mario Gomez-Pacheco5, Mickaël Bourges5, Léo Lamy1,8, Béatrice Thibaud2,3,
Heriniaina Ramanankierana6, Herizo Randriambanona6, Hervé Vandrot7, Pierre Mournet2,3, Eric Giraud1,8
and Jean-François Arrighi1,8* * Correspondence: jean-francois.arrighi@ird.fr
1IRD, Laboratoire des Symbioses Tropicales et Méditerranéennes, UMR LSTM,
Campus International de Baillarguet, F-34398 Montpellier, France
8LSTM, Univ. Montpellier, CIRAD, INRA, IRD, Montpellier SupAgro, Montpellier,
France
Full list of author information is available at the end of the article HAL Id: hal-01850345
https://hal.umontpellier.fr/hal-01850345v1
Submitted on 25 Nov 2019 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 Chaintreuil et al. BMC Plant Biology (2018) 18:54
https://doi.org/10.1186/s12870-018-1260-2 Background To broaden our understanding
of the molecular mechanisms underlying the nitrogen-
fixing symbiosis, there has arisen a fast growing interest in
uncovering the diversity of nodulation processes that are
found in other legume species [5, 6]. p
g
Forward genetics are now expected to allow the identi-
fication of the specific molecular determinants of the
Nod-independent process in A. evenia. To optimize re-
search effort, a reference line was inbred and success-
fully used to generate an SSR-based genetic map of A. evenia [9]. This genetic map uncovered the genome
structure and the distribution of symbiotic genes. It also
provides a basis for a genome sequencing project and
paves the way of the genetic dissection of nodulation in
this reference line. This does not exclude exploring the
naturally occurring variations in nodulation, as a com-
plementary genetic approach, in order to increase our
understanding of symbiotic gene functions and of the
genetic control of symbiosis as successfully performed in
other legumes such as Medicago, Lotus and soybean
[22]. But exploiting genetic diversity requires prior
knowledge of the extent and structure of the variations
occurring in the species of interest. Although the genetic
relationships among the Nod-independent Aeschynomene
species have been analysed using molecular markers, only
two studies have included a small set of accessions for the
diploid A. evenia and the related polyploid A. indica [17,
18]. As a consequence, the variations within and among
the species of the Nod-independent clade remain largely
uncharacterized. g
In this line, the mainly tropical legume genus Aeschynomene
represents a group of prime interest as it contains several
original symbiotic features. The genus Aeschynomene was ori-
ginally known for the ability of different species to develop
stem nodules in addition to the typical root nodules. Stem
nodulation is uncommon in legumes, being shared with a very
few hydrophytic species of the genera Sesbania, Neptunia and
Discolobium, but it is widespread among the semi-aquatic
Aeschynomene species [7–9]. In addition, some bradyrhizobia
isolated from Aeschynomene stem nodules exhibit a photosyn-
thetic activity that was shown to play a key role in stem
nodules by directly furnishing energy to the bacterium that
can be used for biological nitrogen fixation [10, 11]. Background taxonomic revision of American Aeschynomene was pub-
lished in 1955 by Rudd [15], but it predated phylogenetic
studies in plants and included no species native outside the
Neotropics. First molecular studies of Aeschynomene pointed
to new cryptic taxa differing by their ploidy levels [16, 17]. The knowledge gained from the study of the Nod-
independent clade was also used to select Aeschynomene
evenia as a new model legume for the purpose of decipher-
ing the molecular mechanisms of the Nod-independent sym-
biosis [16, 18]. Key attributes of this species include its small,
diploid genome (2n = 20, 415 Mb/1C), its selfing nature and
its prolific seed production. Several tools have been devel-
oped including artificial hybridization and the Agrobacterium
rhizogenes-mediated root transformation, rendering this spe-
cies ideal for molecular genetic studies. First insights were
obtained from RNAseq analysis and reverse genetics by re-
vealing that some symbiotic determinants identified in
Medicago and Lotus are recruited in the Nod-independent
process but several key genes involved in bacterial recogni-
tion, symbiotic infection and nodule functioning were found
not to be expressed during root nodulation [9, 19–21]. g
The legume family (Leguminosae) accounts for ~ 27% of
the world’s primary crop production and is second only to
cereals in economic and nutritional value. It includes many
crops of agronomic importance for grain production, pas-
ture and agroforestry. Many legumes are pioneers plants
improving soil fertility and moderating harsh environments. Such economic and ecological success of the legume family
is, in large part, due to the ability of the vast majority of its
20,000 species to develop symbiotic interactions with
nitrogen-fixing bacteria collectively referred as rhizobia [1]. In this symbiosis, the rhizobia produce signal molecules,
the Nod factors, whose specific recognition by the host
plant is necessary to activate the formation of root nodules
that correspond to symbiotic organs where the rhizobia are
hosted. Inside the nodules, the rhizobia reduce atmospheric
nitrogen (N2) into ammonium (NH4
+), a form of nitrogen
that is usable by the plant for its development. Historically,
two model legumes, Medicago truncatula and Lotus japoni-
cus, have been used to genetically investigate this nodula-
tion process. Such studies have resulted in the identification
and elucidation of the role of many genes that are essential
for the different steps of nodule development and its infec-
tion by the symbiont [2–4]. * Correspondence: jean-francois.arrighi@ird.fr
1 Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Page 2 of 15 Chaintreuil et al. BMC Plant Biology (2018) 18:54 Page 2 of 15 Background Even
more outstanding was the discovery that some of these photo-
synthetic bradyrhizobia lack both the canonical nodABC genes
required for the synthesis of the key Nod factors and a type III
secretion system (T3SS) that is known in other rhizobia to
activate or modulate nodulation [12–14]. This led to
a new paradigm in nodulation studies in which an al-
ternative symbiotic process between rhizobia and le-
gumes efficiently triggers nodule formation in a Nod
(factor)-independent fashion. To enable an efficient use of the natural variation in
genetic studies of nodulation, we surveyed the genetic
diversity occurring in a collection of 226 Nod-independent
Aeschynomene accessions spanning the whole distributional
range of this clade. As a first step, genetic relationships and
differentiation between Nod-independent Aeschynomene
taxa were established using a combination of molecular
phylogenies, cytogenetics and hybridization experiments. Phylogenetic analysis of the genus Aeschynomene revealed
that all the species endowed with a Nod-independent
nodulation process cluster in a single clade where no species
using a Nod-dependent symbiotic process are found [8]. A Page 3 of 15 Chaintreuil et al. BMC Plant Biology (2018) 18:54 This information then served as supportive data for the
analysis of genotype data obtained for the germplasm col-
lection using a set of 54 SSR markers. Patterns of microsat-
ellite
diversity
illuminated
the
genetic
basis
of
the
Aeschynomene taxa and uncovered their genetic differenti-
ation. The presence of an underlying genetic structure was
then compared with geographical distribution data. with A. deamii, a third one comprising A. evenia and its
sister species, and a fourth one containing A. sensitiva
and related species (Fig. 1a). p
g
In this phylogenetic tree, a number of putative species
displayed the same ITS sequence. In particular, two rare
species, A. magna and A. pluriarticulata, tightly clus-
tered with two widespread species, A. denticulata and A. evenia, respectively (Fig. 1a). Similarly, A. evenia and A. indica shared the same ITS signature but they were pre-
viously shown to form a species complex containing
three cytotypes (2×, 4× and 6×) [17]. To sort these,
accessions of this species complex were genotyped with
SSR markers that are indicators of their genome consti-
tution [17]. This led to the additional discovery that the
A. indica 6× accessions of African and Australian origin
displayed different SSR profiles, justifying their distinc-
tion in the phylogenic tree (Fig. 1a, data not shown). Species identification and relationships A collection of 233 accessions was developed to investi-
gate the phylogenetic relationships and the genetic dif-
ferentiation
in
the
Nod-independent
Aeschynomene
clade (Additional file 1: Table S1). It included all the
known species included in the clade and aimed to cover
their distributional range [15, 23, 24]. The nuclear ribo-
somal ITS region was used as a marker of the species
identity and served to reconstruct a phylogeny of the
whole group based on the Neighbor Joining (NJ) method
(Additional files 2 and 3: Tables S2 and S3). To simplify
the resulting tree, accessions showing less than 1% of di-
vergence in their sequence were grouped in the same
clade (Fig. 1a, Additional file 4: Doc. S1). The ITS tree
was composed of four lineages: one grouping A. filosa,
A. rostrata and A. tambacoundensis, a monospecific one a
b
Fig. 1 Phylogenetic and genetic relationships in the Nod-independent Aeschynomene clade. Phylogenetic reconstructions were obtained using
the Neighbor Joining method. a ITS phylogeny. Accessions with ITS sequence divergence < 1% were clustered together. Numbers of accessions
per taxon are indicated in brackets. b Phylogeny based on 5 concatenated low-copy nuclear gene fragments: CYP1, eiF1α, SUI1, SuSy and a gene
homolog to Glyma.07G136800 and Glyma.18G187300. -A, -B and -C indicate the different copies found in polyploid species. The four gene pools
are identified with a circled number and the A. evenia and A. sensitiva groups are framed in a box bordered with a dashed red line. Diploid taxa
are in black and polyploid taxa in blue with ploidy level indicated. Numbers at nodes represent bootstrap values (% of 1000 replicates) b a b b Fig. 1 Phylogenetic and genetic relationships in the Nod-independent Aeschynomene clade. Phylogenetic reconstructions were obtained using
the Neighbor Joining method. a ITS phylogeny. Accessions with ITS sequence divergence < 1% were clustered together. Numbers of accessions
per taxon are indicated in brackets. b Phylogeny based on 5 concatenated low-copy nuclear gene fragments: CYP1, eiF1α, SUI1, SuSy and a gene
homolog to Glyma.07G136800 and Glyma.18G187300. -A, -B and -C indicate the different copies found in polyploid species. The four gene pools
are identified with a circled number and the A. evenia and A. sensitiva groups are framed in a box bordered with a dashed red line. Diploid taxa
are in black and polyploid taxa in blue with ploidy level indicated. Background It
is also noteworthy that two accessions did not fit well
with the description of any known Aeschynomene species
and so we refer to these as Aeschynomene sp. (328) and A. sp. (353) (Fig. 1a, Additional file 1: Table S1). To clarify the
genetic status of these putative new taxa, they were both in-
cluded in a flow cytometry analysis and a chromosome count
(Additional file 1: Table S1, Additional file 5: Figure S1). Cytogenetic data were mapped onto the phylogeny, showing Genetic patterns and behaviours g
To further uncover genetic relationships between taxa,
five low copy nuclear genes -CYP1 (Cyclophilin 1), eiF1α
(eukaryotic translation initiation factor α), SUI1 (transla-
tion factor), SuSy (Sucrose Synthase) and a gene homolog
to Glyma.07G136800 and Glyma.18G187300 identified in
Glycine max - were cloned and sequenced in selected ac-
cessions (Additional file 2: Table S2). For diploid species,
single sequences were obtained, while for polyploid spe-
cies homeologous sequences were isolated for almost all
genes (Additional file 3: Table S3). The five genes treated
separately gave similar NJ trees where the homeologous
sequences for polyploid taxa could be differentiated based
on the differential clustering with the sequences of diploid
taxa (not shown). To provide a unique and well-resolved
NJ phylogeny, the gene sequences were concatenated to-
gether (Fig. 1b). In the resulting tree, the topology of the
branches containing the diploid species was similar to that
of the ITS tree, corroborating the distinctness of A. sp. (328) from other known Aeschynomene species (Fig. 1a,b). For the polyploid taxa, the different genome components
were scattered different part of the phylogeny, revealing
that the two taxa A. sp. (353) and A. virginica, and the
African and Australian A. indica 6× had the same or a
similar genomic constitution (Fig. 1b). To assess the gen-
etic differentiation of these related taxa, they were manu-
ally crossed: A. sp. (353) with A. virginica, the African A. indica 6× with the Australian A. indica 6× and A. indica
4× with A. indica 6× for comparison (Fig. 2). In all cases,
hybrid plants were obtained but they greatly differed in
their fertility compared to their respective parental acces-
sions, as seen by a drastic reduction of both the number
of developed pods per flowering axis and of seeds per pod. To determine the genetic structure of the genomes of dif-
ferent taxa, we conducted a SSR genotyping of the collec-
tion of Aeschynomene accessions. For this, we tested the
set of 500 SSRs previously used when developing a genetic
map for A. evenia [9]. These markers were screened for
polymorphism in four samples that are genetically differ-
ent: two accessions of A. evenia 2× (the reference line
CIAT 22838 and the mapping parent CIAT 8232), one ac-
cession of A. indica 4× (PI 196206) and one of A. indica
6× (LSTM19). Species identification and relationships Numbers at nodes represent bootstrap values (% of 1000 replicates) Chaintreuil et al. BMC Plant Biology (2018) 18:54 Page 4 of 15 As a result, these data suggest that the tested taxa are truly
different Aeschynomene species. that Aeschynomene sp. (328) represented a new 2× taxon
while A. sp. (353) was a 4× taxon, similar to the closely related
A. virginica, and confirming the hexaploid status of the
Australian A. indica 6× (Fig. 1a). Genetic patterns and behaviours sensitiva group was compensated by the use
of an additional set of SSR markers. Of the 64 SSR markers,
54 generated clearly interpretable allele profiles in at least
one of the two groups, 38 SSR markers for the A. evenia
group and 46 SSR markers for the A. sensitiva group, with
30 of them in common (Additional file 6: Figure S2). Allelic
diversity, estimated by the number of alleles Na, varied im-
portantly among the SSR markers used with an average of
12.8 alleles per locus in the A. evenia group (n = 186 acces-
sions) and 4.21 in the A. sensitiva group (n = 40 accession)
(Additional file 10: Table S6). Observed heterozygosities
were very low for each SSR (Ho < 0.1 with one exception at
0.2), indicating that they amplified at single locus for diploid
accessions and homeologous loci for polyploid accessions
(Additional file 10: Table S6). the Aeschynomene taxa, one of the highest being found in A. evenia (mean 5.2 alleles per SSR) (Table 1, Additional file 10:
Table S6). These values reflected the genetic diversity of the
taxa, but they were likely to have been influenced by the
marked variation in sample sizes and by the non-random
selection of the SSR markers. The mean number of alleles
detected for the SSR markers in each taxon were congruent
with their ploidy levels, with (i) 1.00 to 1.06 alleles observed
for diploid taxa, (ii) 1.42 to 1.92 alleles for tetraploid taxa and
(iii) 2.63 to 2.84 alleles for hexaploid taxa (Table 1,
Additional file 12: Table S8). Accessions displaying more al-
leles than expected from their ploidy level were considered
as heterozygous (Additional files 1 and 13: Tables S1, S9). Selfing rates based on the observed heterozygosities, ranged
from 94.5% to 100%, with an average 98.7% score for A. evenia (Table 1, Additional file 13: Table S9). This provided
genetic support for previous observations that the Nod-
independent Aeschynomene species are preferentially autog-
amous [17, 18]. These results supported the appropriateness of these SSRs
for investigating the genetic properties of species. Therefore
they were analysed in a second step to characterize the taxa. Transferability level of the SSR markers from A. evenia,
ranged from 92 to 100% for other taxa of the A. evenia group
and from 72 to 78% for those of the A. sensitiva group
(Table 1, Additional file 11: Table S7). Genetic patterns and behaviours Of these, 64 markers were selected using
two main criteria: (1) the requirement to be polymorphic
between the two A. evenia accessions so as to avoid the
use of invariant SSRs, (2) to amplify a single allele in the
2× accessions, 2 and 3 alleles in the 4× and 6× accessions,
respectively, with the assumption that the distinct alleles
came from the different genomic components of the
polyploid species (Additional files 6, 7 and 8: Figure S2,
Tables S4 and S5). Forty nine out of the 64 selected
markers were previously positioned on different A. evenia
linkage groups, with their distribution representing a
reasonable coverage of the A. evenia genetic map
(Additional file 7: Table S4). Preliminary genotyping experiments revealed that the
SSRs developed from A. evenia sequences had a transfer-
ability rate qualified as (i) modest with A. deamii and A. tambacoundensis, (ii) good with A. sensitiva and A. pratensis and (iii) high with A. denticulata and A. scabra. These data were in accordance with the phylogenetic
distance of the different gene pools relative to A. evenia
(Fig. 1), but this prompted us to restrict our analysis to
the A. evenia and A. sensitiva groups. The corresponding
accessions were then subjected to high-throughput SSR Fig. 2 Hybridization experiments between related taxa. Manual crosses were performed between A. virginica and A. sp (353), A. indica 4× and A. indica
6× -Africa-, A. indica 6× -Africa- and A. indica 6× -Australia. Plant fertility was evaluated for the parental taxa and two independently obtained F1
hybrids based on the number of seeds produced per pod and the number of developed pods per flowering axis. Error bars represent s.d. (n = 30) Fig. 2 Hybridization experiments between related taxa. Manual crosses were performed between A. virginica and A. sp (353), A. indica 4× and A. indica
6× -Africa-, A. indica 6× -Africa- and A. indica 6× -Australia. Plant fertility was evaluated for the parental taxa and two independently obtained F1
hybrids based on the number of seeds produced per pod and the number of developed pods per flowering axis. Error bars represent s.d. (n = 30) Page 5 of 15 Chaintreuil et al. BMC Plant Biology (2018) 18:54 Page 5 of 15 genotyping by capillary sequencing (Additional file 9:
Figure S3). The absence of amplification for some of
them in the A. Genetic patterns and behaviours Analysing the Na par-
ameter revealed different levels of genetic variation among Geographical structure of the Aeschynomene species Geographical structure of the Aeschynomene species
The Nod-independent Aeschynomene clade is mainly a trop-
ical/subtropical group, but the species show distinct geo-
graphical distributions [15, 23, 24]. Out of the 19 taxa
considered here (Table 1), 13 are strictly American, while
three taxa (A. evenia, A. indica 4× and A. sensitiva) have a
wider distribution; two are African (A. indica 6× - Africa-
and A. tambacoundensis) and one Australian (A. indica 6× -
Australia-). This confirmed previous conclusions that Amer-
ica is the centre of origin and diversification of this clade but
that several outliers have subsequently evolved in other con-
tinents [8]. Because some species showed a pronounced gen-
etic differentiation and are part of polyploid species
complexes, we investigated to what extent the corresponding
cytotypes and genotypes are geographically structured. In the
A. sensitiva-A. pratensis species complex, A. sensitiva has a
transatlantic distribution. When mapping accessions globally,
a clear geographical separation of the four A. sensitiva geno-
types was observed. One occupies the Caribbean region, one
found in Colombia, another one the central area of South
America and, more noticeably a fourth one being present
both in coastal East Brazil and in Africa, suggesting a recent
dispersal event (Fig. 5a, Additional file 9: Figure S3,
Additional file 1: Table S1). In contrast to A. sensitiva, the
low genetic diversity observed in A. pratensis 4× revealed no
consistent geographical pattern (Fig. 5A, Additional file 9:
Figure S3, Additional file 1: Table S1). In a search for other relationships, a Factorial Ana-
lysis (FA) was also carried out in DARwin v5 [25]. This approach is more informative regarding distances
among different groups and it also allows comparison
of accessions of different ploidy levels. We focused on
A. evenia and A. sensitiva to get a more detailed view
of the genetic relatedness between the 2× genotypes
and the derived polyploid taxa. FA clearly distin-
guished A. sensitiva and A. pratensis when using the
factorial axes 1 and 2 (Fig. 4a). Factorial axes 2 and 3
separated the four A. sensitiva genotypes and but the
central position of A. pratensis was interpreted has an
absence of a preferential relationship with any of the
A. sensitiva genotypes (Fig. 4b). Therefore, either the
parental A. sensitiva genotype that contributed to the
polyploid genome of A. pratensis was missing or A. In the pantropically distributed A. evenia-A. Genetic diversity and genotype delineation Based on the geographical distributions of the accessions
making up the different Nod-independent Aeschynomene
taxa, most of their genetic diversity and structure were ex-
pected to be uncovered. Therefore, the genotyping data Table 1 Summary of the data obtained for the Aeschynomene taxa
Taxa/ ploidy level
n samples
n genotypes
Cross-species transferability
N Table 1 Summary of the data obtained for the Aeschynomene taxa Table 1 Summary of the data obtained for the Aeschynomene taxa
Taxa/ ploidy level
n samples
n genotypes
Cross-species transferability
NA
n co-present alleles/SSR
Ho
Comment
2× taxa
A. ciliata
5
100%
1.2
1.00
0.000
A. deamii
3
–
–
–
–
A. denticulata
24
3
92%
2.7
1.06
0.055
A. magna conspecific
A. evenia ssp. evenia
44
7
–
5.2
1.01
0.013
A. pluriarticulata conspecific
A. evenia ssp. serrulata
15
2
100%
2.3
1.02
0.019
A. filosa
2
–
–
–
–
A. rostrata
1
–
–
–
–
A. rudis
7
95%
1.9
1.00
0.004
A. scabra
8
100%
1.6
1.00
0.000
A. selloi
3
78%
1.1
1.00
0.000
A. sensitiva
27
4
78%
2.6
1.03
0.028
A. sp (328)
1
72%
1
1.00
0.000
new taxon
A. tambacoundensis
1
–
–
–
–
4× taxa
A. indica 4×
41
6
100%
4.9
1.92
0.019
A. pratensis
9
78%
2.1
1.60
0.000
A. sp (353)
1
100%
1.6
1.66
0.000
new taxon
A. virginica
1
100%
1.5
1.42
0.000
6× taxa
A. indica 6× Africa
16
2
100%
4.8
2.84
0.005
A. indica 6× Australia
24
3
100%
6.9
2.63
0.002
new taxon Chaintreuil et al. BMC Plant Biology (2018) 18:54 Page 6 of 15 Page 6 of 15 obtained for the A. evenia and A. sensitiva groups were
combined and used to estimate pair-wise distances be-
tween all accessions and thereby generate a dissimilarity
matrix in DARwin v5 [25]. From this, NJ trees were calcu-
lated for the 2×, 4×, 6× ploidy levels separately. Indeed, al-
leles of the genomic components of polyploid accessions
cannot be analysed separately and so their inclusion in the
analysis of the diploid accessions would result in their
grouping with only one of the potential progenitors. Considering the 2× NJ tree, well-separated clades were
evident and corresponded to the known species A. evenia,
A. ciliata, A. denticulata, A. rudis, A. scabra, A. serrulata,
A. selloi and A. sensitiva, as well as the newly identified A. sp. (328) (Fig. Genetic diversity and genotype delineation 3a,Additional file 9: Figure S3). Interestingly,
A. pluriarticulata was found to be nested within the A. evenia accessions and the accession of A. magna tightly
clustered with accessions of A. denticulata. This information
together with the phylogenetic and genetic relationships
makes their taxonomic distinctness uncertain. Conversely,
the tree topology showed a clear separation of the two A. evenia subspecies, evenia and serrulata, and several 2× taxa
(A. denticulata, A. evenia ssp. evenia, A. evenia ssp. serrulata
and A. sensitiva) could be subdivided in different clusters de-
lineating genotypes (Fig. 3a,Additional file 9: Figure S3). Among these, genetic diversity and differentiation was the
highest for A. evenia ssp. evenia (here after A. evenia s.s. in
the text), with 7 genotypes identified. Regarding the 4× tree,
A. virginica and A. sp (353) were found to form sister line-
ages and contrasted patterns of genetic differentiation were
observed with a very low genetic variability noted for A. pratensis whereas A. indica 4× was composed of several
well-diverged clusters (Fig. 3b,Additional file 9: Figure S3). In
the 6× tree, the African and Australian A. indica 6× were
genetically distant with the African set subdivided in two
homogenous genotypes while the Australian set contained
three genotypes that formed far more diverse assemblages,
indicative of distinct evolutions (Fig. 3c, Additional file 9:
Figure S3). pratensis was formed before the intraspecific differen-
tiation of A. sensitiva as suggested by the sequence
divergence of the nuclear genes (Fig. 1b). For A. evenia s.s., the FA separated the 7 identified geno-
types but grouped the three A. indica taxa together
when using factorial axes 1 and 2 (Fig. 4c). These
polyploid taxa could be separated along factorial axis
4 and showed preferential affinity with the two un-
separated genotypes (1) and (2) of A. evenia along
factorial axis 2 (Fig. 4d). These observations suggested
a common origin of the three A. indica taxa that
would derive from the same A. evenia genome donor,
this latter being potentially ancestral to the genotypes
1 and 2. obtained for the A. evenia and A. sensitiva groups were
combined and used to estimate pair-wise distances be-
tween all accessions and thereby generate a dissimilarity
matrix in DARwin v5 [25]. From this, NJ trees were calcu-
lated for the 2×, 4×, 6× ploidy levels separately. Genetic diversity and genotype delineation Indeed, al-
leles of the genomic components of polyploid accessions
cannot be analysed separately and so their inclusion in the
analysis of the diploid accessions would result in their
grouping with only one of the potential progenitors. Considering the 2× NJ tree, well-separated clades were
evident and corresponded to the known species A. evenia,
A. ciliata, A. denticulata, A. rudis, A. scabra, A. serrulata,
A. selloi and A. sensitiva, as well as the newly identified A. sp. (328) (Fig. 3a,Additional file 9: Figure S3). Interestingly,
A. pluriarticulata was found to be nested within the A. evenia accessions and the accession of A. magna tightly
clustered with accessions of A. denticulata. This information
together with the phylogenetic and genetic relationships
makes their taxonomic distinctness uncertain. Conversely,
the tree topology showed a clear separation of the two A. evenia subspecies, evenia and serrulata, and several 2× taxa
(A. denticulata, A. evenia ssp. evenia, A. evenia ssp. serrulata
and A. sensitiva) could be subdivided in different clusters de-
lineating genotypes (Fig. 3a,Additional file 9: Figure S3). Among these, genetic diversity and differentiation was the
highest for A. evenia ssp. evenia (here after A. evenia s.s. in
the text), with 7 genotypes identified. Regarding the 4× tree,
A. virginica and A. sp (353) were found to form sister line-
ages and contrasted patterns of genetic differentiation were
observed with a very low genetic variability noted for A. pratensis whereas A. indica 4× was composed of several
well-diverged clusters (Fig. 3b,Additional file 9: Figure S3). In
the 6× tree, the African and Australian A. indica 6× were
genetically distant with the African set subdivided in two
homogenous genotypes while the Australian set contained
three genotypes that formed far more diverse assemblages,
indicative of distinct evolutions (Fig. 3c, Additional file 9:
Figure S3). Geographical structure of the Aeschynomene species indica
species complex, a prominent geographical division be-
tween the different taxa was observed. A. evenia s.s. (2×)
grows both in America and Africa, A. indica 4× is wide-
spread in Asia, including India, and it is also present in
North Eastern America, while the two A. indica 6× taxa
distinctly occur in Africa and Australia (Fig. 5b). At the
intraspecific level, the identified genotypes were found
to represent geographically defined groups. A. evenia
displayed a high level of genetic structure with 5 Ameri-
can and 2 African genotypes. As far as phylogenetic rela-
tionships can be inferred from an SSR-based NJ tree, the Chaintreuil et al. BMC Plant Biology (2018) 18:54 Page 7 of 15 a
b
c
Fig. 3 NJ trees representing the genetic diversity among the Nod-independent Aeschynomene accessions. The trees were developed separately in
DARWIN using the allelic data of 54 SSRs for the 2× (a), 4× (b) and 6× (c) taxa. Well-differentiated taxa are distinctly coloured and identified
genotypes are numbered. Species suspected to be morphological variants are marked with an asterisk a
b a a c c c Fig. 3 NJ trees representing the genetic diversity among the Nod-independent Aeschynomene accessions. The trees were developed separately in
DARWIN using the allelic data of 54 SSRs for the 2× (a), 4× (b) and 6× (c) taxa. Well-differentiated taxa are distinctly coloured and identified
genotypes are numbered. Species suspected to be morphological variants are marked with an asterisk Fig. 3 NJ trees representing the genetic diversity among the Nod-independent Aeschynomene accessions. The trees were developed separately in
DARWIN using the allelic data of 54 SSRs for the 2× (a), 4× (b) and 6× (c) taxa. Well-differentiated taxa are distinctly coloured and identified
genotypes are numbered. Species suspected to be morphological variants are marked with an asterisk Fig. 3 NJ trees representing the genetic diversity among the Nod-independent Aeschynomene accessions. The trees were developed separately in
DARWIN using the allelic data of 54 SSRs for the 2× (a), 4× (b) and 6× (c) taxa. Well-differentiated taxa are distinctly coloured and identified
genotypes are numbered. Species suspected to be morphological variants are marked with an asterisk African genotypes appeared to have diverged after a re-
cent transatlantic migration from the Neotropics (Fig. 5b,
Additional file 9: Figure S3, Additional file 1: Table S1). A. Geographical structure of the Aeschynomene species Two pairss of factorial axes (with the percentage of variation they account for indicated in parenthesis) are
used for each species complex so as to show genetic distinctness and relationships. Taxon colours and genotype numbers are the same as in Fig. 3 standpoint. For the polyploid taxa (4 tetraploids and 2
hexaploids), low-copy nuclear genes and SSR analysis
supported an allopolyploid origin and several genome
donors could be identified [17; this study]. This must fa-
cilitate the identification and distinction of the different
subgenomic components of the polyploid genomes and
this indicated that these polyploid taxa must behave gen-
etically as diploids. Segregation of molecular markers,
however, has not been performed to date at a genome-
wide scale and we therefore cannot exclude that they
might behave as segmental allopolyploids just as recently
reported for peanut and chrysanthemum [27, 28]. Such
dual structure may be found notably in A. pratensis for
which the two expected homeologous versions of the
tested low-copy nuclear genes were not always detected. reactions characterized by the accumulation of brown
compounds, most probably of polyphenol nature as
already described for incompatible interactions [26],
were obvious in nodules of A. indica 6× -Africa-
inoculated with BTAi1 (Fig. 6b, Additional file 14:
Figure S4a). This incompatibility was accompanied by
a low nitrogen-fixing activity and an overall reduction in
plant development (Fig. 6c, Additional file 14: Figure S4b). Geographical structure of the Aeschynomene species indica 4× contained 3 distinct Indian lineages, one
genotype spanning Eastern Asia and the North Eastern
America, a second widespread in Northern Australia but
also occurring in Asia, and a third throughout South Asia
and extending to the Pacific (Fig. 5b). The Australian A. indica 6× was found to have a wide distribution extend-
ing from Eastern to Western Australia. Missing collec-
tion data limited the analysis of the three 6× genotypes
but they did not overlap in distributional area with A. indica 4× (Fig. 5b, Additional file 9: Figure S3, Additional
file 1: Table S1). In contrast, distribution of the African
A. indica 6× was restricted to the Subsahelian zone and the two identified genotypes clearly could be distin-
guished based on their location, one in the western part
of the zone, the other one with a more central position
(Fig. 5b, Additional file 9: Figure S3, Additional file 1:
Table S1). Noteworthy, at a macroscale, there exists an
overlap in distribution of African A. indica 6× with A. evenia s.s, but the latter was more widespread in Africa. To test whether this genetic diversity could support poly-
morphism in nodulation traits, a number of accessions of A. evenia s.s. and A. indica were submitted to root or stem in-
oculation with two photosynthetic nodABC gene-lacking
Bradyrhizobium strains, ORS278 and BTAi1. A high vari-
ation in stem nodule development was observed in A. evenia
when it was inoculated with ORS278 (Fig. 6a). Conversely,
root nodulation was more homogenous but plant defense Chaintreuil et al. BMC Plant Biology (2018) 18:54 Page 8 of 15 b
d a
b
c
d
Fig. 4 Factorial analysis of Nod-independent Aeschynomene taxa. (a) and (b) for the A. evenia-A. indica species complex, (c) and (d) for the A. sensitiva-A. pratensis species complex. Two pairss of factorial axes (with the percentage of variation they account for indicated in parenthesis) are
used for each species complex so as to show genetic distinctness and relationships. Taxon colours and genotype numbers are the same as in Fig. 3 a a b a c d Fig. 4 Factorial analysis of Nod-independent Aeschynomene taxa. (a) and (b) for the A. evenia-A. indica species complex, (c) and (d) for the A. sensitiva-A. pratensis species complex. Discussion This in-depth characterization of a Nod-independent
Aeschynomene
germplasm
collection
identified
four
main lineages and uncovered genetic diversity and struc-
ture at different scales: cytotypes, species and genotypes
(as summarised in Table 1). Interestingly, this group of
Aeschynomene is composed of mainly diploid taxa (13
out of 19), including the model legume A. evenia, which
are expected to be the easiest to handle at a genetic In addition to shedding light on the genetic basis of
the different Aeschynomene species, our analyses were
used to identify different taxa and to delimit their Chaintreuil et al. BMC Plant Biology (2018) 18:54 Page 9 of 15 b
a
Fig. 5 Geographical distribution of Nod-independent Aeschynomene taxa. (a) for the A. sensitiva-A. pratensis species complex and (b) for the A. evenia-A. indica species complex. Accessions with no geographical information are not shown; details of the accession origins are provided in Additional files 1 and
14: Table S1 and Figure S4. Taxon colours and genotype numbers are the same as in Fig. 3 a a b
Fig. 5 Geographical distribution of Nod-independent Aeschynomene taxa. (a) for the A. sensitiva-A. pratensis species complex and (b) for the A. evenia-A. indica species complex. Accessions with no geographical information are not shown; details of the accession origins are provided in Additional files 1 and
14: Table S1 and Figure S4. Taxon colours and genotype numbers are the same as in Fig. 3 b
g. 5 Geographical distribution of Nod-independent Aeschynomene taxa. (a) for the A. sensitiva-A. pratensis species complex and (b) for the A. evenia-A. dica species complex. Accessions with no geographical information are not shown; details of the accession origins are provided in Additional files 1 and
4 Table S1 and Fig re S4 Ta on colo rs and genot pe n mbers are the same as in Fig 3 b Fig. 5 Geographical distribution of Nod-independent Aeschynomene taxa. (a) for the A. sensitiva-A. pratensis species complex and (b) for the A. evenia-A. indica species complex. Accessions with no geographical information are not shown; details of the accession origins are provided in Additional files 1 and
14: Table S1 and Figure S4. Taxon colours and genotype numbers are the same as in Fig. Discussion 6 Variation of nodulation traits observed in accessions of A. evenia and A. indica. (a)Stem nodulation observed in various accessions of A. evenia, 3 weeks post inoculation with ORS278. (b) Root nodule development in accessions of A. evenia and A. indica following inoculation with
Bradyrhizobium ORS278 and BTAi1. 14-dpi nodules were cut to observe the leghemoglobin color and reaction defense (arrow). (c) Comparison of
plant growth (aerial part) after inoculation with Bradyrhizobium ORS278 and BTAi1, at 14 dpi. Scale bar in (a): 5 mm, in (b): 1 mm All these data taken together make our germplasm
collection a valuable genetic resource for the Nod-
independent Aeschynomene group. So how best to fur-
ther exploit it? To decipher the molecular mechanisms
underlying the different original nodulation properties
found in the Nod-independent symbiosis, A. evenia was
recently selected as a model species [16, 18]. The species
is currently being subjected to full genome sequence
analysis and an ongoing mutagenesis project is predicted
to identify new symbiotic genetic determinants in the
near future. Such approaches applied to the historical
model legumes M. truncatula and L. japonicus led to
major advances in the study of the nitrogen-fixing sym-
biosis with the identification of a set of symbiotic genes
involved in the recognition of rhizobial signals, transduc-
tion, infection and nodule organogenesis [2, 4]. But
mutants are usually screened for the loss of their ability
to establish a symbiosis, due to the disruption of gene
function, and they are developed in the frame of the
study of a single plant line-rhizobial strain system. Conversely, the screening of natural populations with
multiple rhizobial strains can reveal some symbiotic phe-
notypes that depend on both the host genetic back-
ground and the rhizobial strain. In fact, studying natural
variation approach has been shown to be a powerful tool
for gaining insights into the genetic basis underlying the
specificity of the symbiotic interaction in three legumes, be reliably considered as a new species belonging to the A. sensitiva group, notwithstanding that this conclusion is based
on a single accession of what is apparently a rare taxon. Given their high variability, the SSR markers are a power-
ful tool to highlight putative subdivisions in different
Aeschynomene species that served to define genotypes. Strik-
ing is that these genotypes, delineated solely on the basis of
marker data, corresponded to geographically based sub-
groups. Discussion 3 were found to form sister clades in the ITS and low-copy nu-
clear gene phylogenies (in accordance with their current
grouping into a single species) but are clearly distinct entities
in the SSR tree (Figs. 1,3). Concordant with this strong
genetic differentiation, the two subspecies of A. evenia previ-
ously were demonstrated not to be cross-compatible [18]. Similarly, A. virginica and A. sp. 353, along with the African
and Australian 6× A. indica taxa, formed separate groups in
the SSR tree and their hybridization generated F1 hybrids
with a marked reduction in fertility (Figs. 2,3). This raises the
question of whether these taxa should be treated at the
subspecies level or as separate species. On the other hand,
the situation was clear for Aeschynomene sp. (328); this taxon
was consistently found to be divergent in the ITS, low-copy
nuclear gene and the SSR trees (Figs. 1,3). Therefore, it can taxonomic boundaries. Indeed, the approach of genotyp-
ing a germplasm collection already has shown to be a
powerful tool resolving taxonomic issues and providing
the basis of a good taxonomic classification in the leg-
ume genus Lens that includes the cultivated lentil [29]. Here, the rare species Aeschynomene pluriarticulata and
A. magna were found to have ITS and low-copy nuclear
gene sequences very similar to those of A. evenia and A. denticulata, respectively (Fig. 1). The NJ SSR trees
further revealed that these two pairs of taxa clustered closely
together. This strongly suggested that A. pluriarticulata and
A. magna are more likely morphological variants of A. evenia
and A. denticulata than distinct species (Fig. 3). But for A. magna, living material is now necessary to confirm this. Conversely, A. evenia ssp. evenia and A. evenia ssp. serrulata Page 10 of 15 Chaintreuil et al. BMC Plant Biology (2018) 18:54 a
b
c
Fig. 6 Variation of nodulation traits observed in accessions of A. evenia and A. indica. (a)Stem nodulation observed in various accessions of A. evenia, 3 weeks post inoculation with ORS278. (b) Root nodule development in accessions of A. evenia and A. indica following inoculation with
Bradyrhizobium ORS278 and BTAi1. 14-dpi nodules were cut to observe the leghemoglobin color and reaction defense (arrow). (c) Comparison of
plant growth (aerial part) after inoculation with Bradyrhizobium ORS278 and BTAi1, at 14 dpi. Scale bar in (a): 5 mm, in (b): 1 mm b c Fig. Discussion Thus, the genetic structure of the Aeschynomene
taxa appeared to mirror the eco-geographic distribution of
the associated genotypes, a situation also described for other
plants including lentil, tomato, pigeonpea and switchgrass
[30–33]. Although not a major aim of this study, such
genetic structure can help to understand the origin and the
migration of some populations. This is notably the case for
A. indica whose natural distribution range is obscure. The
identified genetic clusters, which most prominently corre-
sponded with geographical distribution patterns, likely
reflected real differences within each species. Among the
species studied, A. evenia s.s. had the highest genetic diver-
sity with 7 genotypes, some of them previously being shown
to be fully cross-compatible [18]. It is also noteworthy that
the different Aeschynomene taxa displayed very high selfing
rates. Using lines that tend to be mostly homozygous facili-
tates artificial hybridizations and analysis of confidently seg-
regation patterns in the progeny in order to investigate the
genetic determinism underlying the nodulation traits. Page 11 of 15 Page 11 of 15 Chaintreuil et al. BMC Plant Biology (2018) 18:54 genetic analysis of this differential susceptibility. Variations
in symbiotic traits with naturally-nodulating photosyn-
thetic Bradyrhizobium strains have not been thoroughly
surveyed yet, but the observations made in the present
study using the strains ORS278 and BTAi1 yield promis-
ing preliminary results. By evaluating different genotype-
rhizobial combinations, we predict that symbiotic poly-
morphisms will be found, notably in the induction of
nodulation, nitrogen fixation and also in stem nodulation. Crossable accessions exhibiting polymorphic symbiotic
phenotypes can then be selected for hybridization experi-
ments, subsequent genetic analysis and locus mapping. This will pave the way for the identification of the genes
underpinning these symbiotic responses through forward
or reverse genetics. To assist in these studies, genomic
and genetic data, together with a number of molecular
tools, are being accumulated for the model species A. evenia and it thus represents the easiest system to work
with [16, 20]. Despite the high genetic diversity observed
in A. evenia, the number of available germplasm samples
remains relatively modest since it has not been as
extensively sampled as other legumes of interest [22]. Therefore, the accessions of other Nod-independent taxa
in the same gene pool or in the related one containing A. sensitiva, represent a good complement. In addition, the
expected high level of microsynteny with A. Discussion evenia will
facilitate a synteny-based positional approach, as has been
successfully performed between Pea and Medicago to iden-
tify the gene underlying the SYM2 locus [34, 45]. As a re-
sult, genetic resources developed for the Nod-independent
Aeschynomene clade can be fully exploited for the search of
natural variation in nodulation with Bradyrhizobium. Medicago, Lotus or soybean. The identified symbiotic
polymorphisms were mostly of two types: some plant-
rhizobial strain combinations resulted in non-nodulating
phenotypes (Nod−), others in the production of small
white infected nodules proved to be defective in nitrogen
fixation (Fix−) [34–38]. This indicated that the control of
host-rhizobial strain compatibility occurred at two differ-
ent levels in the symbiotic process. y
p
It is noteworthy that the genetic analysis of natural vari-
ation shed light on the function of some key symbiotic
genes. The LysM-RLK receptors, which were identified
using the mutant approach to be the probable Nod factor
receptors, represent a well-known example. Indeed, a
synteny-based positional cloning identified LYK3 in Medi-
cago as corresponding to the SYM2 gene that controls the
symbiotic infection in a Nod factor structure dependent
manner in the pea ‘Afghanistan’ ecotype [34, 38]. The diver-
sity information has also been exploited in Lotus to further
substantiate that the LysM-RLK receptors mediate specific
recognition of Nod factors [39]. More recently, our current
understanding of the symbiotic mechanisms has been chal-
lenged for the supposed role of NCR peptides that were ini-
tially shown as important effectors of endosymbiont’s
differentiation to nitrogen-fixing bacteroids. Making use of
the differential ability of Sinorhizobium meliloti Rm41 to
form functional or non-nitrogen fixing nodules depending
on the Medicago accession used, two genes, NFS1 and
NFS2, were identified and shown to code for NCR peptides
[36–40]. This broadened the role of the NCR peptides in
the fixation stage by revealing that some of them also con-
trol discrimination against incompatible microsymbionts. A
thorough survey of naturally occurring variation also pro-
vided ground for the discovery of new genetic determinants
of nodulation. This was notably the case in soybean where
this approach led to the identification of two dominant
genes that restrict nodulation in a strain-specific manner,
Rj2/Rfg1 encoding a TIR-NBS-LRR resistance (R) gene and
Rj4 that codes for a thaumatin-like protein [41, 42]. Discussion This
revealed that some host genotypes are able to trigger gene-
for-gene resistance, which is found in plant-pathogen
interactions, to selectively interact with certain symbiotic
strains but to exclude others. Genome size estimation and chromosome counting g
Flow cytometry measurement was performed on leaf
material to estimate genome sizes of various accessions
as already explained [16]. These estimates were based on
the measurements of three plants per accession using
Lycopersicum esculentum (Solanaceae) cv “Roma” (2C =
1.99 pg) as the internal standard. For chromosome num-
ber counts, metaphasic chromosomes were prepared
from root-tips, spread on slides and stained with DAPI
(4′,6-diamidino-2-phenylindole). Chromosomes
were
counted from images obtained with a fluorescent micro-
scope as previously described [16]. Plant DNA extraction underlying genetic determinants. Discoveries of alterna-
tive functions in symbiotic genes identified in other
model legumes or of new genes involved in the recogni-
tion of the still unknown non-Nod bacterial signal, nod-
ule functioning and in the restriction of compatibility
would add a new dimension to our understanding of the
genetic control of nodulation in the Nod-independent
symbiosis. Genomic DNA was extracted from fresh leaves using the
CTAB (Cetyl Trimethyl Ammonium Bromide) method
improved by the addition of β-mercaptoethanol 2% and
PVPP 2%. For herbarium material, a protocol adapted
for fragmented DNA was used, with increased length of
the incubation (90 min), centrifugation (20 min) and
precipitation (15 min) steps was used. DNA quantity
was evaluated by spectrophotometer and DNA samples
normalized to a uniform concentration of 10 ng/μL. Gene sequencing and sequence analysis All the accessions of Aeschynomene used in this study,
their geographical origin and source data are listed in
Additional file 1: Table S1. Seeds were scarified with
sulphuric acid for germination and plants were grown in
pots filled with compost under greenhouse conditions
(temperature:
26-36
°C,
relative
humidity:
70%-80%,
insect-proof screens) as detailed [16]. Interspecific crosses
were performed according to the protocol developed earl-
ier and the nature of the resulting hybrids was checked
using SSR markers (data not shown) [16]. Fertility of the
F1 plants was assessed by recording the number of
successfully developed pods per flowering axis and the
number of seeds in each pod. The nuclear ribosomal internal transcribed spacer region
(ITS: ITS1-5.8S rDNA gene-ITS2) and five low copy
nuclear genes CYP1, eiF1α, Sucrose Synthase, SUI1 and a
gene homolog to Glyma.07G136800 and Glyma.18G187300
identified in Glycine max were amplified with the primers
listed in Additional file 2: Table S2. PCR amplifica-
tions, cloning and sequencing of PCR products were
performed as already described [17, 18]. The DNA se-
quences generated in this study were deposited in
Genbank (Additional file 3: Table S3) and additional
sequences are available in Additional file 4: Doc. S1. For the phylogenetic analyses, the gene sequences
were aligned in ClustalX, version 1.81b and the align-
ments were checked in Genedoc v2.7. Phylogenetic
reconstructions were performed with the MEGA v7
program using the Neighbor Joining approach and the
Tamura 3-parameter model with a 1000 x bootstrap. SSR marker selection and genotyping SSR marker selection and genotyping
A total of 500 primer pairs were initially defined to de-
velop SSR markers for genetic mapping in A. evenia
(Additional file 7: Table S4) [9]. In the present study,
they were again tested on two polymorphic accessions of
A. evenia and two cytotypes of A. indica (Additional file 8:
Table S5). Forward primers all contained a 5′-end M13
tail (5′-CACGACGTTGTAAAACGAC-3′), enabling the
tagging the PCR products during the PCR amplification
with 4 M13 primer-fluorescent dyes 6-FAM™, NED®, VIC®,
or PET® (Applied Biosystems, CA, USA). Amplicon sizes
were analysed using an ABI 3700 automatic capillary se-
quencer (Applied Biosystems) as previously described [9]. 54 SSR markers were organized in 4-SSR multiplexes and
used to genotype the accessions belonging to the A. evenia
and the A. sensitiva groups as detailed in Additional file 6:
Figure S2. Allele scorings were analysed using GeneMapper
4.0 software (Applied Biosystems) and exported as data ta-
bles for two groups of genotyping. Conclusions With the goal of discovering natural variation in the
Nod-independent Aeschynomene legumes, we developed
a large collection of 226 accessions that spans the geo-
graphical distribution of the different taxa in this group,
including the model species A. evenia. These accessions
were subjected to combined analyses of gene sequen-
cing, cytogenetics, hybridization experiments and SSR
genotyping. This work resulted in the delineation of
taxon boundaries and in the discovery of new genotypes. Taxa displaying a significant genetic diversity were char-
acterized by a clear geographically-based genetic struc-
ture. In addition, low-copy nuclear genes and patterns of
microsatellite diversity illuminated the genetic basis of
the Aeschynomene diploid and polyploid taxa that are all
predominantly autogamous and have a predicted simple
disomic inheritance, two attributes favorable for genet-
ics. Such a well-characterized collection of accessions
constitutes a major genetic resource for exploring the
natural variation of nodulation in response to different
Bradyrhizobia
strains
and
for
searching
for
their These illuminating examples show that there is a
mileage to be gained from exploring the natural variation
in nodulation in the Nod-independent Aeschynomene
group. To screen our germplasm collection, many Bradyr-
hizobium strains are available, including photosynthetic
and non-photosynthetic ones [14, 43, 44]. A recent report
revealed important variations in the ability of different
Nod-independent Aeschynomene species to be nodulated
in a T3SS-dependent fashion by the non-photosynthetic
Bradyrhizobium strains STM6978 and USDA61 [14]. Such
marked variations were also observed between different
accessions of A. evenia s.s., providing the basis for a Page 12 of 15 Page 12 of 15 Chaintreuil et al. BMC Plant Biology (2018) 18:54 Plant nodulation and ARA Nodulation tests were carried out using Bradyrhizobium
sp. strains ORS278 and BTAi1 [12]. The strains were
cultivated for 7 days in yeast-mannitol liquid medium at
34 °C. Root inoculation was performed in an in vitro
growth chamber on 7-day-old plants using 1 mL of bac-
terial culture with an optical density at 600 nm adjusted
to 1. Stem inoculation was carried out in a greenhouse
by wrapping the stem of 5-week-old plants with a paper
soaked with bacterial culture for 24 h. Stem nodules
were observed at 21dpi and root nodules at 14dpi. Nitrogen-fixing activity was estimated on the entire
plant by measurement of acetylene reducing activity
(ARA) and microscopic observations were performed
using a stereo-microscope (Nikon AZ100, Champigny-
sur-Marne, France) as previously published [46]. Competing interests Additional file 10: Table S6. Allelic diversity of the SSR markers (XLSX
22 kb) Additional file 10: Table S6. Allelic diversity of the SSR markers (XLSX
22 kb) Funding g
This work was supported by a grant from the French National Research
Agency (ANR-AeschyNod-14-CE19-0005-01) that served for the design of the
study, experimentation and analysis of the data. Availability of data and materials The gene sequences generated in this study have been deposited in
GenBank (accession numbers listed in Additional file 3: Table S3) and
additional sequences are available in Additional file 4: Doc. S1. All the SSR
marker characteristics are included as supplementary information files
(Additional files 7, 8, 10, 11, 12 and 13: Table S4-S9). SSR data analysis Genotyping data files were assembled in a single database
that was used to determine genetic relationships among
the accessions. For this, a distance-based approach was Page 13 of 15 Chaintreuil et al. BMC Plant Biology (2018) 18:54 Page 13 of 15 applied by calculating with a shared allele index a
genetic dissimilarity matrix in DARwin v5 software
[25]. Then, individual relations were separately ana-
lyzed for each ploidy level (2×, 4×, 6×) with a tree
construction based on an unweighted Neighbor Join-
ing method while genetic affinities between diploid
and polyploid species were investigated by a factorial
analysis as implemented in DARwin v5. The genetic
diversity was evaluated by computing the number of
alleles per locus (Na) and the observed heterozygos-
ities Ho for each SSR locus and for different accession
groups (Additional files 12 and 13: Tables S8, S9). USDA (USA). We are also very grateful to the many herbaria and botanists who
provided additional relevant plant material that significantly contributed our
sampling. The present work has benefited from the facilities and expertise of the
cytometry facilities of Imagerie-Gif (http://www.i2bc.paris-saclay.fr/spip.php?rubri-
que184) and of the molecular cytogenetic facilities of the AGAP laboratory (https://
umr-agap.cirad.fr/en/platforms/plateformes/genotyping). Authors’ contributions CC was responsible for obtaining the plant material and the SSR genotyping
data, and together with LB carried out the gene sequencing. RR and PM
participated in the SSR marker development and genotyping. XP, GM, LL and
BT performed computational analysis of the SSR genotyping data. MGP and
MB carried out cytometry analyses. JF and EG handled the nodulation tests. HV, H. Randriambanona and H. Ramanankierna organized field collection of
plant material and contributed to the acquisition and analysis of diversity
data. GPL provided expertise in legume taxonomy and biogeography, and
together with EG reviewed the manuscript. J.F.A. conceived the study,
analyzed the data and wrote the paper. All authors read and approved the
final manuscript. Additional file 2: Table S2. Nuclear genes used for the phylogenetic
analyses. (XLSX 11 kb) Additional file 3: Table S3. GenBank numbers for the sequences used
in the phylogenetic analyses. (XLSX 12 kb) Additional file 4: Doc. S1. ITS sequences obtained for the Nod-
independent Aeschynomene accessions. (DOCX 30 kb) Additional file 5: Figure S1. Chromosome numbers in new
Aeschynomene taxa. Root tip metaphase chromosomes stained in blue
with DAPI (4′,6-diamidino-2-phenylindole). Chromosome numbers are
indicated in brackets. Scale bars: 5 μm. (PPTX 1008 kb) Additional file 6: Figure S2. Schematic representation of the different
steps of the genotyping process from marker selection to data treatment. (PPTX 64 kb) Additional files Additional file 1: Table S1. Accessions used in this study, origin and
characteristics. (XLSX 23 kb) Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Additional file 11: Table S7. Cross-species transferability of the SSR
markers (XLSX 13 kb) Additional file 11: Table S7. Cross-species transferability of the SSR
markers (XLSX 13 kb) Ethics approval and consent to participate The sources of the Aeschynomene accessions used in this study are indicated
in Additional file 1: Table S1. In addition to seedbank and herbaria supply,
some accession were collected for purpose of this study in Madagascar (with
authorization from the Malagasy Ministry of Scientific Research) and in
New-Caledonia (licence n°60,912 from the Northern Province). Collection and
identification of these new accessions were performed by Heriniaina
Ramanankierana, Herizo Randriambanona, Hervé Vandrot and Jean-François
Arrighi. Additional file 7: Table S4. Origin, location and primer sequences for
the SSR markers used for genotyping. (XLSX 13 kb) Additional file 8: Table S5. Repeat motif and allelic amplification
profiles of the SSR selected for genotyping. (XLSX 13 kb) Additional file 9: Figure S3. Detailed NJ trees representing the genetic
diversity among the Nod-independent Aeschynomene accessions. The
trees were developed separately in DARWIN using the allelic data of 65
SSRs for the 2× (a), 4× (b) and 6× (c) taxa. Well-differentiated taxa are
distinctly colored. Identified genotypes are marked with a red dot and
numbered. Accessions are designated with their LSTM code mentioned
in Additional file 1: Table S1 followed by their geographical origin. Species suspected to be morphological variants are marked with an
asterisk. Taxon colours and genotype numbers are the same as in Fig. 3. (PPTX 187 kb) Additional file 9: Figure S3. Detailed NJ trees representing the genetic
diversity among the Nod-independent Aeschynomene accessions. The
trees were developed separately in DARWIN using the allelic data of 65
SSRs for the 2× (a), 4× (b) and 6× (c) taxa. Well-differentiated taxa are
distinctly colored. Identified genotypes are marked with a red dot and
numbered. Accessions are designated with their LSTM code mentioned
in Additional file 1: Table S1 followed by their geographical origin. Species suspected to be morphological variants are marked with an
asterisk. Taxon colours and genotype numbers are the same as in Fig. 3. (PPTX 187 kb) Author details
1 Additional file 12: Table S8. Scoring of mean allele number per SSR
and species (XLSX 15 kb) Additional file 12: Table S8. Scoring of mean allele number per SSR
and species (XLSX 15 kb) 1IRD, Laboratoire des Symbioses Tropicales et Méditerranéennes, UMR LSTM,
Campus International de Baillarguet, F-34398 Montpellier, France. 2CIRAD,
Amélioration Génétique et Adaptation des Plantes Méditerranéennes et
Tropicales, UMR AGAP, Campus de Lavalette, F-34398 Montpellier, France. 3AGAP, Univ. Montpellier, CIRAD, INRA, Montpellier SupAgro, Montpellier,
France. 4Comparative Plant and Fungal Biology Department, Royal Botanic
Gardens, Kew, Richmond, Surrey TW9 3AB, UK. 5Institute of Integrative
Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud. Université Paris-Saclay,
91198 Gif-sur-Yvette, France. 6Laboratoire de Microbiologie de
l’Environnement/Centre National de Recherche sur l’Environnement, 101
Antananarivo, Madagascar. 7IAC, Laboratoire de Botanique et d’Ecologie
Végétale Appliquée, UMR AMAP, 98825 Pouembout, Nouvelle-Calédonie,
France. 8LSTM, Univ. Montpellier, CIRAD, INRA, IRD, Montpellier SupAgro,
Montpellier, France. 1IRD, Laboratoire des Symbioses Tropicales et Méditerranéennes, UMR LSTM,
Campus International de Baillarguet, F-34398 Montpellier, France. 2CIRAD,
Amélioration Génétique et Adaptation des Plantes Méditerranéennes et
Tropicales, UMR AGAP, Campus de Lavalette, F-34398 Montpellier, France. 3AGAP, Univ. Montpellier, CIRAD, INRA, Montpellier SupAgro, Montpellier,
France. 4Comparative Plant and Fungal Biology Department, Royal Botanic
Gardens, Kew, Richmond, Surrey TW9 3AB, UK. 5Institute of Integrative
Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud. Université Paris-Saclay,
91198 Gif-sur-Yvette, France. 6Laboratoire de Microbiologie de Additional file 13: Table S9. Observed heterozygosity (Ho) in
Aeschynomene species (XLSX 14 kb) Additional file 13: Table S9. Observed heterozygosity (Ho) in
Aeschynomene species (XLSX 14 kb) Additional file 14: Figure S4. Comparison of the nodulation properties
of A. evenia s.s. and A. indica. Different accessions were root inoculated
with Bradyrhizobium ORS278 and BTAi1 and analysed at 14dpi. (a)
Number of nodules per accession. (b) Acetylene-reducing activity (ARA). A.U. Arbitrary Unit. Error bars represent s.d. (n = 6). (PPTX 179 kb) Additional file 14: Figure S4. Comparison of the nodulation properties
of A. evenia s.s. and A. indica. Different accessions were root inoculated
with Bradyrhizobium ORS278 and BTAi1 and analysed at 14dpi. (a)
Number of nodules per accession. (b) Acetylene-reducing activity (ARA). A.U. Arbitrary Unit. Error bars represent s.d. (n = 6). (PPTX 179 kb) Consent for publication
Not applicable Competing interests
The authors declare that they have no competing interests. Competing interests
The authors declare that they have no competing interests. Acknowledgements
f We are grateful to the different germplasm banks for provision of seeds: AusPGRIS
(Australia), CIAT (Colombia), ILRI (Ethiopia), IRRI (Philippines), Kew Gardens (UK) and Page 14 of 15 Page 14 of 15 Page 14 of 15 Chaintreuil et al. BMC Plant Biology (2018) 18:54 Chaintreuil et al. BMC Plant Biology (2018) 18:54 Received: 27 October 2017 Accepted: 6 March 2018 References Liu J, Yang S, Zheng Q, Zhu H. Identification of a dominant gene in
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Del Carmen Villegas-Hernandez M, et al. Diversity analyses of Aeschynomene Page 15 of 15 Chaintreuil et al. BMC Plant Biology (2018) 18:54 • We accept pre-submission inquiries
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The Effect of Freezing on Non-invasive Prenatal Testing
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Scientific reports
| 2,019
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cc-by
| 4,995
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Xiaolei Xie1,2, Fuguang Li1, Weihe Tan1, Weiguo Yin2, Feiyan Chen3 & Xiaoyan Guo1 Xiaolei Xie1,2, Fuguang Li1, Weihe Tan1, Weiguo Yin2, Feiyan Chen3 & Xiaoyan Guo1 Plasma cryopreservation is unavoidable in China, due to technical specifications requiring storage of
additional plasma at −80 degrees for three years. However, the effect of freezing on non-invasive
prenatal testing (NIPT) is still uncertain. We collected 144 euploid pregnant samples, 22 on trisomy
21, 4 on trisomy 13, and 3 on trisomy 18, by massively parallel sequencing before and after freezing. Compared with the success rate of 100% of fresh samples, the detection success rates of trisomy 21,
trisomy 13 and euploidy in frozen samples by NIPT were 95.45%, 75% and 95.14%, respectively. Of
these, 9 cases of frozen sample sequencing failed, with 8 cases being due to high GC content. The
chromosome 21 (chr21) z-value of the frozen trisomy 21 samples was lower than that of fresh samples. Meanwhile, freezing reduced the male positive foetal cell-free DNA (cfDNA) fraction, which was
accompanied by an increase in the Unimap-GC level in the massively parallel sequencing data and a
decrease in the Unique reads/Total reads ratio. Laboratory freezing reduced the chr21 z-value of foetal
trisomy 21, which can be explained by a reduction in the foetal cfDNA fraction and effective Unique
reads for NIPT analysis. The Unimap-GC content of the serum samples after freezing was higher, which
can lead to failure of NIPT detection. Received: 29 August 2018
Accepted: 10 April 2019
Published: xx xx xxxx Chromosomal foetal aneuploidies represent a major class of genetic defects, including trisomy 21, trisomy 18,
trisomy 13 and sex chromosome aneuploidies1,2. Most notably, the incidence and hazard of trisomy 21 (Down
syndrome) for families are much higher compared to trisomy 18 and 13, with the current occurrence rate for tri-
somy 21 being one in 417 newborns2. Traditional serum screening is not effective for trisomy 21 detection3. With
the development of next generation sequencing technology, non-invasive prenatal testing (NIPT) for foetal chro-
mosomal aneuploidy serum screening has been validated by various clinical trials, and NIPT has been proven to
be highly sensitive and specific for trisomy 214–6. g yi
y
Foetal cell-free DNA (cfDNA) is found in maternal plasma and serum7. One important clinical application is
the use of cfDNA in maternal blood for non-invasive prenatal diagnosis8,9. Foetal trisomy determination of spe-
cific chromosomes depends on a small increase in the proportion of the chromosome and is detected by a z-test. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Xiaolei Xie1,2, Fuguang Li1, Weihe Tan1, Weiguo Yin2, Feiyan Chen3 & Xiaoyan Guo1 The foetal cfDNA fraction is an important parameter to determine the z value for foetal chromosomal aneuploid
assessment10,11.t During the NIPT laboratory testing process, the remaining maternal plasma samples are often stored at
−80 °C for a long period of time. However, the effect of freezing on NIPT detection is still uncertain. In this study,
we investigated the impact of freezing on the performance of NIPT testing, thus allowing us to understand the
effect of laboratory treatment on trisomy 21 detection sensitivity. Received: 29 August 2018
Accepted: 10 April 2019
Published: xx xx xxxx Results The freezing time of each group was different, with the longest freezing time in the trisomy 13
group, with a median of 76 days, and the shortest freezing time in the euploid group, with a median of 13 days.ih and 25 weeks. The freezing time of each group was different, with the longest freezing time in the trisomy 13
group, with a median of 76 days, and the shortest freezing time in the euploid group, with a median of 13 days. h
l
ll
l l
fi
d
h
f
f l Th f
l g
p
y
g
p
g
p
y
Among the 173 samples, all maternal plasma DNA first sequenced without freezing was successful. The foetal
chromosomal aneuploidy or euploidy, including trisomy 21, trisomy 18, and trisomy 13, was accurately iden-
tified with a detection sensitivity of 100% and a detection specificity of 100%. After freezing serum samples
at −80 °C, the detection success rates of trisomy 21, trisomy 13 and euploidy on NIPT were 95.45%, 75% and
95.14%, respectively. Nine cases of plasma cfDNA for a second sequencing failed, including 1 case of trisomy 21,
1 case of trisomy 13, and 7 cases of euploidy. The median maternal age and gestational weeks for the nine cases in
which sequencing failed were 34.5(range 27–44) and 16 (range 13–25), respectively. There was no specific distri-
bution of maternal age or gestational week in these 9 failed cases. Among these cases, 8 cases were due to high GC
content, and 1 case of trisomy 13 detection failed. All failed results were interpreted as redo by the BambniTest
software. Details are shown in Table 2. Freezing reduces the z value of trisomy 21. The results of NIPT were consistent before and after freez-
ing except for the detection failure of a few samples. Comparing the chromosome 21 (chr21) z value of NIPT
between fresh samples and frozen samples, 80.95% (17/21) of fresh sample values were higher than frozen values,
and 19.05% (4/21) of the values were lower than frozen values (Fig. 1a). Our data show that the chr21 z values
of the fresh sample group were significantly higher than the values of the frozen group by using a paired t test
(Fig. 1b). There was no significant difference between the chromosome 13 (chr13) z value and the chromosome
18 (chr18) z value before and after freezing (Fig. 1c,d). Results The normal euploid foetus exhibited no obvious difference
in fresh and frozen samples (Fig. 1e). Comparing the sequencing data before and after freezing. We next investigated the sequencing data
of 27 positive samples (21 trisomy 21, 3 trisomy13 and 3 trisomy18) before and after freezing. There were no sig-
nificant differences in total reads and uniq-reads between the two groups (see Supplemental Fig. 1), but the ratios
of Uniq-reads/Total-reads in fresh samples were higher than the ratios in frozen samples (Fig. 2a). This suggests
that more effective sequencing data in fresh samples were used to evaluate the NIPT results. At the same time, the
content of Unimap-GC upon freezing increased significantly (Fig. 2b).i p
p
g
gi
y ( g
)
To confirm whether changes in foetal cfDNA fractions occur due to freezing, we further selected and com-
pared the foetal fraction in 18 cases of male positive samples (14 trisomy 21, 2 trisomy13 and 2 trisomy18). The
minimum chromosome Y (chrY) z-value of male positive samples was 22.47, and the foetuses were confirmed to
be male by follow-up. The result showed that the foetal cfDNA fraction in fresh samples was slightly higher than
the fractions in frozen samples by using the paired t test (Fig. 2c). Results Maternal plasma DNA sequencing before and after freezing. Maternal characteristics of the study
population are presented in Table 1. We collected 144 euploid pregnant samples, 22 on trisomy 21, 4 on trisomy
13, and 3 on trisomy 18 for non-invasive prenatal cell-free DNA sequencing before and after freezing. The median
gestational week for trisomy 21 was 17 (range 12–23), which is slightly older than the trisomy13 and 18 groups. In
the clinical routine, traditional serum screening abnormalities would be required for NIPT, so the T21 gestational
week was slightly older. Trisomies 13 and 18 were often accompanied by malformations that can be detected by
B-ultrasound, suggesting early detection of gestational week. The gestational age of all samples was between 12 1Prenatal Diagnosis Center, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People’s
Hospital, Qingyuan, Guangdong, 511518, China. 2Molecular Diagnosis Center, The Sixth Affiliated Hospital of
Guangzhou Medical University, Qingyuan People’s Hospital, Qingyuan, Guangdong, 511518, China. 3Guangzhou
KingMed Company, Guangdong, China. Correspondence and requests for materials should be addressed to X.G. (email: 743090128@qq.com) Scientific Reports | (2019) 9:6962 | https://doi.org/10.1038/s41598-019-42980-7 Scientific Reports | (2019) 9:6962 | https://doi.org/10.1038/s41598-019-42980-7 1 www.nature.com/scientificreports/ n
Median, range
Maternal age (years)
Gestational weeks
Freezing time (days)
Trisomy 21
22
38.5 (15.0–44.0)
17.0 (12.0–23.0)
25.0 (12.0–88.0)
Trisomy 13
4
30.0 (30.0–40.0)
16.0 (16.0–20.0)
76.0 (14.0–78.0)
Trisomy 18
3
36.0 (35.0–41.0)
16.0 (15.0–20.0)
44.0 (38.0–45.0)
Euploid
144
31.0 (16.0–43.0)
17.0 (12.0–25.0)
13.0 (9.0–18.0)
Table 1. Characteristics of pregnant women within the study population. Table 1. Characteristics of pregnant women within the study population. Syndrome
The detection success ratios of NIPT
Fresh samples
Frozen samples
Trisomy 21
100% (22/22)
95.45% (21/22)
Trisomy 13
100% (4/4)
75% (3/4)
Trisomy 18
100% (3/3)
100% (3/3)
Euploid
100% (144/144)
95.14% (137/144)
Table 2. Summary of the syndromes detected before and after freezing. Syndrome
The detection success ratios of NIPT
Fresh samples
Frozen samples
Trisomy 21
100% (22/22)
95.45% (21/22)
Trisomy 13
100% (4/4)
75% (3/4)
Trisomy 18
100% (3/3)
100% (3/3)
Euploid
100% (144/144)
95.14% (137/144)
Table 2. Summary of the syndromes detected before and after freezing. Table 2. Summary of the syndromes detected before and after freezing. Table 2. Summary of the syndromes detected before and after freezing. and 25 weeks. Discussion Non-invasive prenatal testing by massively parallel sequencing has been reported since 200812. Numerous studies
proved that NIPT was highly accurate for detecting foetal chromosomal aneuploidies2,5,6,8, which presents a new
era of prenatal screening. Frozen serum samples are unavoidable in the process of NIPT detection in the labora-
tory. The findings in this study demonstrated that the chr21 z-value of NIPT detection was reduced by laboratory
freezing treatment. Meanwhile, freezing reduced the male foetal cfDNA fraction. It was also shown that the con-
tent of Unimap-GC in frozen samples increased, whereas the Unique reads/Total reads ratio decreased.fh p
p
q
Foetal cfDNA is a key determinant that affects the performance of foetal DNA-based prenatal tests11,13. The
foetal fraction is derived from apoptotic trophoblastic cells in the placenta14. Samples with sufficient foetal frac-
tions that pass quality control metrics can provide an accurate assessment of the chromosomes tested. A variety of
methods are used to calculate foetal DNA concentration15,16. In our study, freezing led to a decrease in foetal DNA Scientific Reports | (2019) 9:6962 | https://doi.org/10.1038/s41598-019-42980-7 2 www.nature.com/scientificreports/ Figure 1. Non-invasive prenatal testing of trisomy 21,13,18 or euploid foetus samples before and after freezing. (a,b) Comparison of the chr21 z-score of trisomy 21 before and after freezing. (c) Comparison of the chr13
z-score of trisomy 13 before and after freezing. (d) Comparison of the chr18 z-score of trisomy 18 before and
after freezing. (e) Comparison of the chr21 z-score of the euploid foetus before and after freezing. Error bars are
the SEM. **P < 0.01, paired t test. Figure 1. Non-invasive prenatal testing of trisomy 21,13,18 or euploid foetus samples before and after freezing. (a,b) Comparison of the chr21 z-score of trisomy 21 before and after freezing. (c) Comparison of the chr13
z-score of trisomy 13 before and after freezing. (d) Comparison of the chr18 z-score of trisomy 18 before and
after freezing. (e) Comparison of the chr21 z-score of the euploid foetus before and after freezing. Error bars are
the SEM. **P<0.01, paired t test. Figure 1. Non-invasive prenatal testing of trisomy 21,13,18 or euploid foetus samples before and after freezing. (a,b) Comparison of the chr21 z-score of trisomy 21 before and after freezing. (c) Comparison of the chr13
z-score of trisomy 13 before and after freezing. (d) Comparison of the chr18 z-score of trisomy 18 before and
after freezing. Discussion (e) Comparison of the chr21 z-score of the euploid foetus before and after freezing. Error bars are
the SEM. **P < 0.01, paired t test. Figure 2. Analysis of the sequencing data on trisomy 21,13 or 18 samples before and after freezing. (a)
Comparison of the ratios of Uniq-reads/Total-reads in 27 positive samples, including 21 trisomy 21, 3 trisomy
13and 3 trisomy 18. (b) Comparison of the content of Unimap-GC in 27 positive samples before and after
freezing. (c) Comparison of the positive male foetal fraction before and after freezing. Error bars are the SEM. **P < 0.01, ****P < 0.0001, paired t test. Figure 2. Analysis of the sequencing data on trisomy 21,13 or 18 samples before and after freezing. (a) Figure 2. Analysis of the sequencing data on trisomy 21,13 or 18 samples before and after freezing. (a)
Comparison of the ratios of Uniq-reads/Total-reads in 27 positive samples, including 21 trisomy 21, 3 trisomy
13and 3 trisomy 18. (b) Comparison of the content of Unimap-GC in 27 positive samples before and after
freezing. (c) Comparison of the positive male foetal fraction before and after freezing. Error bars are the SEM. **P < 0.01, ****P < 0.0001, paired t test. Figure 2. Analysis of the sequencing data on trisomy 21,13 or 18 samples before and after freezing. (a)
Comparison of the ratios of Uniq-reads/Total-reads in 27 positive samples, including 21 trisomy 21, 3 trisomy
13and 3 trisomy 18. (b) Comparison of the content of Unimap-GC in 27 positive samples before and after
freezing. (c) Comparison of the positive male foetal fraction before and after freezing. Error bars are the SEM. **P < 0.01, ****P < 0.0001, paired t test. concentration after cryopreservation. With the reduction of effective sequencing data for NIPT analysis, it is rea-
sonable to speculate that the chr21 z-value decreases after freezing. Further analysis revealed that the 21-trisomy
foetal cfDNA concentration did not decrease with the freezing time, suggesting that freezing may affect the chr21
z-value regardless of the length of freezing time. This reminds physicians and examiner to avoid freezing the
NIPT plasma during transport or storage, especially plasma with low foetal cfDNA concentrations. The freezing
time was different among groups. Trisomy 13 was frozen the longest and showed the worst performance. Discussion Due to
limitation of the number of samples, we cannot make an inference regarding whether the long freezing time leads
to a low detection success rate. Scientific Reports | (2019) 9:6962 | https://doi.org/10.1038/s41598-019-42980-7 3 www.nature.com/scientificreports/ On average, the amount of foetal cfDNA in plasma from a pregnant woman is approximately 2–20%, but there
is a large variance in the fraction of foetal cfDNA between patients17. The higher the percentage of foetal cfDNA,
the more effective NIPT is at distinguishing foetal trisomy from a euploid foetus, especially for trisomy 2116. This study suggests that the high level of foetal cfDNA concentration and more effective data will increase the
detection success rate of trisomy 21, especially the z-value in cut-off regions. It has been speculated that the foetal
fraction should be at least 4% to allow for reliable detection of common trisomies18. Currently, the threshold we
use for accurate detection of trisomy using Illumina’s NextSeq CN500 is a foetal cfDNA fraction of no less than
3.5%. In the 9 cases of NIPT detection failure after freezing, no sample was detected due to the cfDNA concen-
tration being below 3.5%.f g
Foetal cfDNA is affected by many factors, including the gestational age19, maternal weight16 and physical activ-
ity20. The cfDNA in plasma increases as pregnancy progresses, but it decreases with maternal weight gain. There
seemed to be a slight positive correlation between the foetal DNA fraction and the crown-rump length (CRL), log
freeβ-hCG MoM and log PAPP-A MoM16. However, the nuchal translucency (NT) and serum prenatal screening
results in the high-risk and low-risk groups showed no significant differences in the foetal cfDNA fraction21. In
the samples positive for trisomies 13 and 18, the foetal fractions were significantly lower compared with that of
the NIPT-negative cases and trisomy21, indicating that different foetal aneuploidies have varied effects on the
foetal DNA fraction22,23.The higher foetus fractions in trisomy 21 may be one of the reasons that the detection
performance for trisomy 21 was better than that of trisomy 13 or 18. These studies indicate that trisomy 21 is
more susceptible to foetal fraction. Because of the limited number of trisomies 13 and 18, we did not see the effect
of the foetal fraction changes in trisomies 13 and 18 after freezing in this study. Materials and Methods Study design and sample collection. Samples from 173 pregnant women were collected between April
2017 and July 2017 for this study. The research adhered to the tenets of the Declaration of Helsinki on research
involving human subjects. The study design was approved by the ethics committee of the Six Affiliated Hospital
of Guangzhou Medical University. All participants gave informed written consent. The pregnant women were
enrolled as participants and were determined to be high risks for aneuploidy by conventional serum screen-
ing, single serum index abnormalities, abnormal foetal ultrasound or maternal age (≥35 years). All partici-
pants (range 12 to 25 weeks gestation) required NIPT detection to avoid foetal trisomies 21,18 and 13. One
hundred seventy-three pregnant women bearing 99 male foetuses and 74 female fetuses participated in the study. Follow-up information was acquired by telephone. All positive results of NIPT detection were confirmed by pre-
natal diagnosis for chromosome karyotype analysis. The foetus gender was confirmed by follow-up after birth. Ten milliliters of maternal peripheral blood was collected into cell-free DNA BCT tubes (Streck company, La
Vista, USA) for NIPT detection. Maternal plasma processing, cfDNA extraction and sequencing. The peripheral maternal blood
samples were sent to a clinical laboratory within 2 hours. The blood samples were centrifuged first at 1,600 g
for 10 min at 4 °C. The plasma was transferred to microcentrifuge tubes and then centrifuged again at 16,000 g
for 10 min to remove residual cells. Each blood sample was separated into three tubes of plasma (1.2 ml serum/
tube), with the fresh first tube used for a follow-up experiment and the rest stored at −80 °C. On the first day, we
extracted cfDNA in 1.2 ml of serum from pregnant women using the Berry Genomics Nucleic Acid kit (Berry
genomics company, Beijing, China). The cfDNA was dissolved in 40 μL of Tris-HCL solution (10 mM), with a
concentration greater than 0.5 ng/μL. The next day, all cfDNA was used as the input DNA to create a library using
the library prep kit from Berry Genomics. Plasma DNA libraries of 95 samples were indexed using 6 bp indexing
oligos and quantitated by the SYBR fast qPCR kit from Kapa Biosystems (Woburn, MA, USA). The library con-
centration of each sample must be greater than 10pM before sequencing. On the third day, each sample library
was pooled and loaded into a NextSeq flow cell at 3pM DNA concentration. Discussion Future research should be exam-
ined whether increased GC content in cfDNA after cryopreservation means that freezing more likely destroys the
two hydrogen bonds between adenine and thymine, resulting in an increased GC content. y
g
y
g
In conclusion, this study determined that freezing decreased the chr21 z-value of NIPT detection. Laboratory
freezing reduced the foetal cfDNA concentration, accompanied by an increase in the Unimap-GC content in the
massively parallel sequencing data and a decrease in the Unique reads/Total reads ratio. The findings in this study
provide one laboratory factor affecting the accuracy of NIPT detection, especially for trisomy 21, which will help
to further understand cfDNA characteristics. Materials and Methods Clustering and sequencing were con-
ducted according to Illumina’s NextSeq CN500 instructions using the single-ended 45 bp sequencing protocol. The time of the NIPT clinical reports for the fresh samples was no more than 7 working days. The procedure of
NIPT detection in frozen samples was the same as in fresh samples. Sequencing data analysis The male foetal DNA fraction calculation based on unique read counts on the
Y chromosome were reliable15. We calculated the proportion of male foetal DNA based on the calculation method
published by Irena H et al.16.h The male foetal DNA fraction = (% chrY-female % chrY)/(male % chrY- female % chrY), where % chrY repre-
sents the ratio of unique reads of the Y chromosome to the number of unique reads on all chromosomes; female
% chrY is the average of % chrY in 200 pregnant women bearing euploid female foetuses; and male % chrY repre-
sents the average of the ratio of % chrY of two adult male volunteers. Statistical analysis. Data were analysed using GraphPad Prism 6.0 software. Data between two groups were
analysed by a two-tailed paired t test. Values are presented as the mean ± SEM. Statistical significance was set at
**P < 0.01 and ****P < 0.0001. Statistical analysis. Data were analysed using GraphPad Prism 6.0 software. Data between two groups were
analysed by a two-tailed paired t test. Values are presented as the mean ± SEM. Statistical significance was set at
**P < 0.01 and ****P < 0.0001. References 1. Goldwaser, T., Klugman, S. & Cell-free, D. N. A. for the detection of fetal aneuploidy. FERTIL STERIL. 109, 195–200, https://doi. org/10.1016/j.fertnstert.2017.12.019 (2018). 1. Goldwaser, T., Klugman, S. & Cell-free, D. N. A. for the detection of fetal aneuploidy. FERTIL STERIL. 109, 195–200, https://doi. org/10.1016/j.fertnstert.2017.12.019 (2018). g
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(
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2. Norton, M. E. et al. Cell-free DNA analysis for noninvasive examination of trisomy. N Engl J Med. 372, 1589–1597, https:/
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3. Bianchi, D. W. et al. DNA sequencing versus standard prenatal aneuploidy screening. N Engl J Med. 370, 799–808, https://doi
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4. McKanna, T. et al. Fetal fraction-based risk algorithm for non-invasive prenatal testing: screening for trisomies 13 and 18 and
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9. Valderramos, S. G. et al. Cell-free DNA screening in clinical practice: abnormal autosomal aneuploidy and microdeletion re
Am J Obstet Gynecol. 215, 626.e1–626.e10, https://doi.org/10.1016/j.ajog.2016.06.039 (2016). y
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0. Le Conte, G. et al. Cell-free fetal DNA analysis in maternal plasma as screening test for trisomies 21, 18 and 13 in twin pregnancy
Ultrasound Obstet Gynecol. Data Availability
The data generated during and/or analysed during the current study are available from the corresponding author
by reasonable request. y
The data generated during and/or analysed during the current study are available from the corresponding author
by reasonable request. y
The data generated during and/or analysed during the current study are available from the corresponding author
by reasonable request. References 52, 318–324, https://doi.org/10.1002/uog.18838 (2018).h y
p
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10. Le Conte, G. et al. Cell-free fetal DNA analysis in maternal plasma as screening test for trisom
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11. Burns, W. et al. The association between anticoagulation therapy, maternal characteristics, and a failed cfDNA test due to a low fetal
fraction. Prenat Diagn. 37, 1125–1129, https://doi.org/10.1002/pd.5152 (2017). g
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12. Chiu, R. W. et al. Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of
DNA in maternal plasma. Proc Natl Acad Sci USA 105, 20458–20463, https://doi.org/10.1073/pnas.0810641105 (2008). p
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3. Kim, S. K. et al. Determination of fetal DNA fraction from the plasma of pregnant women using sequence read counts. Prenat Diagn
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14. Hahn, S., Huppertz, B. & Holzgreve, W. Fetal cells and cell free fetal nucleic acids in maternal blood: new tools to study abnormal
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5. van Beek, D. M. et al. Comparing methods for fetal fraction determination and quality control of NIPT samples. Prenat Diagn. 37
769–773, https://doi.org/10.1002/pd.5079 (2017). p
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6. Hudecova, I. et al. Maternal plasma fetal DNA fractions in pregnancies with low and high risks for fetal chromosomal aneuploidies
PLOS ONE. 9, e88484, https://doi.org/10.1371/journal.pone.0088484. eCollection 2014 (2014). g
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17. Liang, B. et al. Enrichment of the fetal fraction in non-invasive prenatal screening reduces maternal background interference. Sci
Rep. 8, 17675, https://doi.org/10.1038/s41598-018-35738-0 (2018).h 18. Canick, J. A., Palomaki, G. E., Kloza, E. M., Lambert-Messerlian, G. M. & Haddow, J. E. The impact of maternal plasma DNA fetal
fraction on next generation sequencing tests for common fetal aneuploidies. Prenat Diagn. 33, 667–674, https://doi.org/10.1002/
pd.4126 (2013).f p
19. Wang, E. et al. Gestational age and maternal weight effects on fetal cell-free DNA in maternal plasma. Prenat Diagn. 33, 662–666,
https://doi.org/10.1002/pd.4119 (2013).ht g
0. Schlutter, J. M. et al. The cell-free fetal DNA fraction in maternal blood decreases after physical activity. Prenat Diagn. 34, 341–344
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21. Brar, H., Wang, E., Struble, C., Musci, T. J. & Norton, M. E. The fetal fraction of cell-free DNA in maternal plasma is not affected by
a priori risk of fetal trisomy. J Matern Fetal Neonatal Med. Sequencing data analysis Sequencing data analysis. A analysis of sequencing reads was performed by the BambniTest software
(Berry genomics company, Beijing, China). Sequencing reads were mapped to the non repeat-masked reference
human genome (hg18) using the SOAP bioinformatic algorithm24. Unique reads were the only mapped one chro-
mosome DNA sequences, which were analysed for follow-up counting. The number of unique sequence reads
aligned to each chromosome was counted, and the Unimap-GC content was calculated. To offset the GC bias
generated between runs, the final displayed Unimap-GC was corrected by the BambniTest25. If the Unimap-GC
content was ≥42%, the software will interpret the sample as having high GC content. High GC content and
sequencing detection failure were all defined as redo by the BambniTest software. q
gi
yt
For each chromosome of each sample, the unique chromosome representation (% chrN) value was generated
according to the following equation: % chrN = Unique count for chrN /Total unique count of the sequences Scientific Reports | (2019) 9:6962 | https://doi.org/10.1038/s41598-019-42980-7 4 www.nature.com/scientificreports/ mapped to all the autosomes. The assessment of foetal aneuploidy risk was according to the formula chrN
z-score = (% chrN- mean% chrNreference)/S.D.% chrNreference. The cut-off z-value was 312. Four hundred twelve
pregnant women with euploidy foetuses were selected as the reference set. The mean (% chrNreference) and the
standard deviation (% chrNreference) of the reference set were used for calculating z-scores. In this sequencing plat-
form, the lowest foetal concentration for distinguishing trisomy effectively was 3.5%. Samples below this quality
control standard were recommended to re-collect blood or abandon testing. mapped to all the autosomes. The assessment of foetal aneuploidy risk was according to the formula chrN
z-score = (% chrN- mean% chrNreference)/S.D.% chrNreference. The cut-off z-value was 312. Four hundred twelve
pregnant women with euploidy foetuses were selected as the reference set. The mean (% chrNreference) and the
standard deviation (% chrNreference) of the reference set were used for calculating z-scores. In this sequencing plat-
form, the lowest foetal concentration for distinguishing trisomy effectively was 3.5%. Samples below this quality
control standard were recommended to re-collect blood or abandon testing. Foetal fraction calculation. The male foetal DNA fraction calculation based on unique read counts on the
Y chromosome were reliable15. We calculated the proportion of male foetal DNA based on the calculation method
published by Irena H et al.16.h Foetal fraction calculation. Author Contributions X.L.X. and X.Y.G. designed experiments, analysed and interpreted the data, and wrote the manuscript; F.G.L. and
F.Y.C. performed NIPT detection; WH.T. performed genetic counselling for patients; X.L.X. and W.G.Y. analysed
and interpreted the data. References 26, 143–145, https://doi.org/10.3109/14767058.2012.722731 (2013). 22. Rava, R. P., Srinivasan, A., Sehnert, A. J. & Bianchi, D. W. Circulating fetal cell-free DNA fractions differ in autosomal aneuploidies
and monosomy X. CLIN CHEM. 60, 243–250, https://doi.org/10.1373/clinchem.2013.207951 (2014).f 3. Suzumori, N. et al. Fetal cell-free DNA fraction in maternal plasma is affected by fetal trisomy. J HUM GENET. 61, 647–652, https:/
doi.org/10.1038/jhg.2016.25 (2016). g
j g
4. Liu, C. M. et al. SOAP3: ultra-fast GPU-based parallel alignment tool for short reads. BIOINFORMATICS. 28, 878–879, https://doi
org/10.1093/bioinformatics/bts061 (2012). g
25. Liang, D. et al. Non-invasive prenatal testing of fetal whole chromosome aneuploidy by massively parallel sequencing. Prenat Diagn. 33, 409–415, https://doi.org/10.1002/pd.4033 (2013). Scientific Reports | (2019) 9:6962 | https://doi.org/10.1038/s41598-019-42980-7 5 www.nature.com/scientificreports/ Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-42980-7. Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre-
ative Commons license, and indicate if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons license and your intended use is not per-
mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the
copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2019 Scientific Reports | (2019) 9:6962 | https://doi.org/10.1038/s41598-019-42980-7 6
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https://openalex.org/W2148564836
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https://discovery.ucl.ac.uk/1433074/1/journal.pgen.1000534.pdf
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Identifying Relationships among Genomic Disease Regions: Predicting Genes at Pathogenic SNP Associations and Rare Deletions
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PLOS genetics
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cc-by
| 15,598
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Abstract Translating a set of disease regions into insight about pathogenic mechanisms requires not only the ability to identify the
key disease genes within them, but also the biological relationships among those key genes. Here we describe a statistical
method, Gene Relationships Among Implicated Loci (GRAIL), that takes a list of disease regions and automatically assesses
the degree of relatedness of implicated genes using 250,000 PubMed abstracts. We first evaluated GRAIL by assessing its
ability to identify subsets of highly related genes in common pathways from validated lipid and height SNP associations
from recent genome-wide studies. We then tested GRAIL, by assessing its ability to separate true disease regions from many
false positive disease regions in two separate practical applications in human genetics. First, we took 74 nominally
associated Crohn’s disease SNPs and applied GRAIL to identify a subset of 13 SNPs with highly related genes. Of these, ten
convincingly validated in follow-up genotyping; genotyping results for the remaining three were inconclusive. Next, we
applied GRAIL to 165 rare deletion events seen in schizophrenia cases (less than one-third of which are contributing to
disease risk). We demonstrate that GRAIL is able to identify a subset of 16 deletions containing highly related genes; many
of these genes are expressed in the central nervous system and play a role in neuronal synapses. GRAIL offers a statistically
robust approach to identifying functionally related genes from across multiple disease regions—that likely represent key
disease pathways. An online version of this method is available for public use (http://www.broad.mit.edu/mpg/grail/). Citation: Raychaudhuri S, Plenge RM, Rossin EJ, Ng ACY, International Schizophrenia Consortium, et al. (2009) Identifying Relationships among Genomic Disease
Regions: Predicting Genes at Pathogenic SNP Associations and Rare Deletions. PLoS Genet 5(6): e1000534. doi:10.1371/journal.pgen.1000534 Received February 16, 2009; Accepted May 22, 2009; Published June 26, 2009 Copyright: 2009 Raychaudhuri 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: 2009 Raychaudhuri et al. Abstract This is an open-access article distributed under the terms of the Creative C
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are cred Funding: For this project, SR was supported by a T32 NIH training grant (AR007530-23), an NIH Career Development Award (1K08AR055688-01A1), an American
College of Rheumatology Bridge Grant, and through the BWH Rheumatology Fellowship program, directed by Simon Helfgott. MJD is supported by a U01 NIH
grant (U01 HG004171). MJD and RJX are supported by an R01 NIH grant (R01 DK083759). ACYN is supported through Research Fellowship Award from the Crohn’s
and Colitis Foundation of America. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: soumya@broad.mit.edu (SR); mjdaly@broad.mit.edu (MJD) These disease regions often overlap multiple genes – though only
one is typically relevant to pathogenesis and the remaining are
spuriously implicated by proximity. The difficulty of this task is
heightened
by
the
limited
state
of
cataloged
interactions,
pathways, and functions for the vast majority of genes. However,
undefined gene relationships might often be conjectured from the
literature, even if they are not explicitly described yet. Identifying Relationships among Genomic Disease
Regions: Predicting Genes at Pathogenic SNP
Associations and Rare Deletions Soumya Raychaudhuri1,2,3*, Robert M. Plenge1,3,4, Elizabeth J. Rossin1,2,5, Aylwin C. Y. Ng6,7,
International Schizophrenia Consortium, Shaun M. Purcell2,8,9, Pamela Sklar2,8,9,10, Edward M. Scolnick 2,8,10, Ramnik J. Xavier6,7, David Altshuler1,2,11,12,13, Mark J. Daly1,2* 1 Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America, 2 Center for Human Genetic
Research, Massachusetts General Hospital, Boston, Massachusetts, United States of America, 3 Division of Rheumatology, Immunology and Allergy, Brigham and Women’s
Hospital, Harvard Medical School, Boston, Massachusetts, United States of America, 4 Harvard Medical School – Partners HealthCare Center for Genetics and Genomics,
Boston, Massachusetts, United States of America, 5 Harvard-MIT Health Sciences and Technology, Cambridge, Massachusetts, United States of America, 6 Center for
Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts, United States of America, 7 Gastroenterology Unit, Massachusetts General
Hospital, Boston, Massachusetts, United States of America, 8 Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, Massachusetts,
United States of America, 9 Psychiatric and Neurodevelopmental Genetics Unit, Massachusetts General Hospital, Boston, Massachusetts, United States of America,
10 Department of Psychiatry, Massachusetts General Hospital, Boston, Massachusetts, United States of America, 11 Department of Molecular Biology, Massachusetts
General Hospital, Boston, Massachusetts, United States of America, 12 Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America,
13 Diabetes Unit, Massachusetts General Hospital, Boston, Massachusetts, United States of America PLoS Genetics | www.plosgenetics.org June 2009 | Volume 5 | Issue 6 | e1000534 Author Summary We propose, instead, to use a
flexible metric of gene relatedness that not only captures clearly
established close gene relationships, but also has the ability to
capture potential undocumented or distant ones. Such a metric
may be a more powerful tool to approach this problem rather then
relying on incomplete databases of gene functions, interactions, or
relationships. Encouraged by GRAIL’s ability to recognize biologically
meaningful connections, we tested its ability to distinguish true
disease regions from false positive regions in two practical
applications in human genetics. First, in Crohn’s disease, we start
with a long list of putative SNP associations from a recent GWA
(genome-wide association) meta-analysis [24]. We demonstrate
that a substantial fraction of these SNPs contain highly related
genes—far beyond what can be expected by chance. We
demonstrate that many of these SNPs subsequently validate in
an independent replication genotyping experiment. Second, in
schizophrenia, we previously identified an over-representation of
rare deletions in schizophrenia cases compared to controls [8]. Despite the statistical excess, it is challenging to identify exactly
which case deletions are causal, given the relatively high
background rate of rare deletions in controls. Using GRAIL
however, we are able to demonstrate that a subset of case deletions
contain related genes. We further demonstrate that these genes are
highly and significantly enriched for central nervous system (CNS)
expressed genes. In stark contrast, GRAIL finds no excess
relatedness among genes implicated by case deletions. To
this
end,
we
use
established
statistical
text
mining
approaches to quantify relatedness between two genes – specifically,
gene relatedness is the degree of similarity in the text describing
them within article abstracts. The published literature represented
in online PubMed abstracts encapsulates years of research on
biological mechanisms. We and others have shown the great utility
of statistical text mining to rapidly obtain functional information
about genes, including protein-protein interactions, gene function
annotation, and measuring gene-gene similarity [19–22]. Text is
an abundant and underutilized resource in human genetics, and
currently a total of 140,000 abstracts from articles that reference
human genes are available through PubMed [23]. Additional
valuable information can be seamlessly gained by including more
than 100,000 references from orthologous genes; many important
pathways have been more thoroughly explored in model systems
than in humans. We have developed a novel statistical method to evaluate the
degree of relatedness among genes within disease regions: Gene
Relationships Among Implicated Loci (GRAIL). Identifying Common Function Across Disease Regions content across regions. We assume that each region contains a
single pathogenic gene; therefore narrow regions with one or just a
few genes are more informative than expansive regions with many
genes, since they are likely to have many irrelevant genes. To take
advantage of this, we have designed GRAIL to set a lower
threshold in considering relatedness for those genes in narrow
regions, allowing for more distant relationships to be considered;
on the other hand it sets a more stringent threshold for genes
located in expansive mutligenic regions and considers only the
very closest of relationships. This strategy prevents large regions
with many genes from dominating the analysis. Author Summary Modern genetic studies, including genome-wide surveys
for disease-associated loci and copy number variation,
provide a list of critical genomic regions that play an
important role in predisposition to disease. Using these
regions to understand disease pathogenesis requires the
ability to first distinguish causal genes from other nearby
genes spuriously contained within these regions. To do
this we must identify the key pathways suggested by
those causal genes. In this manuscript we describe a
statistical approach, Gene Relationships Across Implicated
Loci (GRAIL), to achieve this task. It starts with genomic
regions and identifies related subsets of genes involved in
similar biological processes—these genes highlight the
likely causal genes and the key pathways. GRAIL uses
abstracts from the entirety of the published scientific
literature about the genes to look for potential relation-
ships between genes. We apply GRAIL to four very
different phenotypes. In each case we identify a subset
of highly related genes; in cases where false positive
regions are present, GRAIL is able to separate out likely
true positives. GRAIL therefore offers the potential to
translate disease genomic regions from unbiased genomic
surveys into the key processes that may be critical to the
disease. In this paper we apply GRAIL to four phenotypes. In each case
GRAIL is able to identify a subsets of genes enriched for
relatedness – more than expected by random chance. We
demonstrate enrichment for relatedness among true disease
regions rigorously based on both GRAIL’s theoretically derived
p-value and also based on parallel analysis of either (1) carefully
selected random regions matched for gene content and size or (2)
experimentally derived false positive disease regions. GRAIL is able to identify subsets of highly related genes among
validated SNP associations. First we use GRAIL to identify related
genes from SNPs associated with serum lipid levels; GRAIL
correctly identifies genes already known to influence lipid levels
within the cholesterol biosynthesis pathway. In comparison to
randomly selected matched SNP sets, the set of lipid SNPs
demonstrate significantly more relatedness. Second, we use
GRAIL to identify significantly related genes near height-
associated SNPs; these genes highlight plausible pathways involved
in height. In comparison to randomly selected matched SNP sets,
the set of height SNPs also demonstrate significantly more
relatedness. expression data, carefully constructed gene networks based on
multiple information sources, predefined gene sets and pathways,
and disease-related keywords. Author Summary Given only a collection
of disease regions, GRAIL uses our text-based definition of
relatedness (or alternative metrics of relatedness) to identify a
subset of genes, more highly related than by chance; it also assigns
a select set of keywords that suggest putative biological pathways. It uses no information about the phenotype, such as known
pathways or genes, and is therefore not tethered to potentially
biased pre-existing concepts about the disease. PLoS Genetics | www.plosgenetics.org Introduction An emerging challenge in genomics is the ability to examine
multiple disease regions within the human genome, and to
recognize a subset of key genes that are involved in a common
cellular process or pathway. This is a key task to translate
experimentally
ascertained
disease
regions
into
meaningful
understanding
about pathogenesis. The
importance
of this
challenge has been highlighted by advances in human genetics
that are facilitating the rapid discovery of disease regions in the
form of genomic regions around associated SNPs (single nucleotide
polymorphisms) [1–6] or CNVs (copy number variants) [7–10]. y
p
y
y
The general strategy of using function to prioritize genes in
disease regions has been substantially explored [11–18]. However,
predicted disease genes have not, in general, been easily validated. Thus far, published approaches have utilized a range of codified
gene
information
including
protein-interaction
maps,
gene June 2009 | Volume 5 | Issue 6 | e1000534 1 June 2009 | Volume 5 | Issue 6 | e1000534 Identifying Common Function Across Disease Regions Figure 1. Gene Relationships Among Implicated Loci (GRAIL) method consists of four steps. (A) Identifying genes in disease regions. For
each independent associated SNP or CNV from a GWA study, GRAIL defines a disease region; then GRAIL identifies genes overlapping the region. In
this region there are three genes. We use gene 1 (pink arrow) as an example. (B) Assess relatedness to other human genes. GRAIL scores each gene
contained in a disease region for relatedness to all other human genes. GRAIL determines gene relatedness by looking at words in gene references;
related genes are defined as those whose abstract references use similar words. Here gene 1 has word counts that are highly similar to gene A but not
to gene B. All human genes are ranked according to text-based similarity (green bar), and the most similar genes are considered related. (C) Counting
regions with similar genes. For each gene in a disease region, GRAIL assesses whether other independent disease regions contain highly significant
genes. GRAIL assigns a significance score to the count. In this illustration gene 1 is similar to genes in three of the regions (green arrows), including
gene A. (D) Assigning a significance score to a disease region. After all of the genes within a region are scored, GRAIL identifies the most significant
gene as the likely candidate. GRAIL corrects its significance score for multiple hypothesis testing (by adjusting for the number of genes in the region),
to assign a significance score to the region. doi:10.1371/journal.pgen.1000534.g001 Figure 1. Gene Relationships Among Implicated Loci (GRAIL) method consists of four steps. (A) Identifying genes in disease regions. For
each independent associated SNP or CNV from a GWA study, GRAIL defines a disease region; then GRAIL identifies genes overlapping the region. In
this region there are three genes. We use gene 1 (pink arrow) as an example. (B) Assess relatedness to other human genes. GRAIL scores each gene
contained in a disease region for relatedness to all other human genes. GRAIL determines gene relatedness by looking at words in gene references;
related genes are defined as those whose abstract references use similar words. Here gene 1 has word counts that are highly similar to gene A but not
to gene B. All human genes are ranked according to text-based similarity (green bar), and the most similar genes are considered related. (C) Counting
regions with similar genes. Summary of statistical approach y
pp
GRAIL relies on two key methods: (1) a novel statistical
framework that assesses the significance of relatedness between
genes in disease regions (2) a text-based similarity measure that
scores two genes for relatedness to each other based on text in
PubMed abstracts. Details for both are presented in the Methods. The GRAIL statistical framework consists of four steps (see
Figure 1). First, given a set of disease regions we identify the genes
overlapping them (Figure 1A); for SNPs we use LD (linkage
disequilibrium) characteristics to define the region. Second, for In addition to a flexible text-based metric of relatedness,
GRAIL’s ability to successfully connect genes also leverages a
statistical framework that carefully accounts for differential gene June 2009 | Volume 5 | Issue 6 | e1000534 2 Identifying Common Function Across Disease Regions Identifying Common Function Across Disease Regions each overlapping gene we score all other human genes by their
relatedness to it (Figure 1B). In this paper we use a text-based
similarity
measure;
alternative
measures
of
relatedness,
for
example similarity in gene annotations or expression data, could
be easily applied instead [25,26]. Third, for each gene we count
the number of independent regions with at least one highly related
gene (Figure 1C); here the threshold for relatedness varies between
regions depending on the number of genes within them. We assign a p-value to that count. Fourth, for each disease region we select
the single most connected gene as the key gene. We assign the
disease region that key gene’s p-value after adjusting for multiple
hypothesis testing (if there are multiple genes within the region)
(Figure 1D). This final score is listed in this paper as pmetric where
the metric is text, expression, or annotation based. Very low ptext scores
for one region indicate that a gene within it is more related to
genes in other disease regions through PubMed abstracts than Figure 1. Gene Relationships Among Implicated Loci (GRAIL) method consists of four steps. (A) Identifying genes in disease regions. For
each independent associated SNP or CNV from a GWA study, GRAIL defines a disease region; then GRAIL identifies genes overlapping the region. In
this region there are three genes. We use gene 1 (pink arrow) as an example. (B) Assess relatedness to other human genes. GRAIL scores each gene
contained in a disease region for relatedness to all other human genes. GRAIL determines gene relatedness by looking at words in gene references;
related genes are defined as those whose abstract references use similar words. Here gene 1 has word counts that are highly similar to gene A but not
to gene B. All human genes are ranked according to text-based similarity (green bar), and the most similar genes are considered related. (C) Counting
regions with similar genes. For each gene in a disease region, GRAIL assesses whether other independent disease regions contain highly significant
genes. GRAIL assigns a significance score to the count. In this illustration gene 1 is similar to genes in three of the regions (green arrows), including
gene A. (D) Assigning a significance score to a disease region. Identifying Common Function Across Disease Regions After all of the genes within a region are scored, GRAIL identifies the most significant
gene as the likely candidate GRAIL corrects its significance score for multiple hypothesis testing (by adjusting for the number of genes in the region) Evaluating relationships between known associated
SNPs: lipid levels and height expected by chance. Simulations on random groups of SNPs
demonstrate that the ptext values approximately estimate Type I
error rates, being approximately uniformly distributed under the
null hypothesis (see Figure S1). However, we recommend the use
of careful simulations or controls rather than actual theoretical p-
values to reinforce the significance of GRAIL’s findings – as we do
in the examples below. g
We first applied GRAIL to a set of 19 validated SNPs associated
with triglyceride, LDL, and/or HDL levels [5,6]. Since 14 SNPs (out
of 19) are near genes that are known members of lipid metabolism
pathways, we hypothesized that GRAIL should be able to identify
these genes accurately. A total of 87 genes were implicated by the 19
associated SNPs. Of the 14 SNPs near compelling candidate genes,
13 obtained ptext scores,0.01 (Figure 2A, Table S1). GRAIL correctly
identified those genes implicated in lipid metabolism from each of
these 14 regions. To asses the significance of these findings, we
applied GRAIL to 1000 random matched SNP sets; each set
consisted of 19 SNPs randomly selected from a commercial
genotyping array which implicated a similar total of 87610 genes. In contrast to lipid associated SNPs, not a single matched random set
contained 13 SNPs that obtained ptext scores#0.01; on average
matched sets had 0.26 (maximum 6) SNPs with ptext#0.01 (Figure 2A). Thus, there is substantial enrichment for highly connected genes
captured by true lipid associated SNPs. p
The text-based similarity metric is based on standard approach-
es used in statistical text mining. To avoid publications that report
on or are influenced by disease regions discovered in the recent
scans, we use only those PubMed abstracts published prior to
December 2006, before the recent onslaught of GWA papers
identifying novel associations. This approach effectively avoids the
evaluation of gene relationships being confounded by papers
listing
genes
in
regions
discovered
as
associated
to
these
phenotypes. In addition to including primary abstract references
about genes listed in Entrez Gene, we augment our text
compendium with references to orthologous genes listed in
Homologene [23]; this increases the number of articles available
per gene from 6 to 12 (see Table 1). We note that the distribution
of articles per gene is skewed toward a small number of genes with
many references; 0.4% of genes are referenced by .500 articles,
while 26% of genes are referenced by ,5. Evaluating relationships between known associated
SNPs: lipid levels and height In fact 2,034 genes
could not be connected to any abstracts at all. For each abstract we
convert free text into vectors of word counts [19]. For each gene
we define a word vector that consists of averaged word counts
from document references to it. Pairwise gene relatedness is then
the correlation between the vectors of word counts between two
genes. Two genes that are referenced by abstracts using the same
sorts of words will receive a high similarity score, whereas two
genes that have abstract references that largely use a different
vocabulary will receive a low score (Figure 1B). Importantly, genes
do not need to be co-cited in the same document to be identified as
highly similar. Despite relatively comprehensive lipid biology annotation, GO
does not identify relationships between regions as effectively as
published text (Figure 2A). A total of 12 out of the 19 associated
SNPs obtained pannotation,0.01. Relationships between highest
scoring candidate genes are explained by several shared GO
codes including: GO:0008203 (‘cholesterol metabolic process’),
GO:0016125
(‘sterol
metabolic
process’),
GO:0006629
(‘lipid
metabolic process’), GO:0008202 (‘steroid metabolism’), and
GO:0005319 (‘lipid transporter activity’). Gene expression does
not identify relationships between regions as effectively as text,
either (Figure 2A). A subset of 4 associated SNPs obtain
pexpression,0.01. The regions with the most significantly connected
genes have similar tissue-specific expression profiles. The highest
expression is in four samples taken from adult and fetal liver
tissues, known to play a major role in cholesterol metabolism. While associated SNPs are less connected with these alternative
metrics, they do seem to leverage the appropriate functional
variables and provide valuable phenotypic information. After regions are scored with GRAIL, PubMed text can be used
to identify keywords that may provide insight into the underlying
biological pathways. We define these keywords as those words that
most strongly link the significant genes in each region, that is the
words with overall greatest weight across all of the text vectors
from those genes. We next applied GRAIL to 42 validated SNP associations to
adult height in recent GWA studies [2–4]. This application tests
GRAIL’s ability to connect genes in the absence of functional
literature connecting the phenotype to the relevant pathways. In
contrast to lipid metabolism, all associated common SNPs were
identified in 2007 and 2008 and the underlying biological
pathways involved in height are still poorly understood. For each gene in a disease region, GRAIL assesses whether other independent disease regions contain highly significant
genes. GRAIL assigns a significance score to the count. In this illustration gene 1 is similar to genes in three of the regions (green arrows), including
gene A. (D) Assigning a significance score to a disease region. After all of the genes within a region are scored, GRAIL identifies the most significant
gene as the likely candidate. GRAIL corrects its significance score for multiple hypothesis testing (by adjusting for the number of genes in the region),
to assign a significance score to the region. doi:10.1371/journal.pgen.1000534.g001 June 2009 | Volume 5 | Issue 6 | e1000534 PLoS Genetics | www.plosgenetics.org 3 Identifying Common Function Across Disease Regions Evaluating relationships between known associated
SNPs: lipid levels and height The scatter plot on the right illustrates ptext values for actual SNPs associated with height
(blue dots). Black horizontal line marks the median ptext value. We assessed the same SNP with similarity metrics based on gene annotation (green
dots) and gene expression correlation (purple dots). On the right we list for each ptext threshold the number of expected SNPs less than the threshold
based on matched sets, and the number of observed SNPs less than the threshold among height associated SNPs. doi:10.1371/journal.pgen.1000534.g002 Figure 2. SNPs associated with lipid metabolism and height contain genes related to each other. (A) 19 SNPs associated with lipid
metabolism. The y-axis plots the ptext values on a log scale, with increasing significance at the top. The histogram on the left side of the graph
illustrates values for matched SNP sets. 88.6% of those SNPs have ptext values that are .0.1. The scatter plot on the right illustrates ptext values for
actual serum cholesterol associated SNPs (blue dots). Black horizontal line marks the median ptext value. We assessed the same SNP with similarity
metrics based on gene annotation (green dots) and gene expression correlation (purple dots). (B) 42 SNPs associated with height. Similar plot for 42
height associated SNPs. The histogram on the left of the graph illustrates ptext values for random SNP sets carefully matched to height-associated SNP
set. 86.5% of those SNPs have ptext values that are .0.1. The scatter plot on the right illustrates ptext values for actual SNPs associated with height
(blue dots). Black horizontal line marks the median ptext value. We assessed the same SNP with similarity metrics based on gene annotation (green
dots) and gene expression correlation (purple dots). On the right we list for each ptext threshold the number of expected SNPs less than the threshold
based on matched sets, and the number of observed SNPs less than the threshold among height associated SNPs. doi:10.1371/journal.pgen.1000534.g002 The 42 height SNPs implicated a total of 185 genes (Table S2). Of these 42 regions, 13 obtained ptext scores,0.01 (Figure 2B). For
comparison, we used GRAIL to score 1000 matched SNP sets; as
before each set consisted of 42 SNPs randomly selected from a
commercial array and implicated a total of 185610 genes. Not a
single
random
set
contained
13
SNPs
that
obtained
ptext
scores#0.01; on average matched sets had 0.77 (maximum 10)
SNPs with ptext scores,0.01. Evaluating relationships between known associated
SNPs: lipid levels and height This
insures
independence
between
association
results
and
the
functional literature from before 2007 that is mined in this study. In most cases the key genes are not yet known. Since the GRAIL framework can be easily used with any gene
relatedness metric, we also devised and tested two alternative
metrics derived from Gene Ontology (GO) annotations [27] and
an mRNA expression atlas consisting of expression measurements
across multiple human tissues (The Novartis Gene Expression
Atlas) [28]. These metrics are described in greater detail in
Methods. Table 1. Text resources. Refs/gene
Genes
Articles
References
mean
median
mean
median
Standard
18,690
137,395
260,658
13.9
6
1.9
1
Homologs
15,990
138,720
434,690
27.1
13
3.1
1
Combined
18,875
259,638
599,537
31.7
12
2.3
1
We obtained text from abstracts relevant to human genes from PubMed on December 2006. In the first row we list the number of human genes with any references
listed, the total number of abstracts referencing them, and the total number of gene references. We then list the mean and median number of abstract references per
gene, and also the mean and median number of gene references per abstract. We used Homologene to identify human gene orthologs, and obtained text for those
genes; information about those genes and references is listed in the second row. We combined the two pools of gene references to create a large combined database
of 18,875 genes with 599,537 references to 259,638 articles, described in the third row. doi:10.1371/journal.pgen.1000534.t001 Table 1. Text resources. We obtained text from abstracts relevant to human genes from PubMed on December 2006. In the first row we list the number of human genes with any references
listed, the total number of abstracts referencing them, and the total number of gene references. We then list the mean and median number of abstract references per
gene, and also the mean and median number of gene references per abstract. We used Homologene to identify human gene orthologs, and obtained text for those
genes; information about those genes and references is listed in the second row. We combined the two pools of gene references to create a large combined database
of 18,875 genes with 599,537 references to 259,638 articles, described in the third row. doi:10.1371/journal.pgen.1000534.t001 June 2009 | Volume 5 | Issue 6 | e1000534 4 Identifying Common Function Across Disease Regions Figure 2. Evaluating relationships between known associated
SNPs: lipid levels and height Thus, we present clear statistical
evidence that GRAIL identifies genes with non-random functional
connections among associated loci. to prospectively identify true disease regions, based on the
relatedness of genes within them, from false positive regions. We
tested GRAIL’s ability to distinguish disease regions from a longer
list of results containing a large number of false positive regions as
well in two separate human genetics applications. A recent GWA meta-analysis in Crohn’s disease identified 74
independent SNPs as nominally significant (p,561025) [24]. While the excess beyond chance suggested many of these regions
were likely true positives, up to half of these regions should by
necessity be unrelated to Crohn’s and simply represent the tail of
the null distribution. Thus we sought to explore whether GRAIL
could identify a subset of these SNPs that implicate an inter-
connected set of genes, and whether those represented true
associations that could be validated. Strikingly, the top five keywords linking the genes were
‘hedgehog’, ‘histone’, ‘bone’, ‘cartilage’, and ‘growth’ (see Table
S3 for a more complete list). Of note, ‘height’, does not emerge as
a keyword since these genes had not been previously related to
height. For comparison, the top five keywords for lipid metabolism
associated SNPs were ‘lipoprotein’, ‘cholesterol’, ‘lipase’, ‘apoli-
poprotein’, and ‘triglyceride’ (Table S3). These results are
particularly noteworthy as this analysis uses only a simple list of
SNPs implicated by GWA studies—no specific biological pathways
or mechanisms or phenotype details are assumed. In a now published replication genotyping of the 74 SNPs, 30
replicated convincingly when tested in independent samples
(defined as having one-tailed association p-values,0.0007 in
replication samples and two tailed association p-values,561028
overall),
confirming
true
positive
associations,
whereas
22
convincingly failed to replicate (defined as overall association p-
value rising to .1024); the remaining 22 regions had intermediate
levels of significance following replication (and can be considered
as yet unresolved associations) [24]. Evaluating relationships between known associated
SNPs: lipid levels and height SNPs associated with lipid metabolism and height contain genes related to each other. (A) 19 SNPs associated with lipid
metabolism. The y-axis plots the ptext values on a log scale, with increasing significance at the top. The histogram on the left side of the graph
illustrates values for matched SNP sets. 88.6% of those SNPs have ptext values that are .0.1. The scatter plot on the right illustrates ptext values for
actual serum cholesterol associated SNPs (blue dots). Black horizontal line marks the median ptext value. We assessed the same SNP with similarity
metrics based on gene annotation (green dots) and gene expression correlation (purple dots). (B) 42 SNPs associated with height. Similar plot for 42
height associated SNPs. The histogram on the left of the graph illustrates ptext values for random SNP sets carefully matched to height-associated SNP
set. 86.5% of those SNPs have ptext values that are .0.1. The scatter plot on the right illustrates ptext values for actual SNPs associated with height
(blue dots). Black horizontal line marks the median ptext value. We assessed the same SNP with similarity metrics based on gene annotation (green
dots) and gene expression correlation (purple dots). On the right we list for each ptext threshold the number of expected SNPs less than the threshold
based on matched sets, and the number of observed SNPs less than the threshold among height associated SNPs. doi:10.1371/journal.pgen.1000534.g002 Figure 2. SNPs associated with lipid metabolism and height contain genes related to each other. (A Figure 2. SNPs associated with lipid metabolism and height contain genes related to each other. (A) 19 SNPs associated with lipid
metabolism. The y-axis plots the ptext values on a log scale, with increasing significance at the top. The histogram on the left side of the graph
illustrates values for matched SNP sets. 88.6% of those SNPs have ptext values that are .0.1. The scatter plot on the right illustrates ptext values for
actual serum cholesterol associated SNPs (blue dots). Black horizontal line marks the median ptext value. We assessed the same SNP with similarity
metrics based on gene annotation (green dots) and gene expression correlation (purple dots). (B) 42 SNPs associated with height. Similar plot for 42
height associated SNPs. The histogram on the left of the graph illustrates ptext values for random SNP sets carefully matched to height-associated SNP
set. 86.5% of those SNPs have ptext values that are .0.1. PLoS Genetics | www.plosgenetics.org Identifying Common Function Across Disease Regions In contast, we did not identify any regions with
significantly related genes in the corresponding list of deletions; out
of a total 124 regions, none obtained ptext scores,0.001 (see Table
S8). This represents a significant enrichment within the cases
(p = 0.01, one Fisher’s exact text). As a further test of GRAIL, we then evaluated the next most
significant 74 associated SNPs that emerged from the Crohn’s
disease GWA meta-analysis (association p-values ranging from
561025 to 261024). Out of the 75 regions, 8 are not near any
gene, and we did not score them. The remaining 67 regions were
tested with GRAIL for relationships to the 52 replicated and
indeterminate regions that emerged following replication. Two
emerge with highly significant GRAIL scores: rs8178556 on
chromosome 21 (IFNAR1, ptext = 1.761024) and rs12928822 on
chromosome 16 (SOCS1, ptext = 8.261024) suggesting these inde-
pendent regions may lead to novel associated SNPs for Crohn’s
disease (see Table S7). We then sought independent assessment of the biological
relationship of the genes highlighted by GRAIL by examining the
extent to which these genes demonstrate preferential expression in
CNS tissues using a publicly available tissue atlas [30]. Here we
define preferential expression as median CNS tissue expression
significantly greater than in other tissues (p,0.01 by one-tailed Table 2. High scoring regions from a Crohn’s disease GWA meta-analysis. Table 2. High scoring regions from a Crohn’s disease GWA meta-analysis. Identifying Common Function Across Disease Regions any knowledge of the contemporaneous replication genotyping
experiment. Each region contained between 1 and 34 genes,
except for two regions that contained no genes and were not
scored. GRAIL identified 13 regions as significant (achieving ptext
scores,0.01), as with the previous examples far in excess of
chance. We next applied GRAIL to recently published sets of rare
deletions seen in schizophrenia cases and matched controls. Multiple groups have recently demonstrated that extremely rare
deletions, many of which are likely de novo, are notably enriched
in schizophrenia [8–10,29]. However, since rare deletions occur
frequently in healthy individuals as well, many of these case
deletions will also be non-pathogenic. In fact, we previously found
that large (.100 kb), gene overlapping, singleton, deletions were
present in 4.9% of cases but also in 3.8% of controls, suggesting
that over two-thirds of these deletions are not relevant to disease
[8]. We identified 165 published de-novo or case-only deletions of
.100 kb overlapping at least one gene; a total of 511 genes are
deleted or disrupted by these deletions [8,9,10]. Additionally, we
identified 122 regions similar control-only deletions; a total of 252
genes are deleted or disrupted by these deletions. Of those 13 regions, 10 were among the set that convincingly
validated in subsequent replication (Table 2)—the remaining three
had indeterminate levels of significance. By contrast, only 20 of 63
SNPs remaining SNPs validated (Table S4). Disease regions that
replicate have more significant GRAIL scores than those that
failed (p = 0.00064, one-tailed rank-sum test, Figure 3A). As with
randomly selected SNP lists, the distribution of scores for the 21
failed regions was indistinguishable from a random (uniform)
distribution of p-values (Figure 3B). Using these Crohn’s results, we have compared GRAIL’s
performance to four other competing algorithms that also use
functional information to prioritize genes, and GRAIL’s perfor-
mance is superior at predicting true positive associations (see Text
S1, Figure S2, Table S5, Table S6). We applied GRAIL separately to both the case and control sets
of deletions. In the case deletions, we identified a subset containing
highly connected genes (Figure 4A). Specifically, 12 of the 165
regions obtain ptext scores,0.001 with text-similarity (Table 3). The
top
keywords
suggest
some
common
biological
underlying
functions: ‘phosphatase’, ‘glutamate’, ‘receptor’, ‘cadherin’, and
‘neurons’. Here we list a subset of the 74 regions that emerged from a Crohn’s disease GWA meta-analysis that GRAIL assigned the most compelling ptext scores to. The first three
columns list information about the associated SNP. The fourth column lists the combined p-value of association from a GWA meta-analysis and subsequent replication.
The fifth column indicates whether the region was validated, indeterminate, or failed in replication. Those regions that represent novel findings, not previously
published are also indicated. The sixth column lists the number of genes in the disease region, and the seventh column lists the candidate gene identified by GRAIL. The
eighth column lists the regions ptext score.
doi:10.1371/journal.pgen.1000534.t002 Genetic associations to Crohn’s disease and
schizophrenia—predicting disease regions After successfully applying GRAIL to validated associations for
two phenotypes, we hypothesized that GRAIL could also be used We applied GRAIL prospectively to these 74 nominally
associated SNPs. GRAIL was initially operated independent of PLoS Genetics | www.plosgenetics.org June 2009 | Volume 5 | Issue 6 | e1000534 June 2009 | Volume 5 | Issue 6 | e1000534 5 Identifying Common Function Across Disease Regions Identifying Common Function Across Disease Regions SNP
Chr
Position (HG17)
passociation
Replication Study Result
N (genes)
Implicated Gene
ptext
rs2066845
16
49314041
1.5E-24
VALIDATED
3
NOD2
0.00010
rs10863202
16
84545499
1.4E-05
INDETERMINATE
4
IRF8
0.00058
rs10045431
5
158747111
1.9E-13
VALIDATED-NOVEL
1
IL12B
0.00066
rs11465804
1
67414547
3.3E-63
VALIDATED
1
IL23R
0.00094
rs2476601
1
114089610
7.3E-09
VALIDATED-NOVEL
8
PTPN22
0.0014
rs762421
21
44439989
7.0E-10
VALIDATED-NOVEL
1
ICOSLG
0.0023
rs2188962
5
131798704
1.2E-18
VALIDATED
9
IRF1
0.0026
rs917997
2
102529086
1.1E-05
INDETERMINATE
5
IL18RAP
0.0027
rs11747270
5
150239060
1.7E-16
VALIDATED
3
IRGM
0.0032
rs2738758
20
61820069
2.7E-06
INDETERMINATE
10
TNFRSF6B
0.0038
rs9286879
1
169593891
7.7E-10
VALIDATED-NOVEL
4
TNFSF18
0.0042
rs2301436
6
167408399
5.2E-13
VALIDATED-NOVEL
3
CCR6
0.0052
rs4263839
9
114645994
1.3E-10
VALIDATED
2
TNFSF8
0.008
rs3828309
2
233962410
1.2E-32
VALIDATED
4
USP40
0.019
rs744166
17
37767727
3.4E-12
VALIDATED-NOVEL
2
STAT3
0.023
rs7758080
6
149618772
4.4E-06
INDETERMINATE
4
SUMO4
0.033
rs7161377
14
75071147
2.3E-05
INDETERMINATE
1
BATF
0.09
Here we list a subset of the 74 regions that emerged from a Crohn’s disease GWA meta-analysis that GRAIL assigned the most compelling ptext scores to. The first three
columns list information about the associated SNP. The fourth column lists the combined p-value of association from a GWA meta-analysis and subsequent replication. The fifth column indicates whether the region was validated, indeterminate, or failed in replication. Those regions that represent novel findings, not previously
published are also indicated. The sixth column lists the number of genes in the disease region, and the seventh column lists the candidate gene identified by GRAIL. The
eighth column lists the regions ptext score. doi:10.1371/journal.pgen.1000534.t002 June 2009 | Volume 5 | Issue 6 | e1000534 PLoS Genetics | www.plosgenetics.org 6 Identifying Common Function Across Disease Regions Figure 3. GRAIL predicts Crohn’s disease SNPs. (A) Validated versus Failed SNPs. Prior to replication, GRAIL scored Crohn’s SNPs that emerged
from a meta-analysis study. Results from follow-up testing either validated Crohn’s SNPs, or identified those SNPs that failed. We produce a scatter
plot of the significance of text-based similiarty (ptext) for validated regions (green) versus regions that failed to replicate (red). Black horizontal lines
mark the median ptext values. The distribution of scores for failed SNPs resembles a random distribution of p-values. The distribution of scores for
validated SNPs is significantly different; almost K of these SNPs obtain ptext scores,0.1. (B) Histogram of text-based scores for Crohn’s disease
candidate regions. Identifying Common Function Across Disease Regions Here we plot a histogram of ptext scores for 74 Crohn’s disease SNPs. Validated SNPs (green) have ptext values that are enriched for
significant values. Indeterminate SNPs (yellow) have a subset of ptext values that are significant. Failed SNPs (Red) have all of their ptext scores.0.1. doi:10.1371/journal.pgen.1000534.g003 Figure 3. GRAIL predicts Crohn’s disease SNPs. (A) Validated versus Failed SNPs. Prior to replication, GRAIL scored Crohn’s SNPs that emerged
from a meta-analysis study. Results from follow-up testing either validated Crohn’s SNPs, or identified those SNPs that failed. We produce a scatter
plot of the significance of text-based similiarty (ptext) for validated regions (green) versus regions that failed to replicate (red). Black horizontal lines
mark the median ptext values. The distribution of scores for failed SNPs resembles a random distribution of p-values. The distribution of scores for
validated SNPs is significantly different; almost K of these SNPs obtain ptext scores,0.1. (B) Histogram of text-based scores for Crohn’s disease
candidate regions. Here we plot a histogram of ptext scores for 74 Crohn’s disease SNPs. Validated SNPs (green) have ptext values that are enriched for
significant values. Indeterminate SNPs (yellow) have a subset of ptext values that are significant. Failed SNPs (Red) have all of their ptext scores.0.1. doi:10.1371/journal.pgen.1000534.g003 This approach could have widespread application to follow-up
GWA study results and offers a mechanism to prioritize the
hundreds of SNPs that are expected to achieve an intermediately
significant level of association (1025,p,1023). As far as we are
aware – this is the first successful prediction of the outcome of a
GWA validation study. rank-sum test). Considering the entire set, case-deletions are not
enriched for genes preferentially expressed in the CNS (22% are
preferentially expressed in the CNS, compared to 25% of control-
deletion genes). However, considering the subset of genes
indentified by GRAIL (ptext,0.01), 60% (9 of 15 genes) are
preferentially CNS expressed. Furthermore, the fraction of genes
with preferential CNS expression correlates inversely with the
significance of the GRAIL score (Figure 4B). Regions that GRAIL
assigns
non-significant
scores
to,
do
not
demonstrate
any
compelling enrichment for CNS expressed genes. GRAIL offers the greatest value in situations where disease
regions are being considered that are difficult to validate, for
example rare deletions. The ability to genetically validate any
individual rare deletion is challenged given the limited power
afforded by the size of available patient collections. Identifying Common Function Across Disease Regions In schizo-
phrenia the excess of rare deletions has now been well documented
– but it had been difficult to connect these rare deletions to a
specific pathway. We identified a subset of related genes that have
functions that are plausibly related to schizophrenia. As other
diseases emerge where rare variants play a role in the genetic
architecture, our approach may provide a crucial first step to put
context to genetic findings. PLoS Genetics | www.plosgenetics.org Discussion We have presented an automated text-based strategy to take a
list of disease regions and identify those regions with significantly
inter-related genes. In the process it recognizes the likely candidate
gene in each disease region. It makes no assumptions about the
phenotype being studied or underlying pathways that might be
presumed to be relevant to a disease state. While in principle a
diligent investigator could potentially examine the literature
related to all potentially associated genes and arrive at the same
conclusions, they are unlikely in practice be able to work with the
same efficiency and objectivity as the approach outlined here. In
the
schizophrenia
application,
for
example,
we
objectively
interpret and analyze the relationship between over 500 genes. PLoS Genetics | www.plosgenetics.org Identifying disease genes within a region SP110. IRGM is not readily connected to these genes in a well-
defined pathway and, in-fact, is not referenced together with them
in any abstracts; furthermore no IRGM interactions are listed in
Entrez at all. Yet they all are involved in the host response to
Mycobacterium
and
possibly
other
intracellular
infections
by
macrophages. The top keywords describing the connections
between IRGM and these genes were ‘macrophages’, ‘tuberculosis’
and ‘mycobacterium’. The IRGM gene has been shown exper-
imentally to eliminate intracellular Mycobacterium tuberculosis via
autophagy [31]. The IRF1 homolog studies in mouse have
demonstrated its role in intra-cellular nitrous oxide production,
necessary to fight Mycobacterium infections [32]. Individuals with
loss of function IL12B mutations have been found with increased
susceptibility to Mycobacterium infections [33] and knock out mice
have demonstrated increased susceptibility to infection [34,35]. A
SP110 mouse homolog has been shown to mediate innate
immunity in fighting intra-cellular Mycobacterium tuberculosis infec-
tion [36]. GRAIL is able to identify this common underlying
similarity between these genes, and assign a significant score to
IRGM, while at the same time revealing what may be an important
pathway
in
Inflammatory
Bowel
Disease. Other
strategies
depending on interaction networks or functional databases may
struggle to detect these relationships. GRAIL systematically identifies a single gene within a disease
region as the likely disease gene. We highlight two interesting
examples from the height data of previously unrecognized
potentially causative genes. The first example is the rs42046
SNP on chromosome 7 region implicating five genes. The genetic
studies that identified this region had suggested CDK6 as the likely
causative gene [2–4]. However, GRAIL found greatest evidence in
support of PEX1 (uncorrected ptext = 0.0084). When we compare
the most compelling of these genes, PEX1, to candidates from the
other 41 SNPs with our text-based metric, we found it to be most
related to a gene in a height-associated SNP on chromosome 8,
PEX2 (PXMP3). The protein products of both PEX1 and PEX2 are
involved in peroxisome biogenesis and are implicated in a genetic
disease associated craniofacial and skeletal abnormalities (Zellwe-
ger’s syndrome) [37–39]. While it may be a coincidence that these
two closely related genes are associated by chance, it is certainly
possible that peroxisome biogenesis represents a previously
unrecognized height pathway. Connecting seemingly unrelated genes through text Connecting seemingly unrelated genes through text
The main strength of GRAIL is its ability to link genes through
text that may not yet have an established common pathway or
process. Consider the IRGM gene association to Crohn’s disease –
for
which
GRAIL
found
strong
evidence
(uncorrected
ptext = 0.0011). GRAIL’s text-based similarity metric recognize
the significant connections between IRGM and four other
validated or intermediate region genes: IRF1, IL12B, IRF8, and We present data that GRAIL can identify common SNPs that
subsequently validate in replication genotyping. We have demon-
strated superior performance in this application to other methods. June 2009 | Volume 5 | Issue 6 | e1000534 7 Identifying Common Function Across Disease Regions Figure 4. GRAIL identifies a subset of highly connected genes within rare deletions found in Schizophrenia cases. (A) Case deletions
versus control deletions. Here we plot the results of the separate GRAIL analyses conducted on the deletions observed in schizophrenia cases and
controls. Case deletion ptext scores are displayed in red; control deletion ptext scores are displayed in green. The line in each category in the middle of the
box represents the median GRAIL ptext score. The box represents the 25–75% range. The bars represent the 5–95% range. Additional scores outside the
range are individual plotted. (B) Text-based GRAIL significance score tracks with CNS specific expression. We partition case-only deletions by their GRAIL
scores. For each range of GRAIL ptext scores, we assess the candidate genes selected by GRAIL for CNS expression. The upper portion of this plot
illustrates the fraction of those candidate genes that demonstrate preferential CNS expression along with 95% confidence intervals. The blue line
represents the total fraction of genes that are preferentially CNS expressed. For the most compelling GRAIL scores, the candidate genes are significantly
enriched for CNS expression compared to what would be expected from a random group of genes. The lower portion of the plot is a histogram. doi:10.1371/journal.pgen.1000534.g004 Figure 4. GRAIL identifies a subset of highly connected genes within rare deletions found in Schizophrenia cases. (A) Case deletions
versus control deletions. Here we plot the results of the separate GRAIL analyses conducted on the deletions observed in schizophrenia cases and
controls. Case deletion ptext scores are displayed in red; control deletion ptext scores are displayed in green. The line in each category in the middle of the
box represents the median GRAIL ptext score. Connecting seemingly unrelated genes through text The box represents the 25–75% range. The bars represent the 5–95% range. Additional scores outside the
range are individual plotted. (B) Text-based GRAIL significance score tracks with CNS specific expression. We partition case-only deletions by their GRAIL
scores. For each range of GRAIL ptext scores, we assess the candidate genes selected by GRAIL for CNS expression. The upper portion of this plot
illustrates the fraction of those candidate genes that demonstrate preferential CNS expression along with 95% confidence intervals. The blue line
represents the total fraction of genes that are preferentially CNS expressed. For the most compelling GRAIL scores, the candidate genes are significantly
enriched for CNS expression compared to what would be expected from a random group of genes. The lower portion of the plot is a histogram. doi:10.1371/journal.pgen.1000534.g004 PLoS Genetics | www.plosgenetics.org Identifying Common Function Across Disease Regions Identifying Common Function Across Disease Regions development and migration may contribute to the pathophysiol-
ogy of schizophrenia [42]. Many of the highest scoring genes
recovered by GRAIL within the deleted regions in cases (Table 3)
are localized to the postsynaptic membrane/signaling complex
that propagate signals resulting in changes synapse function and
downstream gene expression/transcription. The DLG2 gene
product interacts at postsynaptic sites to form a multi-meric
scaffold for the clustering of receptors, ion channels, and
associated signaling proteins. MAGI1 and MAGI2 both encode
post-synaptic scaffolding molecules involved in cell adhesion and
signaling [43,44]. Furthermore, glutamatergic neurotransmission
is implicated through the selection of GRM1, GRM7, and GRM8. Many of the most significant candidate genes identified by GRAIL
are involved in neuronal development, cell-cell adhesion and axon
guidance. CNTN5 is an immunoglobulin super-family membrane-
anchored neuronal protein that is also an adhesion molecule [45]. It may play a role in the developing nervous system [46]. The
SDK1 gene expresses a synaptic adhesion protein [47] that guides
axonal terminals to specific synapses in developing neurons. The
PTPRM encodes a neuronally expressed protein tyrosine phos-
phatase that mediates cell-cell aggregation and is involved in cell-
cell adhesion [48,49]. Table 3. Rare or de novo schizophrenia case deletions. CHR
Start
Stop
ptext
Candidate Gene
7
77,788,564
78,591,795
0.0000013
MAGI2
11
*83,680,969
83,943,977
0.0000016
DLG2
3
65,781,878
65,975,330
0.0000057
MAGI1
11
99,153,400
99,286,239
0.000013
CNTN5
4
*87,919,851
88,032,640
0.000025
PTPN13
18
8,054,730
8,257,748
0.000035
PTPRM
3
*7,177,597
7,314,117
0.000087
GRM7
3
7,043,889
7,145,741
0.000087
GRM7
6
146,418,079
146,525,433
0.00013
GRM1
7
125,707,286
126,050,230
0.00015
GRM8
9
9,485,226
9,644,834
0.00024
PTPRD
7
3,759,288
4,087,229
0.00033
SDK1
3
*197,224,662
198,573,215
0.0011
DLG1
15
27,015,263
28,173,703
0.0014
TJP1
5
31,250,352
32,213,541
0.0033
PDZD2
19
10,231,490
10,493,592
0.0048
ICAM5
2
112,407,513
112,512,196
0.014
MERTK
5
19,570,562
19,843,415
0.018
CDH18
6
145,876,484
146,009,981
0.019
EPM2A
7
145,321,439
145,461,533
0.019
CNTNAP2
5
63,115,468
63,431,545
0.023
HTR1A
14
66,287,336
66,470,393
0.025
GPHN
18
56,109,430
56,255,536
0.029
MC4R
5
106,805,717
107,026,020
0.03
EFNA5
2
233,029,864
233,134,571
0.031
ALPPL2
20
2,923,491
3,618,945
0.034
PTPRA
1
72,287,807
72,439,333
0.037
NEGR1
7
94,306,868
94,497,412
0.044
PPP1R9A
7
157,378,450
157,569,847
0.046
PTPRN2
1
*144,943,150
146,292,286
0.047
ACP6
Here we list all of the deletions that GRAIL identified as most related to other
deleted genes (ptext,0.05). Limitations to assessing gene relatedness with text g g
While we have shown the promise of text-based similarity in
identifying regions and the genes within them that are part of a
larger biological pathway, we note that this strategy’s effectiveness
is wholly contingent on the completeness of the scientific text. It
could be biased towards subsets of genes and pathways that are
particularly well studied, and against poorly studied ones. In many
of the cases that we illustrate, there are regions that could not be
connected – for example, GRAIL fails to connect 5 validated
Crohn’s SNPs that obtain ptext scores.0.5 (Figure 3B). These
regions might have been missed since the relevant gene is either
poorly studied, or even if the gene is well studied, the relevant
function of that gene is not well documented in the text. An
alternative possibility is that the SNP is tagging non-genic
regulatory elements. Additionally, the SNP may be the first
discovered representative association for a critical pathway, not
represented by other SNP associations – and therefore cannot be
connected to them. In this case future discoveries will clarify the
significance of that association. In fact, a mutation in the KY gene causes spinal scoliosis in a
mouse model [40], and the KY protein product interacts with
sarcomeric cytoskeletal proteins [41]. While these literature-based
hypotheses may be obvious to a few specialized researchers, the
strength of GRAIL is that it is able to suggest these connections in
a systematic and objective manner from the entirety of the
published literature. In fact, a mutation in the KY gene causes spinal scoliosis in a
mouse model [40], and the KY protein product interacts with
sarcomeric cytoskeletal proteins [41]. While these literature-based
hypotheses may be obvious to a few specialized researchers, the
strength of GRAIL is that it is able to suggest these connections in
a systematic and objective manner from the entirety of the
published literature. Competing methods The most critical technical difference between GRAIL and
other strategies is that it does not use any strict definitions of gene
functions or interactions, but rather uses a metric of relatedness
that allows for a relatively broad range of freedom with which to
connect genes. While GRAIL will certainly identify relationships
between genes known to be in a common pathway, it goes beyond
that, and can allow less strict evidence. In fact, it is even able to
identify relatedness between genes that have no established
common pathways or article co-citations! In contrast, other
strategies start with static gene relationships—such as (1) pre-
constructed molecular networks [12,16] or sets of gene with
common function [11,15] or (2) a subset of functions identified as
relevant to disease either by the user [17] or by mining the
published text [14]. In a head to head match up against four other
methods that we were able to obtain implemented versions of,
GRAIL demonstrated superior performance in predicting Crohn’s
associated SNPs (see Text S1, Figure S2, Table S5, and Table S6). Identifying Common Function Across Disease Regions For each deletion we list the chromosome, the
range of the deletion, the GRAIL p-value for the region, and the best candidate
gene in the region identified by GRAIL. Most genomic coordinates are listed in
HG17. *HG18 coordinates. doi:10.1371/journal.pgen.1000534.t003 PLoS Genetics | www.plosgenetics.org Identifying disease genes within a region The second example is the
rs10935120 SNP on chromosome 3, implicating three genes; the
genetic study that had identified this gene had suggested ANAPC13
as the likely candidate in the region [4]. However, GRAIL
identified the KY gene as the most likely disease gene (ptext = 0.04). June 2009 | Volume 5 | Issue 6 | e1000534 8 Scoring regions for functional relatedness We then proceed outwards in each
direction to the nearest recombination hotspot [51]. The interval
between those two hotspots, which would include the SNP of
interest and all SNPs in LD, is defined as the disease region. The
associated SNP could feasibly be tagging a stronger SNP signal
from another SNP in that region. All genes that overlap that
interval are considered implicated by the SNP. If there are no
genes in that region, the interval is extended an additional 250 kb
in either direction; we chose 250 kb as that distance since that is a
range in which non-coding variants might express gene regulation
[52]. For each query and seed CNV we define an interval that
represents the deleted or duplicated region—all genes that overlap
that interval are associated with the CNV for testing. After testing gene g across NSEED seed regions for related genes,
the probability of a score exceeding cg under the null, pg, can be
approximated: pg~p cwcg
~
X
Nseed
nhit~0
p cwcg,nhit
~
X
Nseed
nhit~0
p nhit
ð
Þp cwcg nhit
j
Where nhit is the number of seed regions connected to gene g. Since
under the null model the probability of a connected region by
chance is always pf, we can estimate its probability distribution of
nhit with a Poisson distribution: p nhit
ð
Þ~ NSEEDpf
nhite{NSEEDpf
nhit! Step 2. Ranking gene relatedness. For each gene near a
query region, we rank all human genes for relatedness. Ranking
may be based on text similarity, or other metrics (see below for
examples). Rank values range from 1 (most related) to NG (least
related), where NG is the number of available human genes, in our
application is 18,875 (see Table 1). Since, cg, is the sum of the log of nhit independent uniformly
distributed values ranging from 0 to 1, for a fixed value of nhit we
can calculate the distribution of cg with a cumulative gamma
distribution: Since, cg, is the sum of the log of nhit independent uniformly
distributed values ranging from 0 to 1, for a fixed value of nhit we
can calculate the distribution of cg with a cumulative gamma
distribution: Step 3. Scoring candidate genes against regions. To
avoid double counting nearby regions, we first combine any seed
regions sharing one or more genes. Genes deleted in schizophrenia suggest relevant
neuronal processes We consider closely the subset of related genes identified by
GRAIL from rare deletions in patients with schizophrenia. Schizophrenia is a disorder characterized by hallucinations,
delusions, cognitive deficits and apathy. The molecular basis of
the symptom complex associated with the disorder is largely
unknown. However accumulating evidence suggest that dysregu-
lation
of
synaptic
activity
and
abnormalities
in
neuronal In cases where there is no apparent published connection
between associated genes, other similarity metrics based on
experimentally derived data, such as gene expression, protein- June 2009 | Volume 5 | Issue 6 | e1000534 June 2009 | Volume 5 | Issue 6 | e1000534 9 Identifying Common Function Across Disease Regions Identifying Common Function Across Disease Regions Identifying Common Function Across Disease Regions testing: testing: protein interactions and transcription factor binding sites could
also complement the text-based approaches presented here. In
fact, we demonstrate how annotation-based metrics or gene
expression-based metrics are able to identify a subset of the
associated SNPs in lipid metabolism. As these and other metrics
are optimized, they could be used in conjunction with the novel
GRAIL statistical framework that we present here to help
understand gene relationships. pg,s~1{ 1{pg,s,uncorrected
ns Now we identify those seed regions where pg,s is less than a pre-
specified threshold pf as regions connected to gene g. For all
applications presented here pf is arbitrarily set to 0.1. The number
of seed regions containing at least one gene exceeding this
threshold, nhit, can be approximated under a random model with
a Poisson distribution. Scoring regions for functional relatedness The Gene Relationships Among Implicated Loci (GRAIL) has
four basic steps that are outlined below. It has two input sets of
disease regions: (1) a collection of NSEED seed regions (SNPs or
CNVs) and (2) a collection of NQUERY query regions. Genes in query
regions are evaluated for relationships to genes in seed regions, and
query regions are then assigned a significance score. In most
applications we are examining a set of regions for relationships
between implicated genes, the query regions and the seed regions are
identical. In other circumstances where we have a set of putative
regions that are being tested against validated ones, the putative
regions are defined as query regions, and the validated ones are
defined as seed regions. wg,s~
{log
pg,s
pf
pg,sƒpf
0
pg,swpf
(
) Under a random model, if pg,s,pf , pg,s should range approximately
uniformly from 0 to pf. Therefore, under these circumstances wg,s
can be modeled approximately with a gamma distribution. For each candidate gene, g, we tally the number of seed regions
that contain a highly related gene into a weighted count, cg: cg~
X
i
wsi,g~
X
pg,si vpf
{log psi,g
pf
Step
1. Defining
disease
regions
and
identifying
overlapping genes. For each query and seed SNP we find the
furthest neighboring SNPs in the 39 and 59 direction in LD
(r2.0.5, CEU HapMap [50]). We then proceed outwards in each
direction to the nearest recombination hotspot [51]. The interval
between those two hotspots, which would include the SNP of
interest and all SNPs in LD, is defined as the disease region. The
associated SNP could feasibly be tagging a stronger SNP signal
from another SNP in that region. All genes that overlap that
interval are considered implicated by the SNP. If there are no
genes in that region, the interval is extended an additional 250 kb
in either direction; we chose 250 kb as that distance since that is a
range in which non-coding variants might express gene regulation
[52]. For each query and seed CNV we define an interval that
represents the deleted or duplicated region—all genes that overlap
that interval are associated with the CNV for testing. Step
1. Defining
disease
regions
and
identifying
overlapping genes. For each query and seed SNP we find the
furthest neighboring SNPs in the 39 and 59 direction in LD
(r2.0.5, CEU HapMap [50]). Methods We assign a greater weight to those cases where there is greater
similarity; that is in the cases where pg,s is particularly small: Keywords To assign keywords to a collection of query regions, we first
identify the single candidate genes with the best GRAIL ptext from
each
region. We
then
eliminate
those
regions
where
the
uncorrected GRAIL score for the gene is ptext.0.2. We restrict Gene expression based relatedness wij~
1zlog tfij
log2
NDOC
dfi
tfijw0
0
tfij~0
(
) wij~
1zlog tfij
log2
NDOC
dfi
tfijw0
0
tfij~0
(
) To calculate gene relatedness based on expression we download-
ed the Novartis Gene Expression Atlas [28]. The data set consists of
measurements for 33,689 probes across 158 conditions. Probes were
averaged into 17,581 gene profiles. Gene relatedness was calculated
as the correlation between expression vectors. where NDOC is the total number of documents, and dfi (or
document frequency) is the number of documents the term i
appears in. This scheme emphasizes rare words, and de-
emphasizes more common words. Lipid and height applications We applied GRAIL to score 19 lipid-associated SNPs and
separately to score 42 height-associated SNPs. Specific SNPs are
listed in Table S1 and Table S2. We used the SNP sets as both the
seed and the query set to look for relatedness between genes across
regions. We scored SNPs separately using text, annotation, and
expression similarity metrics. We compiled the best candidate
genes and scores for the SNP regions. For every gene, we define an averaged term-vector, which is an
average of weighted term vectors from gene references and
homologous gene references. Abstracts are weighted according to
the number of genes they reference; articles referencing many
genes are down-weighted to mitigate their influence: gik~
X
j [ ref k
ð Þ
wij
1
1zlog2 nref ,j
Identifying Common Function Across Disease Regions keywords to those that appear in .500 documents, contain .3
letters, and have no numbers. For each term, i, we calculate a
score which is the difference between averaged term frequencies
among candidate genes and all genes: keywords to those that appear in .500 documents, contain .3
letters, and have no numbers. For each term, i, we calculate a
score which is the difference between averaged term frequencies
among candidate genes and all genes: Putting this together: Putting this together: Putting this together: pg~
X
Nseed
nhit~0
NSEEDpf
nhite{NSEEDpf
nhit! X
nhit{1
i~0
cg
i
i! e{cg
! "
# s ið Þ~
mean
k [ candidate genes gik
ð
Þ{
mean
k [ all genes gik
ð
Þ Step 4. Scoring regions. Finally, for each query region we
identify the best scoring gene within it. A significance score for the
query region, pq, is based on the p-value of that gene, pg, corrected
for multiple hypothesis testing. Assuming the region has nq genes
within it: The top twenty highest scoring terms are selected as keywords. The top twenty highest scoring terms are selected as keywords. Crohn’s disease application Prior to replication, we had access to 74 independent SNP
regions that had emerged from a meta-analysis of Crohn’s Disease. All 74 SNPs were used as both the query set and as the seed set into
GRAIL. We assessed whether those SNPs that replicated had
different text-based significance values than those that fail to
replicate. To identify additional regions of interest, we identified
the next 75 most significant regions in the Crohn’s disease meta-
analysis – they were used in GRAIL as a query set; for the seed set
included all SNPs that did not fail in replication. where gik is a the weighted count of term i for gene k, j is a
document reference for gene k, and document j references nref,j
genes. For a given gene i these gik terms define a gene-text vector. The gene text vectors are normalized, so that their euclidean
length is 1. Pairwise gene relatedness can be calculated as the dot
product between two normalized term vectors for genes. Assessing gene relatedness with text-based similarity We measure relatedness between genes using similarity in
published text from gene references. We first obtain article
abstracts
from
Pubmed. We
downloaded
all
abstracts
on
December 16, 2006. For each gene, we identified and downloaded
abstract references listed in Entrez Gene [23]; additionally, we
downloaded Entrez Gene abstract references for gene orthologs
listed in Homologene [53]. We removed those articles referencing
more than 10,000 genes. Only the title (TI) and abstract (AB) fields
were included for further text processing. We defined a vocabulary
consisting of only those terms appearing in 40 or more abstracts,
and fewer than 130,000; this resulted in a vocabulary of 23,594
terms. For each abstract j we create a vector of term frequencies,
tfij, representing the number of times each term i appears within it. Term frequencies are transformed into weights, wij, according to a
standard inverse document frequency scheme [54]: gij~
log2
NG
gfi
GOij~1
0
GOij~0
(
) gij~
log2
NG
gfi
GOij~1
0
GOij~0
(
) where gij represented the weighted code i for gene j, NG is the total
number of genes, and gfi (or GO frequency) is the number of genes
annotated with the term i. Gene relatedness was the correlation
between these weighted annotation vectors. Annotation based relatedness We defined a relatedness metric between genes based on
similarity in Gene Ontology annotation terms [27]. We downloaded
Gene Ontology structure and annotations on December 19, 2006. In addition to human gene GO annotations, we added orthologous
gene
annotations. Since
GO
is
a
hierarchically
structured
vocabulary, for each gene annotation we also added all of the
more general ancestral terms. This resulted in a total of 843,898
annotations for 18,050 genes with 10,803 unique GO terms; this
corresponds to a median of 40 terms per gene. We weighted
annotations proportionally to the inverse of their frequency, so
common annotations received less emphasis. We used a weighting
scheme analogous to the one we used for word weighting: pq~1{ 1{pg
nq
where pg~min pg’ g’ [ q
j
Scoring regions for functional relatedness For a given gene g in a query
region, we examine the degree of similarity to any of the ns genes
in a given seed region s. To ensure independence, we only look at a
seed region s, if it does not share a single gene with the query region
that gene g is contained in. p cwcg nhit
j
~FGamma ?,nhit,1
ð
Þ{FGamma cg,nhit,1
Since nhit is always an integer, the FGamma term can be simplified: FGamma cg,nhit,1
~1{
X
nhit{1
i~0
cg
i
i! e{cg We identify in each region s, the rank of the most similar (or
lowest ranking) gene in it to gene g, Rg,s. We convert the rank to a
proportion: Therefore, we can be further simplified: Therefore, we can be further simplified: pg,s,uncorrected~Rg,s
NG p cwcg nhit
j
~FGamma ?,nhit,1
ð
Þ{FGamma cg,nhit,1
~
X
nhit{1
i~0
cg
i
i! e{cg To transform this proportion to a uniformly distributed entity
under the null, we recognize that Rg,s was the lowest rank selected
from ns genes – and we correct accordingly for multiple hypothesis June 2009 | Volume 5 | Issue 6 | e1000534 PLoS Genetics | www.plosgenetics.org 10 Identifying Common Function Across Disease Regions PLoS Genetics | www.plosgenetics.org June 2009 | Volume 5 | Issue 6 | e1000534 Identifying Common Function Across Disease Regions were in cases only or in controls only, were at least 100 kb large,
and included at least one gene. We obtained singleton deletions
online published by the International Schizophrenia Consortium
(2008) at [8]. We obtained de novo deletions published by Xu et al
(2008) from Table 1 [10]. We obtained singleton deletions
published in Walsh et al (2008) from Table 2 [9]. We identified
a total of 165 case-only deletions and 122 control-only deletions. We applied the GRAIL algorithm separately to case and controls. We speculated that the case deletions might hit genes from a
common pathway and GRAIL p-values may therefore be enriched
for significant scores. On the other hand, we hypothesized that
control deletions might be located effectively at random, and so no
particular pathway or common function should necessarily be
enriched in this collection. Table S1
19 Lipid regions scored with Text based GRAIL
strategy. Here we scored 19 SNPs, associated with lipid metabolism. In the first three columns we list information about the SNP. In the
fourth column we list the number of genes in the SNP associated
regions. In the fifth column we list the highest scoring gene in the
associated region based on GRAIL using a text-based metric. In the
sixth column we list the ptext values for the associated regions. We
have bolded those candidate genes that are known likely causative
gene. The seventh and eight columns list similar results for GRAIL
with an GO annotation-based metric. The ninth and tenth columns
list similar results for GRAIL with an expression-based metric. Found
at:
doi:10.1371/journal.pgen.1000534.s003
(0.15
MB
DOC) Table S2
42 Height regions scored with Text based GRAIL
strategy. Here we scored 42 SNPs, associated with height. In the first
three columns we list information of the SNP. In the fourth column
we list the number of genes in the SNP associated regions. In the
fifth column we list the highest scoring gene in the associated region
for the SNP based on GRAIL using a text-based metric. In the sixth
column we list the ptext values for the associated regions. The seventh
and eight columns list similar results for GRAIL with an annotation-
based metric. The ninth and tenth columns list similar results for
GRAIL with an expression-based metric. Software An online version of this method is available (http://www. broad.mit.edu/mpg/grail/). Evaluation against other published methods We compared GRAIL’s performance in its ability to prospec-
tively predict Crohn’s associations to five other published methods. The selection of these methods, and the evaluation is detailed in
Text S1. Identifying Common Function Across Disease Regions Found
at:
doi:10.1371/journal.pgen.1000534.s004
(0.28
MB
DOC) To examine genes for tissue specific expression in the CNS
system, we obtained a large publicly available human tissue
expression microarray panel (GEO accession: GSE7307) [30]. We
analyzed the data using the robust multi-array (RMA) method for
background correction, normalization and polishing [55]. We
filtered the data excluding probes with either 100% ‘absent’ calls
(MAS5.0 algorithm) across tissues, expression values ,20 in all
samples, or an expression range ,100 across all tissues. To
represent each gene, we selected the corresponding probe with the
greatest intensity across all samples. The data contained expression
profiles for 19,088 genes. We included expression profiles from
some 96 normal tissues and excluded disease tissues and treated
cell lines. We averaged expression values from replicated tissues
averaged into a single value. To assess whether genes had
differential expression for CNS tissues, we compared the 27 tissue
profiles that represented brain or spinal cord to the remaining 69
tissue profiles with a one-tailed Mann-Whitney rank-sum test. Genes obtaining p,0.01 were identified as preferentially ex-
pressed. Table S3
Keywords for Lipid and Height SNPs. We identified
keywords associated with lipid and height associated SNPs; here
we list the top 20. Table S3
Keywords for Lipid and Height SNPs. We identified
keywords associated with lipid and height associated SNPs; here
we list the top 20. Found
at:
doi:10.1371/journal.pgen.1000534.s005
(0.06
MB
DOC) Found
at:
doi:10.1371/journal.pgen.1000534.s005
(0.06
MB
DOC) Table S4
Crohn’s Disease SNPs from a meta-analysis of GWA
studies. Here we list GRAIL results and summarize genotyping
results for Crohn’s disease SNPs. These 74 SNPs emerged from a
meta-analysis and as a result of replication genotyping, they were
either validated (A), indeterminate (B), or failed (C). For each of
the regions we list the SNP ID and the chromosome in the second
and third column. In the fourth column we list the final combined
association significance score of the SNP to the Crohn’s disease. In
the fifth, sixth, and seventh columns we list GRAIL results
including the number of genes in the region, the best candidate
gene, and the text-based significance score for the region. Found
at:
doi:10.1371/journal.pgen.1000534.s006
(0.21
MB
DOC) Schizophrenia application We identified singleton deletions or confirmed de novo deletions
reported by one of three groups. We selected those deletions that June 2009 | Volume 5 | Issue 6 | e1000534 PLoS Genetics | www.plosgenetics.org 11 Identifying Common Function Across Disease Regions Identifying Common Function Across Disease Regions Supporting Information Pato11
Carlos Paz Ferreira27
Ben Pickard7
Jonathan Pimm6
Shaun M. Purcell1,2,3,4
Vinay Puri6
Digby Quested19¤
Douglas M. Ruderfer1,2,3,4
Edward M. Scolnick2,3
Pamela Sklar1,2,3,4
David St Clair12
Jennifer L. Stone1,2,3,4
Patrick F. Sullivan13
Emma F. Thelander9
Srinivasa Thirumalai18
Draga Toncheva15
Margaret Van Beck7
Peter M. Visscher14
John L. Waddington17
Nicholas Walker22
H. Williams5
Nigel M. Williams5 Table S7
Other promising regions in Crohn’s Disease GWA
meta-analysis. Information about the top six regions identified by
GRAIL from the next 75 most significant regions from the
Crohn’s GWA study. All associations are indeterminate, and
association p-values are taken from the GWA meta-analysis - these
regions have not yet been replicated. Found
at:
doi:10.1371/journal.pgen.1000534.s009
(0.05
MB
DOC) Table S8
Rare or de novo schizophrenia control deletions. Here
we list all of the deletions that GRAIL identified as most related to
other deleted genes (ptext,0.05). For each deletion we list the
chromosome, the range of the deletion, the GRAIL p-value for the
region, and the best candidate gene in the region identified by
GRAIL. Most genomic coordinates are listed in HG17. * HG18
coordinates. Found
at:
doi:10.1371/journal.pgen.1000534.s010
(0.06
MB
DOC) Text S1
A. Random SNP groups; B. Comparison of GRAIL to
other related algorithms. Found
at:
doi:10.1371/journal.pgen.1000534.s011
(0.09
MB
DOC) International Schizophrenia Consortium
3 International Schizophrenia Consortium
Kristen Ardlie3
M. Helena Azevedo28
Nicholas Bass6
Douglas H. R. Blackwood7
Celia Carvalho11
Kimberly Chambert2,3
Khalid Choudhury6
David Conti11
Aiden Corvin8
Nick J. Craddock5
Caroline Crombie21
David Curtis20
Mark J. Daly2,3,4
Susmita Datta6
Stacey B. Gabriel3
Casey Gates3
Lucy Georgieva5
Michael Gill8
Hugh Gurling6
Peter A. Holmans5
Christina M. Hultman9,10
Ayman Fanous11
Gillian Fraser21
Elaine Kenny8
George K. Kirov5
James A. Knowles11
Robert Krasucki6
Joshua Korn3,4
Soh Leh Kwan12
Jacob Lawrence6
Paul Lichtenstein9
Antonio Macedo28
Stuart Macgregor14
Alan W. Maclean7
Scott Mahon3
Pat Malloy7
Kevin A. McGhee7
Andrew McQuillin6
Helena Medeiros11
PLoS Genetics | www.plosgenetics.org International Schizophrenia Consortium
Kristen Ardlie3
M. Helena Azevedo28
Nicholas Bass6
Douglas H. R. Blackwood7
Celia Carvalho11
Kimberly Chambert2,3
Khalid Choudhury6
David Conti11
Aiden Corvin8
Nick J. Craddock5
Caroline Crombie21
David Curtis20
Mark J. Daly2,3,4
Susmita Datta6
Stacey B. Gabriel3
Casey Gates3
Lucy Georgieva5
Michael Gill8
Hugh Gurling6
Peter A. Holmans5
Christina M. Hultman9,10
Ayman Fanous11
Gillian Fraser21
Elaine Kenny8
George K. Kirov5
James A. Knowles11
Robert Krasucki6
Joshua Korn3,4
Soh Leh Kwan12
Jacob Lawrence6
Paul Lichtenstein9
Antonio Macedo28
Stuart Macgregor14
Alan W. Maclean7
Scott Mahon3
Pat Malloy7
Kevin A. Acknowledgments We would like to thank Drs. Eric Lander, Russ Altman , Chris Cotsapas,
Joerg Ermann, Elizabeth W. Karlson, Kasper Lage, Guillaume Lettre,
Roland Nilsson, and Ayellet Segre for insightful feedback and comments. We also thank Jesse Ross for assistance in constructing the web server. We also thank Jesse Ross for assistance in constructing the web server. 1 Department of Psychiatry, Massachusetts General Hospital and
Harvard Medical School, Boston, Massachusetts, United States of
America, Frank Middleton23
Vihra Milanova16
Christopher Morley23
Derek W. Morris8
Walter J. Muir7
Ivan Nikolov5
N. Norton5
Colm T. O’Dushlaine8
Michael C. O’Donovan5
Michael J. Owen5
Carlos N. Pato11
Carlos Paz Ferreira27
Ben Pickard7
Jonathan Pimm6
Shaun M. Purcell1,2,3,4
Vinay Puri6
Digby Quested19¤
Douglas M. Ruderfer1,2,3,4
Edward M. Scolnick2,3
Pamela Sklar1,2,3,4
David St Clair12
Jennifer L. Stone1,2,3,4
Patrick F. Sullivan13
Emma F. Thelander9
Srinivasa Thirumalai18
Draga Toncheva15
Margaret Van Beck7
Peter M. Visscher14
John L. Waddington17
Nicholas Walker22
H. Williams5
Nigel M. Williams5 2 Stanley Center for Psychiatric Research, Broad Institute of MIT and
Harvard, Cambridge, Massachusetts, United States of America, Supporting Information Table S5
Algorithms to prioritize candidate genes. Our search
of the literature identified nine algorithms that could be used to
prioritize genes for replication. Four methods require no user-
specified disease information (supervised), and five require some
disease information from the user. We list in each row the name of
the disease, the website, the necessary genetic data, the functional
data used to prioritize genes, the disease-specific information that
must be included, and the availability of the method. Found
at:
doi:10.1371/journal.pgen.1000534.s007
(0.09
MB
DOC) Figure S1
GRAIL p-value scores for random SNPs. We scored
100 random groups of 50 SNPs with GRAIL. The y-axis is the
fraction of SNPs in the group with values below the threshold, the x-
axis lists the specific threshold. For each threshold, we plot the
distribution of the fraction of the 50 SNPs below that threshold as a
box plot. The bar is the median - the mean value is explicitly listed
below the box-plot. The box at each threshold lists the 25%–75%
range. The error-bars line depicts the 1.5 inter-quartile range. The
black dots illustrate outliers outside the 1.5 inter-quartile range. Found at: doi:10 1371/journal pgen 1000534 s001 (0 39 MB PDF) Table S6
Performance measures for prioritization algorithms. We used five algorithms (column 1) to score putatively associated
SNPs from the Crohn’s meta-analysis. After calculating an ROC
curve for each algorithm, we calculated the AUC (column 2). We
also calculated a p-value with a one-tailed rank-sum test
comparing the median rank of the validated SNPs to the median
rank of the failed SNPs (column 2). Found at: doi:10.1371/journal.pgen.1000534.s001 (0.39 MB PDF) Figure S2
Sensitivity versus specificity for prioritization algorithms. We used 5 algorithms to score the 74 most promising putative SNP
associations from the Crohn’s meta-analysis study. We assessed each
algorithm’s ability to predict those SNP associations that ultimately
validated in follow-up genotyping. For each algorithm, we created a
received-operator curve (ROC). Found
at:
doi:10.1371/journal.pgen.1000534.s008
(0.04
MB
DOC) Found at: doi:10.1371/journal.pgen.1000534.s002 (0.40 MB PDF) PLoS Genetics | www.plosgenetics.org June 2009 | Volume 5 | Issue 6 | e1000534 June 2009 | Volume 5 | Issue 6 | e1000534 12 Identifying Common Function Across Disease Regions Identifying Common Function Across Disease Regions Frank Middleton23
Vihra Milanova16
Christopher Morley23
Derek W. Morris8
Walter J. Muir7
Ivan Nikolov5
N. Norton5
Colm T. O’Dushlaine8
Michael C. O’Donovan5
Michael J. Owen5
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mediates innate immunity to tuberculosis. Nature 434: 767–772. 14. Identifying Common Function Across Disease Regions 27 Department of Psychiatry, Sao Miguel, Azores, Portugal, 27 Department of Psychiatry, Sao Miguel, Azores, Portugal, 19 West London Mental Health Trust, Hammersmith and Fulham
Mental Health Unit and St Bernard’s Hospital, London, United Kingdom, 28 Department of Psychiatry, University of Coimbra, Coimbra,
Portugal, 28 Department of Psychiatry, University of Coimbra, Coimbra,
Portugal, 28 Department of Psychiatry, University of Coimbra, Coimbra,
Portugal, 20 Queen Mary College, University of London and East London and
City Mental Health Trust, Royal London Hospital, Whitechapel, London,
United Kingdom, ¤ Current address: Department of Psychiatry, University of Oxford,
Warneford Hospital, Headington, Oxford, United Kingdom ¤ Current address: Department of Psychiatry, University of Oxford,
Warneford Hospital, Headington, Oxford, United Kingdom ¤ Current address: Department of Psychiatry, University of Oxford,
Warneford Hospital, Headington, Oxford, United Kingdom 21 Department of Mental Health, University of Aberdeen, Aberdeen,
United Kingdom, Author Contributions 22 Ravenscraig Hospital, Inverkip Road, Greenock, United Kingdom, Conceived and designed the experiments: SR RMP EJR SMP PS DA
MJD. Performed the experiments: SR EJR ACYN International
Schizophrenia Consortium. Analyzed the data: SR ACYN EMS RJX
MJD. Contributed reagents/materials/analysis tools: SR International
Schizophrenia Consortium EMS DA MJD. Wrote the paper: SR RMP
EJR ACYN SMP PS EMS RJX DA MJD. Critically read and contributed
to the final manuscript: SR RMP SJR ACYN SMP PS EMS RJX DA
MJD. 23 State University of New York – Upstate Medical University,
Syracuse, New York, United States of America, 24 Washington VA Medical Center, Washington D. C., United States of
America, 25 Department of Psychiatry, Georgetown University School of
Medicine, Washington D. C., United States of America, 26 Department of Psychiatry, Virginia Commonwealth University,
Richmond, Virginia, United States of America, International Schizophrenia Consortium
3 McGhee7
Andrew McQuillin6
Helena Medeiros11
PLoS Genetics | www.plosgenetics.org 1 Department of Psychiatry, Massachusetts General Hospital and
Harvard Medical School, Boston, Massachusetts, United States of
America, 1 Department of Psychiatry, Massachusetts General Hospital and
Harvard Medical School, Boston, Massachusetts, United States of
America, 2 Stanley Center for Psychiatric Research, Broad Institute of MIT and
Harvard, Cambridge, Massachusetts, United States of America, 3 Broad Institute of Harvard and Massachusetts Institute of Technology,
Cambridge, Massachusetts, United States of America, 4 Center for Human Genetic Research, Massachusetts General
Hospital, Boston, Massachusetts, United States of America, p
5 School of Medicine, Department of Psychological Medicine, School of
Medicine, Cardiff University, Cardiff, United Kingdom, 5 School of Medicine, Department of Psychological Med Medicine, Cardiff University, Cardiff, United Kingdom, 6 Molecular Psychiatry Laboratory, Department of Mental Health
Sciences, University College London Medical School, Windeyer Institute
of Medical Sciences, London, United Kingdom, 7 Division of Psychiatry, School of Molecular and Clinical Medicine,
University of Edinburgh, Edinburgh, United Kingdom, 8 Neuropsychiatric Genetics Research Group, Department of Psychiatry
and Institute of Molecular Medicine, Trinity College Dublin, Dublin,
Ireland, 9 Department of Medical Epidemiology and Biostatistics, Karolinska
Institutet, Stockholm, Sweden, 10 Department of Neuroscience, Psychiatry, Ullera˚ker, Uppsala
University, Uppsala, Sweden, y,
pp
,
,
11 Center for Genomic Psychiatry, University of Southern California,
Los Angeles, California, United States of America, 12 Institute of Medical Sciences, University of Aberdeen, Foresterhill,
Aberdeen, United Kingdom, 13 Departments of Genetics, Psychiatry, and Epidemiology, University
of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United
States of America, 14 Queensland Institute of Medical Research, Brisbane, Queensland,
Australia, 15 Department of Medical Genetics, University Hospital Maichin Dom,
Sofia, Bulgaria, 16 Department of Psychiatry, First Psychiatric Clinic, Alexander
University Hospital, Sofia, Bulgaria, y
p
g
17 Molecular and Cellular Therapeutics and RCSI Research Institute,
Royal College of Surgeons in Ireland, Dublin, Ireland, 7 Molecular and Cellular Therapeutics and RCSI Research I 18 West Berkshire NHS Trust, Reading, United Kingdom, June 2009 | Volume 5 | Issue 6 | e1000534 June 2009 | Volume 5 | Issue 6 | e1000534 13 References Shiratsuchi T, Futamura M, Oda K, Nishimori H, Nakamura Y, et al. (1998)
Cloning and characterization of BAI-associated protein 1: a PDZ domain-
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for molecular biology. Genome Biol 6: 224. PLoS Genetics | www.plosgenetics.org June 2009 | Volume 5 | Issue 6 | e1000534 June 2009 | Volume 5 | Issue 6 | e1000534 14 Identifying Common Function Across Disease Regions Identifying Common Function Across Disease Regions 48. Del Vecchio RL, Tonks NK (2005) The conserved immunoglobulin domain
controls the subcellular localization of the homophilic adhesion receptor protein-
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outgrowth-promoting activity and restricted expression in the brain regions.
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Opin Cell Biol 15: 621–632. Identifying Common Function Across Disease Regions 44. Hirao K, Hata Y, Ide N, Takeuchi M, Irie M, et al. (1998) A novel multiple PDZ
domain-containing molecule interacting with N-methyl-D-aspartate receptors
and neuronal cell adhesion proteins. J Biol Chem 273: 21105–21110. 49. Sugino K, Hempel CM, Miller MN, Hattox AM, Shapiro P, et al. (2006)
Molecular taxonomy of major neuronal classes in the adult mouse forebrain. Nat
Neurosci 9: 99–107. 50. (2005) A haplotype map of the human genome. Nature 437: 1299–1320 45. Kamei Y, Takeda Y, Teramoto K, Tsutsumi O, Taketani Y, et al. (2000)
Human NB-2 of the contactin subgroup molecules: chromosomal localization of
the gene (CNTN5) and distinct expression pattern from other subgroup
members. Genomics 69: 113–119. 51. Myers S, Bottolo L, Freeman C, McVean G, Donnelly P (2005) A fine-scale map
of recombination rates and hotspots across the human genome. Science 310:
321–324. 46. Ogawa J, Lee S, Itoh K, Nagata S, Machida T, et al. (2001) Neural recognition
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outgrowth-promoting activity and restricted expression in the brain regions. J Neurosci Res 65: 100–110. 52. Stranger BE, Nica AC, Forrest MS, Dimas A, Bird CP, et al. (200 52. Stranger BE, Nica AC, Forrest MS, Dimas A, Bird CP, et al. (2007) Population
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Opin Cell Biol 15: 621–632. 54. Manning CM, Schutze H (1999) Foundations of Statistical Natural Language
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https://openalex.org/W1982691950
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https://zenodo.org/records/2270450/files/article.pdf
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Italian
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Sugli spazii che ammettono un gruppo continuo di movimenti
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Annali di matematica pura ed applicata
| 1,903
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public-domain
| 17,221
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(*) Memorie della Soeiet~ Italiana delle Seienze, 1897: Sugli syazii a trc dbnznsioni,
che ammettonQ, ece. Ques~a Memoria sara in seguito indieata con (A). Avverto pure ehe
la elassiea Theorie der Trans/ormationsgruppen del LIF~-ENGI,:L sar£ indicata in seguito
soltanto col home di Lm. Sugli spazii che
un gruppo
continuo
ammettono
di movimenti. (Di GUIDO FuB~NJ, ~t Pisa.) (2) Come seppi, dope che questo lavoro era gi£ terminate, in una Memoria, che io
non conoscevo, del BtscoNcI•I, inserita nel Nuovo Cimento (Aprile 1901) : Suuna classifi-
cazione dei proble~ni dinamiei, quesLo teorema era gi£ state enunciate: perb la dimostra-
zione del BIS0ONClNI in reMt'£ vorrebbe invece prorate che(< ogni gruppo continue con p
~rasformazioni infiniLesime [ndipendenLi si pub ridurre a un gruppo su¢ variabili>> ed
percib completamente sbagliata (cfr. LIE; vo]. I, § 109), perch6 questa proposizione non
vera in generale. Le mie co:~siderazioai rendono rigorosi gli altri bei risultati del BI-
SCONCINI, MEMORIA I. Questo lavoro e un altro che mi riservo di pubblicare fra breve trat. tano della teoria generale degli spazii che ammettono un gruppo continuo di
movimenti, delle proprietb, di questi gruppi, dei loro sottogruppi finiti diseon-
tinui, e della determinazione degli spazii a quattro dimensioni che ammettono
un tale gruppo di movimenti. I metodi di cut il prof. BiA~cm si servt (*) per
determinare tutti gli spazii a tre dimensioni con un gruppo continuo di mo-
vimenti non illuminano abbasianza sul problema generale: uno dei risultati
del presente lavoro b appunto quelIo di dare un metodo generale per risol-
vere con sole quadrature il problema, e che, applieato al caso particolare di
spazii a tre dimensioni, permetterebbe di trovare rapidamente i risultati del
prof. Bix~cm. Ma nb il metodo generale, nb il metodo del prof. Btx~cm
generalizzato possono pot condurre senza una interminabile serie di ealeoli
all'effettiva determinazione di tali spazii quando il numero delle loro dimen-
sioni ~ maggiore di tre: la Memoria seguente svolgerh per il easo di quattro
dimensioni un metodo assai pih rapido, e eomodo. p
p
Lo studio infine dei sottogruppi finiti diseontinui di movimenti ammessi
da tall spazii condurr~, fra l'altro, a notevoli rappresentazioni degli spazii
dei tipi (Viii) e (IX) del prof. BiA:~cm sulla sfera e sulla pseudosfera. 4O Fubini:
Sugli spazii cite ammettono Un altro risultafo del presente lavoro che spore possa presentare qual-
che interess% ~ di dare le condizioni necessarie e sufficienti affinch~ un gruppo
si possa considerate come gruppo di movimenti: condizioni~ da cui si pub
trarre qualche proprietk degna di nora per taluni di tali gruppi. Mi sia permesso infine di ringraziare qui vivamente l'illustre prof. LEvi-
CIWTA, che acconsentl a leggere questo lavoro, prima che fosse pubblicato. § 1. Noi vogliamo trovare anzitutto dei teoremi generali relativi alla
ricerca degli spazii che ammettono un gruppo continue di movimenti: prin-
ciple della nostra ricerca sar~ di distinguere il problema in due parti: ]a
prima relativa alla rieerca dei gruppi, che si possono considerare come gruppi
di movimenti di uno spazi% la seconda re]ativa alla determinazione di uno
spazio, di cui sia note il gruppo corrispondente. MEMORIA I. Il teorema fondamentale della prima parte delle nostre ricerche ~ il se-
guente : Se un gru~vpo Gm trasforma in s~ uno spazio Sn~ e sele variet~ mi-
time invarianti sono delle V,,-t,, ognuna di queste Vn-~ ~ pure trasformata
i~ s~ da un gruppo proprio ad m parametri, eil gruppo G., si pub, con un
opportune cangiamento di variabili, ridurre a un gruppo transitive sun -- k
variabili (*). ,'t)
Per dimostrare il nostro teorema, prendiamo una varietk V,,_~ composta di
Vn-~ invariantij tale cio~ che per ogni punto di V,~L1 passi almeno una Vn-h
invariante contenuta nella it.,
--,,-1 e scegliamo come coordinata (n
k+ 1) esima di
un punto A di S~ la distanza geodetica ynJ,+, dal punto stesso alla --.-iv(~) in
discorso. Le y~_~÷, = cost. sono delle varieth. Vn-, geodeticamente parallele
alla zr,~)
invarianti, ossia composte di variet~
--,,-1 e quindi sono come la ~'(~)
~'~t -- 1
minime invarianti. Immaginiamo era nella _V('._ ~ scelta una .Tr(~),,_~ composta pure
di V,~-k invarianti e consideriamo ]a varieth V (~,,,-1 generata dalle geodetiche dello
Tr~)
nei punti di V (~'
Le trasfcrmazioni di G.,
spazio ambiente nermali a v n_ 1
n-2. TT(D ,
pure
mutando la V ('),~-1 e ]a V `~',~-2 in s~ stesse, e le geodetiche normali a .~_. un gruppo continuo di movlmenti. 41 lr(~)
~ ben eerto che]a V (~
~ una variet~ in-
in geodetiche normali a -n-l,
,,-1
variant% ossia che una V~_~ invariante ehe passa per un suo punto giace
tutta in eqsa. La V~,)_~ dovr~ quindi tagliare ogni variet~ y~-k+,---~ coat. pure
in variet~ iuvarianti. Posto questo, osserviamo che per determinare la posi-
zione di un punto di S~, baster~ conoscerne la proiezione geodetica Ao) su
W(1) ,,_, e la coordinata y~_~+,. E not siamo cosl ricondotti per determinare un sistema di coordinate in
S~, a cercare un sistema di coordinate per ]a VI~)_~, la quale varie~ ~, come
lo spazio ambient% trasformata in s~ dal gruppo G,~. E noi percib opere-
remo sulla V~'~)_I, come abbiamo operato in S~. MEMORIA I. Not determineremo cio~ la
posizione di un punto Ao) di p'.-1Tr(') nel seguente modo: Prenderemo una va-
T/C1)
riet~t --._~lz(~, tutta contenuta in --,,_~ e composta di variet~ minime invarianti
e individueremo un punto A(0 di V~L1 dando la sua distanza geodetiea
y,--k+~ da]la V(,~L~ (cio~ ]a distanza misurata qulla geodetica di V~L, tirata
dal punto in discorso normalmente alla V (~) ~
.-2j e la posizione della qua pro-
iezione A(~) sul]a VI~L2 stessa. E
come coordinata (n-- k + 2) esima di un
punto quatunque A diS, prenderemo appunto ]a y,,_~+~ test~ definit% reIa-
tiva alla qua proiezione Ao) sulla V~L~. Or% per un ragionamento gik usato,
ben chiaro the ]e y,-k+~ = coat. sono nella W(~)~ 1 variet~t invarianti, perch~
geodeticamente parallele alla VC~)_2 entro TT~,
--~,_,; e per quanto abbiamo no-
tato saranno pure invarianti quelle variet~ di S, generate dalle geodetiche
di S, normali a V(,~L1 net punti di una di queste variet~ y,,-.~+2 = cost, ossia
anehe in tutto lo spazio ambienle S. le y,-a+~ ----- cost. sono variet~ (a n--1
dimensioni) invarianti. Ci siamo dunque ridotti a determinare la posizione di un punto A(~) di
V'~)
E anche qui, procedendo con lo stesso metodo, prenderemo una --.-a,
n -
2
•
TT~I~
~,on~enuta nella V~I>o, e invariante; e definiremo la posizione di un punto
Ao) di V(+~>o dandone la distanza geodetica yn-~+~ (cio~ misurata lungo una
geodetica di v-<, o normale alla r/-(,) ~ dalla v'c,
~,_o
-,~_~
-~_~ stessa e la posizione della sua
proiezione A(~) sulla V<,',)_a • E definiremo pot come coordinata (n -- k + 3) e~im~
di un punto qualunque A di S~ la y,~-~+s, test~ definit% relativa alla pro-
iezione A(~) su V<~'2_~. della proiezione Ao) del punto A sulla V¢+~)_~. Per una
osservazione gi~ usat% le y~_n+~ ~ cost. (che nella --n-2TT(I) sono variet~ inva-
rianti, perch~ geodeticamente parallele alla V(~l~)__s) sono varietY, pure inva-
rianti in T~-~)
--n-~ e quindi anche nello spazio ambiente S~. Cosl si prosegue fino ad arrivare a una VI~)._7~+~ invariante, in cut la
posizione di un suo punto qualunque A(~-') si definisee dandone la dis~anza Annali di Matematica, Serie III, tomo VIII. 6 Fu b in i: Sugli spazii che ammettono 42 TT(1)
Tr(1)
invariante della v,-~+l e la posizione della sua
geodetica y, da una ~,,-k
proiezione A(~) sulla V',1/_1~ stessa. MEMORIA I. E si definisce come coordinata n e~i'~ di un
punto A di S~ lay,,, testb definita, relativa a quel punto A(g-o di VC,~2x.+~,
a eui si perviene mediante le successive proiezioni considerate. Nella Tz¢~,
rn--k
si assume poi un sistema qualsiasi di coordinate y,, y~,.., y,-k; e per le
prime n -- k coordinate di un punto A di S, si scelgono appunto le y,... y,-~
relative al corrispondente punto A ¢~) di V~,IL~ a cui si arriva mediante suc-
cessive proiezioni. p
Le osservazioni da noi fatte ci dicono senz'altro che le y,-~+i = cost. (i ~ 1, 2,..., k) sono in S, varieth invarianti, e che quindi le yn-~+J ----- COSt. ~... ~ yn ~
cost. sono precisamente le varieth minime invarianti. sono precisamente le varieth minime invarianti. Quale aspetto assume ora il nostro gruppo con questo sistema di varia. bill? Intanto, siccome le y,_h+i--cosf, sono, come si diss% varietK inva-
rianti ~ certo cheit nostro gruppo non trasforma le y~-k~, y,-k~.~7-.. Y-
ossia che se le trasformazioni di Gm sono date dalle: y't ~- ft (y, .. • yn-k y.-h+,
• • • y.)
(t =
1, 2,.... n) (*) y't ~- ft (y, .. • yn-k y.-h+,
• • • y.)
(t =
1, 2,.... n) (*)
(dove left conterranno m parametri arbitrarii, sar~ certamente: y't ~- ft (y, .. • yn-k y.-h+,
• • • y.)
(t =
1, 2,.... n) (*
(dove left conterranno m parametri arbitrarii, sar~ certamente: (dove left conterranno m parametri arbitrarii, sar~ certamente: f,-k+i= y,-l,+i
(i = 1, 2,... k). Ma noi diciamo di pih che le f,...f,-~
non contengono y,-I,+t.., y,
e dipendono ciob soltanto (oltre ehe dai parametri del gruppo) dalle variabili
y,.. • y,-~. In altre parole: noi dimostreremo che le prime ,, n--/c ,, coordinate
y'~...y',k di quel punto Ao, dove un punto A di coordinate y,...y,-k
y,_~,+,.., y, ~ condotto da una trasformazione F qualsiasi di G,~ non di-
pendono dai valori di y,,-h~-,.., y,~ ossia cher
conduce il punto che ha
per prime coordinate proprio y,...y,~_h~ e ehe ha tutte le altre coordinate
uguali a zero, nel punto, le cui prime coordinate sono y'~...y',-k e di cui
tutte le altre coordinate sono nulle. (*) Dove indichiamo con Y't i valori trasformati delle yt. (*) Ricordiamo perb esplicitamente (a scanso di ogni equivoco) che, come faremo ve-
dere piO tarJi~ queste condizioni sono semi)re soddisfatte da un tale gruppo. MEMORIA I. Posto questo, not ora procederemo a dare a]cune proprieth di
un gruppo, che sono condizioni necessarie e sufi3cienti, affinch~ esso si possa
considerare come un gruppo di movimenti. E comineieremo anzitutto dalla
considerazione dei gruppi a trasformazioni infinitesime linearmente indipen-
denti; e rammenteremo che il Prof. BIA:NcltI dimostrb (e pih sotto io ne darb
una dimostrazione intuitiva, chg ne spiega l' intima ragione di essere) ehe
ogni gruppo G~ transitivo su n variabili g ammesso da qualehc spazio a n
dimensioni; not dimostreremo anzi che eib g veto per qualsiasi gruppo a n
variabili con trasformazioni infinitesime linearmente indipendenti. Not per
ora, a proposito di questi gruppi, non possiamo che dare il teorema seguente:
Condizione necessaria e sufficiente affinch~ un gruppo Gm a trasforma-
zioni i~finitesime linearmente indipendenti sun + m variabili si possa con-
siderare come gruppo di movimenti di uno spazio ad n + m dimensioni ~ ehe
il gruppo sia simile a un gru?po semplicemente transitivo sum lettere ; cib
chesi pub anche esprimere dicendo che Gm'deve essere un sottogruppo di un
gruppo semplicemente transitivo con n + m lettere ed n + m parametri (*). La condizione g evidentemente necessaria; infatti il gruppo Gm ha come
variet~ minime invarianti delle V,~: e~ se S~+,~ g uno spazio the ammette
G~, si vede tosto prendendo in esso quel sistema di coordinate, the fu deft-
nito al paragrafo precedente che il gruppo Gm sar~ trasformato in un gruppo
simile ehe opera transitivamente sum lettere. Che questa condizione sia anche
sufficiente, si dimostra facilmente cos): Se G~ g simile a un gruppo the opera
transitivamente sum variabili y, y~... y,~, si introducano come coordinate
appunto le y, y~... ym insieme ad altre n funzioni qualsiasi delle coordinate
iniziali ym+,...ym+~ tali the y,...ym ym+,...y,~,~ siano indipendenti tra § 2. Posto questo, not ora procederemo a dare a]cune proprieth di
un gruppo, che sono condizioni necessarie e sufi3cienti, affinch~ esso si possa
considerare come un gruppo di movimenti. E comineieremo anzitutto dalla
considerazione dei gruppi a trasformazioni infinitesime linearmente indipen-
denti; e rammenteremo che il Prof. BIA:NcltI dimostrb (e pih sotto io ne darb
una dimostrazione intuitiva, chg ne spiega l' intima ragione di essere) ehe
ogni gruppo G~ transitivo su n variabili g ammesso da qualehc spazio a n
dimensioni; not dimostreremo anzi che eib g veto per qualsiasi gruppo a n
variabili con trasformazioni infinitesime linearmente indipendenti. A (~-~) sulla V(~L~, e se una trasformazione F di G,, conduce i punti A ed
A (~) rispettivamente in B e in B', il punto B' coincide con quel punto B(h)
di V~)~ chesi dedurrebbe da B con le accennate successive proiezioni. E
v~,) sar~
infatti, essendo -,,_,11(') invariante, la geodetiea per A normale a --,~-1
17-(1)
portata nella geodetica per B normale a--n-1 e quindi anche A(') sari~
V(t)
portato nella proiezione B(')di B su rn_l;
C0SI A (~) sarg portato nella
T/-(t)
C0Si eontinuando arriveremo alla di-
proiezione B(2) di B(') su --,~-2 ece.; e
mostrazione dell'asserto, cio~ che F conduce A (k) in B(k) e che quindi i punti
B', B(~) coincidono. MEMORIA I. O in altre parole io dimostrerb chese Ao) ~ la proiezione di un punto A
di S, sulla g ('
A(O la proiezione di .4(0 sulla --,,-2TT(~), ..., A (k) quella di
n--1 un gruppo continuo di movimenti. 43 A (~-~) sulla V(~L~, e se una trasformazione F di G,, conduce i punti A ed
A (~) rispettivamente in B e in B', il punto B' coincide con quel punto B(h)
di V~)~ chesi dedurrebbe da B con le accennate successive proiezioni. E
v~,) sar~
infatti, essendo -,,_,11(') invariante, la geodetiea per A normale a --,~-1
17-(1)
portata nella geodetica per B normale a--n-1 e quindi anche A(') sari~
V(t)
portato nella proiezione B(')di B su rn_l;
C0SI A (~) sarg portato nella
T/-(t)
C0Si eontinuando arriveremo alla di-
proiezione B(2) di B(') su --,~-2 ece.; e
mostrazione dell'asserto, cio~ che F conduce A (k) in B(k) e che quindi i punti
B', B(~) coincidono. A (~-~) sulla V(~L~, e se una trasformazione F di G,, conduce i punti A ed
A (~) rispettivamente in B e in B', il punto B' coincide con quel punto B(h)
di V~)~ chesi dedurrebbe da B con le accennate successive proiezioni. E
v~,) sar~
infatti, essendo -,,_,11(') invariante, la geodetiea per A normale a --,~-1
17-(1)
portata nella geodetica per B normale a--n-1 e quindi anche A(') sari~
V(t)
portato nella proiezione B(')di B su rn_l;
C0SI A (~) sarg portato nella
T/-(t)
C0Si eontinuando arriveremo alla di-
proiezione B(2) di B(') su --,~-2 ece.; e
mostrazione dell'asserto, cio~ che F conduce A (k) in B(k) e che quindi i punti
B', B(~) coincidono. A (~-~) sulla V(~L~, e se una trasformazione F di G,, conduce i punti A ed
A (~) rispettivamente in B e in B', il punto B' coincide con quel punto B(h)
di V~)~ chesi dedurrebbe da B con le accennate successive proiezioni. E
v~,) sar~
infatti, essendo -,,_,11(') invariante, la geodetiea per A normale a --,~-1
17-(1)
portata nella geodetica per B normale a--n-1 e quindi anche A(') sari~
V(t)
portato nella proiezione B(')di B su rn_l;
C0SI A (~) sarg portato nella
T/-(t)
C0Si eontinuando arriveremo alla di-
proiezione B(2) di B(') su --,~-2 ece.; e
mostrazione dell'asserto, cio~ che F conduce A (k) in B(k) e che quindi i punti
B', B(~) coincidono. ,
( )
§ 2. MEMORIA I. Not per
ora, a proposito di questi gruppi, non possiamo che dare il teorema seguente: Condizione necessaria e sufficiente affinch~ un gruppo Gm a trasforma-
zioni i~finitesime linearmente indipendenti sun + m variabili si possa con-
siderare come gruppo di movimenti di uno spazio ad n + m dimensioni ~ ehe
il gruppo sia simile a un gru?po semplicemente transitivo sum lettere ; cib
chesi pub anche esprimere dicendo che Gm'deve essere un sottogruppo di un
gruppo semplicemente transitivo con n + m lettere ed n + m parametri (*). La condizione g evidentemente necessaria; infatti il gruppo Gm ha come
variet~ minime invarianti delle V,~: e~ se S~+,~ g uno spazio the ammette
G~, si vede tosto prendendo in esso quel sistema di coordinate, the fu deft-
nito al paragrafo precedente che il gruppo Gm sar~ trasformato in un gruppo
simile ehe opera transitivamente sum lettere. Che questa condizione sia anche
sufficiente, si dimostra facilmente cos): Se G~ g simile a un gruppo the opera
transitivamente sum variabili y, y~... y,~, si introducano come coordinate
appunto le y, y~... ym insieme ad altre n funzioni qualsiasi delle coordinate
iniziali ym+,...ym+~ tali the y,...ym ym+,...y,~,~ siano indipendenti tra Fubini: Sugli spazii che ammettono 44 loro. Le
trasforma.zioni infinitesime di
Gm insieme alle trasformazioni
genereranno un gruppo Fro+,,, semplicemente transitiv%
y,~+t ' " ' " ' ~ y.~+~
e di cui Gm ~ un sottogruppo. Per il citato teorema del prof. BiA~cm F,,~.,
ammesso da qualche S,,+~, che ammetter~ percib anche G~. q
p
Nella seconda parte di questa dimostrazione abbiamo visto incidental-
mente che se un G,~ ~ simile a un gruppo transitivo surn lettere, esso
anehe sottogruppo di un F,,~ transitivo in S,,+,,. ¥ieeversa se G~ ~ sotto-
gruppo di un F,,+, transitivo in S,,+~, esso sar~ certo un gruppo di movimento
di ciaseuno degli spazii, che ammettono F,,+,~ e quindi, per quanto abbiamo
dimostrato, sarh simile a un gruppo semplicemente transitivo sum lettere. Noi pia tardi dimostreremo che ogni gruppo a trasformazioni infinite-
sime linearmente indipendenti pub essere considerato come gruppo di movi-
menti: il presente teorema ci dar~ pcrcib atcune interessanti proprietY, ge-
nerali di gruppi siffatti; cib che permette di .~gevolarne di molto ]a ricerca. § 3. MEMORIA I. Abbiamo cosl studiato i gruppi a trasformazioni infinitesime li-
nearmente indipendenti, che posstmo essere considerati (tutti)come gruppi di
movimenti e abbiamo visto che in fin dei conti essi si riducono tutti ai gruppi
transitivi, in cui it numero dei parametri eguaglia quello de]le variabili. Ora
ci proporremo l~ questione di riconoscere quando un gruppo a trasformazioni
infinitesime linearmente dipendenti pub essere considerato come gruppo di
movimenti. Noi ora daremo una prima risoluzione di ql, esto problema, av-
vertendo perb ehe noi vi ritorneremo pih oltre, con altri metodi, e ottenendo
una risoluzione pih elegante ed intuitiva. p
g
Anzitutto osserveremo che per il teorema fondamentale del § 1, seil
nostro gruppo ~ un gruppo G~+~ a ,, n + m ,, parametri con sole n trasfor-
mazioni infinitesime linearmente indipendenti, esso (avendo delle V~ per va-
riet~ minime invarianti) sarh certo simile a un gruppo transitivo su n varia-
bill, e si potr~ considerare come gruppo di movimenti di una varietY, ad n
dimensioni. Noi supporremo senz'altro che il gruppo sia gi~ stato ridotto
sotto questa forma, che diremo forma normale del gruppo, con un opportuno
cangiamento di variabiIi. Siano dunque Xt=~/)(x,
. x,) ~
(t=l, 2,...n+m)
r:i
"
"
~ Xr le trasformazioni infinitesime del nostro gruppo Gn+m e sia le trasformazioni infinitesime del nostro gruppo Gn+m e sia n
ds'~
~a~dxidx~ un gruppo continuo di movimenti. 45 un gruppo continuo di movimenti. 45 l'elemento lineare di uno spazio S,, che ammetta G,+,~. Le formule di Kir.-
Ia~G danno che '
~" /=0
(l, r=
1, 2,...,
n). (1) (1) E noi le seriverem% ponendo E noi le seriverem% ponendo (l)
v
=:,
av,=: . alr~
+ak~-] (l)
v
=:,
av,=: . alr~
+ak~-] sotto la forma W ~l) O_ ai~ + a~. -~- 0,
ossia
Xz (aik) "q- a']~ = 0. (1)
Ox,. (1) I1 prof. Bia~crxI osservb the in virth delle (1)' si ha the I1 prof. Bia~crxI osservb the in virth delle (1)' si ha the Xl [X, (aik) + -'"'
.ikl--Xt[Sl(aik)+a'[~]
(1, t=l,
2,..., n+m)
(2) Xl [X, (aik) + -'"'
.ikl--Xt[Sl(aik)+a'[~]
(1, t=l,
2,..., n+m)
(2) (2) identicamente nullo: cib ehe del resto ~ intuitivo perch~ se un S,, ammette
due trasformazioni infinitesime Xt, Xt esso deve ammettere anehe la (Xt, X,). Per ipotesi m delle trasformazioni infinitesime del gruppo sono combinazione
lineare delle altre n, le quali sono poi linearmente indipendenti. MEMORIA I. :Noi ammet-
teremo cite queste ultime sieno le X,~ X,,..., X,~ e porrem% indieando con
?~il delle funzioni di x,, x,,.., zn st
X,,+i = ~=' ~,I" X,
(i = 1, 2,... m). E il sistema delle equazioni di Klr,r,i~(~ sarh da noi scritto nella forma: Xl (aik) + -i~-(" --~- 0
(l = 1, 2,... n)
(3)
"'+"-
=
2,... m). (4)
Xn+t (aik) -- Z ~i ° Xl (aik) 2r- am
,*ik =
1=1 (3) (4) Cib ~ chiaramente lecito perch~ se alla (4) aggiungiamo Cib ~ chiaramente lecito perch~ se alla (4) aggiungiamo st
~,b 1 rc, ft)
,,-fill st
~,b 1 rc, ft)
,,-fill somm~ evidentemente nulla in causa delle (3), otieniamo appunto l'equazione
di Kit, r~xs(~ relativa a X,,+~ ed a ai~, coma 5 scritta nella (1)', E ora poich~
identicamente somm~ evidentemente nulla in causa delle (3), otieniamo appunto l'equazione
di Kit, r~xs(~ relativa a X,,+~ ed a ai~, coma 5 scritta nella (1)', E ora poich~
identicamente tl
-- "~ "' Xt (aik)
0
x.+, (..O
= Fubini: Sugli spazii che ammettono 46 le (4) diventano le (4) diventano
ossia
ossia
n
*'dn+t)
l~=l
a (l)
0
(4)'
r:l
~ Xk
r7>
+
i"
(4)"
^ "^~*)\
n)
,~,. ai~ ~a, ~ ~7 ~
~}, 6 ~, |
(t, i, k, r ~- 1, 2,... ,,.~+ah,.. ~-)=0
(t=l,
2,... m). (4)'" (4)' ossia r:l
~ Xk
r7>
+
i"
(4)" (4)" ossia ^ "^~*)\
n)
,~,. ai~ ~a, ~ ~7 ~
~}, 6 ~, |
(t, i, k, r ~- 1, 2,... ,,.~+ah,.. ~-)=0
(t=l,
2,... m). (4)'" (4)'" Il nostro sistema di equazioni resta cosl mutato nel sistema di equazloni
formato dalle (3) 0 dalle (4)"'. Ora dalle (3), essendo il determinante delle
~, (r, 1
1, 2,... n) differente da zero si possono trarre le derivate delle
aih in funzione delle alk stesse; e, per l'osservazione tests citata del pro-
fessore Bxx~cm si vede ehe affineh~ il sistema delle" (3)~ (4)'" sia integrabile
basta ehe 1. ° Le (4)"' considerate come equazioni lineari a]gebriche per le aik
ammettono in un punto almeno una soluzione (che ci dar~ i valori iniziali
delle aln). Si dovrb~ poi solo esaminare (volendoci restringere a spazii reali)
se le solite disuguaglianze sono soddisfatte. g
g
2. MEMORIA I. ° I primi membri delle (4)'" derivate rispetto a x,, x~,.., x,~ suc-
cessivamente dieno per le (3) e le (4)"' stesse risultati identicamente nulli. Questa seconda condizione equivale anehe (essendo il determinante delle ~
diverso da zero) alt'altra eondizione ehe applicando ai primi membri de]!e (4)"'
le X,... Xn si ottenga in virth delle (3) e delle (4)'" stesse un risultato iden-
ticamente nullo. Noi ora vogliamo far vedere che questa seeonda eondizione h sempre
soddisfatta. Infatti per l'osservazione del prof. Bra~rcm ~ sempre per le (3) e le (4)"
identieamente : _ Xn t [Xz(aik) + alk j ~
0
It=l,
2,... m] [i, k, l=l,
2,... n] ossia ossia Xt [X,,,t (aih)] -- X~+, [Xl (ai~)] + Xz (a~.~ +°) -- -v-
/'ae)
xxn+~ k iki ----- O. (5) (5) Xt [X,,,t (aih)] -- X~+, [Xl (ai~)] + Xz (a~.~ +°) -- -v-
/'ae)
xxn+~ k iki ----- O. (5) un gruppo continuo di mov6nenti. 47 Si ha pure identieamente: Si ha pure identieamente: p
Xz [X~ (a~,)] -- X,. [Xz (.i~,)] + X, [ai'/.] -- X~ r..,,~ = 0
Lt~ik]
(I~ r~ i~ k~---1~ 2~...~ n)
a ]
(I~ r~ i~ k~---1~ 2~...~ n) ossia • It)
--
Ct)
fr)
~,. Xz [x~ (a;k)]
~, x,, [xz (a~)] + ¢' X~ (a,~) -- ~' X~ (a'f~) = o
ossia : • It)
--
Ct)
fr)
~,. Xz [x~ (a;k)]
~, x,, [xz (a~)] + ¢' X~ (a,~) -- ~' X~ (a'f~) = o
ossia : • It)
--
Ct)
fr)
~,. Xz [x~ (a;k)]
~, x,, [xz (a~)] + ¢' X~ (a,~) -- ~' X~ (a'f~) = o
ossia : Xz [~o;' X¢ (ai~)]
XI (77)) X~ (aik) -- _(t,
•
-
v,. X~ [Xt (a~k)] +
'~'
~7' X~ (a~i) -- o. E, poich~ per le (3) sarh pure identicamente (in. virth delle (3) e delle (4)"') sarh pure identicamente (in. virth delle (3) e delle (4)"') Xz ~ "
X~ [Xt (ai~)] + Xl "
(''
--
.~G r~,,,~
0
t?,. X. (aik)]
97'
"'
--
t~ a',7]
~,. w,kJ =
e quindi anehe identicamente: [Xz ~'~
X, [X~ (a,k)] + Xz r,,,w,,~
/
L~,' u,:k] -- ~(~)X,, (aTe)
0.. (6)
r=l [Xz ~'~
X, [X~ (a,k)] + Xz r,,,w,,~
/
L~,' u,:k] -- ~(~)X,, (aTe)
0.. (6)
r=l (6) Sottraendo dalla (5) la (6) troviamo l'identith: Xl Xn+t(a/k)--
~rXr(a/k)
-- Xn+t-- ~ ~(t,,~ X~ Xl(aik) -~-
r=l
r-=l
r=l
r=l E poieh~ ~ identicamente: E poieh~ ~ identicamente: r=l troveremo senz'altro identieamente: /
tt
\
\
r----I
/ che ~ appunto quanto si voleva dimostrare. (Si ricordi che i primi membri
delle (4)' e delle (4)"' sono identici.) Prima ora di venire a parlare della 1) eondizlone vogliamo fare un'os-
servazione generale. 48 Fubini:
Sugli spazii che ammettono Se un gruppo F qualsiasi si pub considerare come gruppo di movimenti
di uno spazio a n dimensioni, esso pub essere anche considerato come gruppo
di movimenti di uno qualche spazio a un numero n + t di dimensioni (t in-
tero qualsiasi). ossia q
Supposto infaifi il gruppo ridotto a forma normale in n o rneno varia-
bili~ e se d s 2 ~- ~ aik d x~ d ~k
(i, k = 1, 2,... n)
i,k l'elemento di uno spazlo a n dimensioni che ammette il nostro gruppo, al-
lora~ come tosto si verifiea l'elemento di uno spazlo a n dimensioni che ammette il nostro gruppo, al-
lora~ come tosto si verifiea ds'---- ~aikdxid
x~ + ~almdxzdx,n
i,k
Im
(i, k-----l, 2,... n) (/, m-----n+ 1,... n+t) x~ + ~almdxzdx,n
Im
(i, k-----l, 2,... n) (/, m-----n+ 1,... n+t) dove le al~, non dipendono dalle x,, x2,.., x~ ~ l'elemento lineare di uno
spazio a n + t dimensioni che ammette il nostro gruppo. Dunq ue : Affinch$ un dato gruppo, gih ridotto a forma normale in n variabili,
possa essere ammesso come gruppo di movimenti da uno spazio (che natu-
ralmente non potrh avere meno di n dimensioni) D condizione necessaria e
sufficiente che le (4)'" considerate come equazioni lineari algebriche helle aik
sieno in un punto risolubili rispetto alle ail,. Dunque questa condizione si
esprime semplicemente con l'annullarsi di una matrice. Ora osserviamo il prim() membro delle (4)"' e vediamo fosto ehe il ter-
~(l, ~ 77 ~
-
~ un termine de'l determinante che si ottiene moltiplicando
mine
ai~ ~,. ~ xk
con le note regole, in modo opportuno i] determinante delle aik per il deter-
minante delle ~(l'e per l'Iaeobiano delle ~(~). Quindi abbiamo: Condizione necessaria e sulficiente affinchD il nostro gruppo gi& ridotto
a forma normale possa essere considerato come gruppo di movimenti ~ che
si possa trovare un determinante non nullo simmetrico [ ai~ [, il prodotto del
quale per il determinante [ ~'~'[
, l = 1 7 2,... n) e per l'Iacobiano delle
• ,~,!) (r = 1, 2,... n) ottenuto in modo opportuno con le note regole sia, almeno
in un punto, semisimmetrico. E cib per ciascun valore particolare di t. in un punto, semisimmetrico. E cib per ciascun valore particolare di t. Se ora n ~ dispari, il determinante semisimmetrieo cosi ottenuto ~ hullo;
• • •
. *
¢~ (t)
='=
0 (x, x, . . . x,)
non sono funzioni indipendenti. Se ora n ~ dispari, il determinante semisimmetrieo cosi ottenuto ~ hullo;
• • •
. *
¢~ (t)
='=
0 (x, x, . . . Annag eli Matematica~ Serle III, tomo VIII. ossia x,)
non sono funzioni indipendenti. un gruppo continuo di movimenti. 49 Quindi in particolare:
l
h Quindi in particolare: Se un S~ ammette un G,,+, transitivo, esiste certamente nel caso che n
sia dispari, qualche trasformazione infinitesima permutabile con G~+,. p
q
f
f
p
§ 4. Sia ora dato un gruppo, gib. ridotto sotto forma normale, cio~
con tante trasformazioni infinitesime linearmente indipendenti quante sono le
variabili su cui opera il gruppo. Noi vogliamo esaminare come si semplifi-
cano in questo caso le equazioni di KxLLm(~. Siano '~
~
(/-----1~ 2,..., n--~-m)
r=l le trasformazioni infinitesime del gruppo e sia le trasformazioni infinitesime del gruppo e sia ds~--~ai~dxidx~
(i, k~l,
2,..., n-I-t) l'elemento lineare di uno spazio a , n-f-t ,, dimensioni che ammetta il no-
stro gruppo. l'elemento lineare di uno spazio a , n-f-t ,, dimensioni che ammetta il no-
stro gruppo. g
pp
Se noi seriviamo le equazioni di KmLxN(~ noi riconoseiamo faCilmente: 1. ° :Per le ai~ (i, k ~ 1, 2 7... n) queste equazioni assumono proprio
la stessa forma, come se noi volessimo cereare gli spazii ad n dimensioni
ammessi dal gruppo. g
pp
2. o Per le alk(i, k~---n-{-1, n+2~...,
n~t)
le equazioifi di KIn-
LInG diventano semplicemente: ~
~ aih ~
O. .'~1 E poich~ delle Xz proprio n sono linearmente indipendenti, si ha sem-
plicemente : aih --0
(r= 1, 2,..., n)
~Xr e le equazioni di KILLISe dicono soltanto che le aik in discorso sono funzioni
soltanto di Xn~,,..., X~+t. e le equazioni di KILLISe dicono soltanto che le aik in discorso sono funzioni
soltanto di Xn~,,..., X~+t. 3. ° Per le aik (i ~ n, k > n) le equazioni di KILHSa diventano: ~ -- 0
~,
O x~
r the ~ un sistema di equazioni ehe non eontiene nessuna aik del primo o del
secondo tipo. Anzi questo sistema di equazioni si seinde per eiaseun valore
di k in t sistemi di equazioni distinti, l'uno relativo alle a6~+,, il secondo
alle aim+~ eee. (i~ 17 2,..., n). Annag eli Matematica~ Serle III, tomo VIII. 7 50 Fubi~i: Sugli spazii che ammeltono Le equazioni relative alle ai~ (i, k ~ n) si semplificano, ossia si possono
ridurre a un nuniero minore di incognite mediante le equazioni lineari (4)"'~
mentre hello stesso tempo per ciaseuna delle aik da determinarsi si ottengono
soltanto ~t equazioni. ossia Queste osservazioni riescono utili specialmente per il
caleolo effettivo degli elementi lineari da determinarsi. § 5. Ora not vogliamo dimostrar% servendoci dei risultati finora ot-
tenuti~ il seguente teorema fondamentale : Con la sola risoluzione di effuazioni algebriche si possono determi~a~'e
tutti i gruppi 7 che si posso~o co~siderare, come 9ruppi di movimenti, tro-
vando le loro tras[ormazioni infinitesime; con sole quadrature si posso~o
determinate Ie trasformazioni finite di tall gruppi~ e gli elemeuti lineari
degli spazii loro corrispondenti. g
p
p
La dimostrazione di questo teorema ~ semplicissima: basra a tale scopo
la semptiee ossei'vazione che ogni gruppo eontinu% ehe si possa eonsiderare
come gruppo di movimenti~ si pub, con un cangiamento di variabili ridmore
sotto forma normale, cio~ si pub far si che possegga tante trasformazioni in-
finitesime linearmente indipendenti quante sono le variabili su cut opera ef-
fettivamente: in aitre parole si pub ridurre a essere transitivo sulle variabili
ehe esso effettivamente trasforma. Ora not sappiamo che con la risoluzione
di sole equazioni algebriche si possono trovare tutte 18 possibili composizioni
realmente distinte dei gruppi a un numero qualsiasi di parametri. Di pih
(LIE, Kap. 27, 29) con la sola risoluzione di equazioni algebriehe si possono
trovare tutti i gruppi eontinui transitivi, appena se ne eonosea la eomposi-
zione, e non si considerino distinti due gruppi simili. E eosi dimostrata sen-
z'altro la prima parte del nostro teorema. Esaminando la forma delle tra-
sformazioni infinitesime dei nostri gruppi si potrebbe dimostrare anehe la terza
parte del nostro teorema; mail proeedimento pifi breve a tale seopo g il
seguente: Si rieordi anzitutto ehe con sole quadrature si possono trovare le
equazioni finite del nostro gruppo, perehg il gruppo ~ transitivo (LI~ 1. c.),
cib ehe intanto dimostra la seeonda parte del nostro teorema. Atlora distin-
guiamo due easi: seeondoehe le dimensioni dello spazio da determinarsi sono
in numero uguale o maggiore del numero detle -¢ariabili, su eui il nostro
gruppo, ehe col proeedimento preeedente g stato evidentemente ottenuto sotto
forma normale, opera effettivamente. Comineiamo a trattare il primo easo:
sia eiog uguale il numero n delle dimensioni dello spazio da determinarsi
e delle variabili su eui opera (transitivamente) il gruppo. Sia A un punto
generieo dello spazio e siano a(,°h valori qualsiasi ehe soddisfano alle equa- un gruppo continuo di movimenti. 5! ossia zioni lineari (4)"' (supposte naturalmente risolubili) (dove alle xi si sostitui-
seano le coordinate (x¢ °') del punto A) e, se v.ogliamo restringerei a spazii
reali, anche alle solite disuguaglianze, se questo b possibile. Sia ora B un
punto qualunque dcllo spazio, in cui si vogliono trovare i valori delle ai~. Siceome il gruppo opera transitivamente nello spazio ambiente esiste almeno
una trasformazione T del gruppo ehe trasporta il punto B nel punto A;
essa porter~ anche un intorno qualunque di B in un intorno di A; di pih
essa si potrb, ottenere con la sola risoluzione di equazioni finite. Sieno x~ 'J le
coordinate di B, e siano al),i valori finora ineogniti delle alh nel punto B. La T, stabilendo una corrispondenza biunivoca tra gli intorni di B o di A,
stabilisee una corrispondenza biunivoea tra i punti x~"+ d x~ '~ e i punti
• y+ d x~ °', corrispondenza determinabile con differenziazioni ; le d x~ °~ ven-
gono date come espressioni lineari omogenee helle d x~ '~, a coefficienti eo-
stanti. Sostituiamo nella ~o,~-'°' d_,~'°'d x~ ) alled x~; i loro valori cosi determi-
nati, otterremo una forma quadratica helle d~ '-')_~ , che deve essere identiea
"~ d x~'; d x~; csprimendo questa identith, si hanno senz'altro i valori
con z a~i,,
delle a~ nel punto qualunque xi" dello spazio ambiente, espressi in funzione
delle costanti iniziali aii?2. Passiamo ora al caso ehe il numero n + m delle dimensioni dello spazio
sia maggiore del numero n delle variabili (x,, x.~,.., x,,), su cui opera effet-
tivamente il gruppo, cosiceh~ nello spazio in diseorso il gruppo non opera
pih transitivamente. E sia ds'---~'a~l,,dxidx~
(i~ k=l,
2,..., n+m)
i,k ds'---~'a~l,,dxidx~
(i~ k=l,
2,..., n+m)
i,k l'elemento lineare dello spazio. Per le a;, (i, k-- n -~- 1,..., n -t- m) nutla vi
sarebbe da dire, poich~ noi sappiamo gi~ che esse sono flmzioni (del resto
arbitrarie) delle x~,, x,,~,...,
x~+,, (§ 4). ossia In ogni modo si osservi che
x
affinche il nostro spazio ammetta effettivamente il gruppo in discorso, ~ con--
dizione necessaria e sut~ciente che una trasformazione T del gruppo che
porta un punto B~x~"
di una variet~ V~ invariante in un altro punto
A ~ x~' della stessa V~ e quindi stabilisce anehe una corrispondenza biuni-
voca ira gli intorni dei due punti~ porti anche la forma ,,,/,,d
i,k
nella forma
(x?') d
d x
. ,,,/,,d
i,k
(x?') d
d x
. ,,,/,,d
i,k ,,,/,,d
i,k
nella forma
(x?') d
d x
. (x?') d
d x
. 52 Fubini: Sugli spazii che ammettono Allora prendiamo una variefft qualsiasi V,~ a m dimensioni ehe abbia
uno e un sol punto eomune con eiaseuna delle variefft V,~ minime invarianti
e fissiamo in ciascun punto della Vm dei valori arbitrarii per le aik aneora
da de~erminarsi, purch~ natmralmente essi soddisfino alle solite equazioni li-
neari: eib ehe eorrisponde all'introduzione di funzioni arbitrarie. Per poi de-
terminate i valori al.}~ delle a~.l~ in un altro punto B qualsiasi di S,~+m basra
trovare quel punto A, in eui la varietb~ invariante V,~ passante per B incontra
la Vm, e in eui le aie rieevono dei valori noti .(o)
~,.~ e applieare quindi ai
punti A, B il precedente proeedimento. II nostro teorema ~ cos). dimostrato
in generale. g
Naturalmente non con ogni speciale seelta delle funzioni e eostanti ar-
bitrarie su deseritte si otterr~ un tipo di spazio realmente distinto: anzi per
le eonsiderazioni generali del § 1, noi possiamo supporre, senza diminuire la
generalith an+~,g ~-- 0
(an+3,~ ~-- O)x,,+,=x.+,~=o
(k~l,
2,..., n, n+2,...,
n+m)
(k =1 = n + 2)
=I=, + 3)
are+t,n+l
=== 1
(an+~,n+3)x.+,=eo.+~=o
~
1 7 eee. an+~,g ~-- 0
(an+3,~ ~-- O)x,,+,=x.+,~=o
(k~l,
2,..., n, n+2,...,
n+m)
(k =1 = n + 2)
=I=, + 3)
are+t,n+l
=== 1
(an+~,n+3)x.+,=eo.+~=o
~
1 7 eee. (an+~,n+3)x.+,=eo.+~=o
~
1 7 eee. dove le uguaglianze scritte in basso fuori delle parentesf indicano su quali
variet~ si possono supporre senz'altro verifieate le equazioni stesse. {} 6. Ora noi ei vogliamo ehiedere qual'~ il significato delle re]azioni
lineari (4)"', a cui i valori iniziali delle air devono sempre soddisfare. A. questa domanda risponde subito la seguente eonsiderazione. Se noi ei rife-
riamo p. es. ossia al primo dei casi test~ trattati e rieordiamo il metodo di cui
noi ei siamo serviti, notiamo tosto the per trovare i valori delle ail, in un
punto B in funzione dei valori delle aik in un punto A, ei siamo serviti di
una trasformazione T ehe eonducesse il punto B nel punto A. 0ra nel caso
di trasformazioni infinitesime linearmente dipendenti osserviamo ehe i punti
A, B non individuano la trasformazione T, ma che anzi di tall trasforma-
zioni ve ne sara, in generale pill di una: eh~ se S ~ la trasformazione pih
generale ehe laseia fisso A, tutte le trasformazioni TS (o S T a seeonda
della notazione ehe si usa) eondueono il punto B nel punto A. E poieh5
le (4)"' ei danno le uniehe eondizioni a eui devono soddisfare i valori ini-
ziali delle aih~ g ben ehiaro ehe il loro signifieato ~ questo, ehe eio~, qual- un gruppo continuo di movimenti. 53 siasi trasformazione TS venga usata, i valori delle ai~ nel punto B siano
sempre quelli the si otterrebbero eonsiderando la T. In altre parole esse si-
gnifieano, che ogni trasformazione S del gruppo che lascia fisso il punto A
deve trasformare in s~ la forma 2 ai~-~°)d #o, d x~;. Del resto risulta senz'altro
ehiaro dal procedimento usato test~ chese questa condizione ~ soddisfatta,
a]lora i valori che si ottengono in un punto B sono perfefitamente determi-
nati, eil gruppo ~ ammesso da uno spazio, il cui elemento lineare ha dei
coefficienti, che nel punto A assumono il valore ~,,~'°'. Possiamo dunque espri-
mere sotto forma pitt elegante e coneisa le condizioni necessarie e suffieienti
amnch~ un gruppo possa essere considerato come gruppo di movimenti. 6bndizione necessaria e sufficiente affinch~ un gruppo G ridotto o no
sotto forma normale si possa conside~:are come gruppo di movimenti ~ che
quel suo sottogruppo F che lascia fisso un pu~to A determinato, in cui il
gruppo sia regolare, sia tale che esista almeno un sistema di quadriche omo-
tetiche in guisa che un punto infinitamente vicino ad A non esca mai per le
trasformazioni di F da quella di queste .quadriche, che passa per ess% o~
in altre parole F trasformi in s~ ciascuna quadrica di un sistema di qua-
driche omotetiche infinitamente vicine ad A (*). (~) Si noti the cos[ immaginiamo il gruppo operante in uno spazio euclideo. ossia Se il gruppo ~ transitivo baster~ chiaramente che questo avvenga in un
solo punto (perch~ allora avviene in tutti); se ~ intransitivo cib dow'~ acca-
dere in un pu~to di ciascuna varieth minima invariante. Questa distinzione
evidentemente superflua, seil gruppo ~ gik r~dotto a forma normale. p
g
pp
g
Se queste condizioni sono soddisfatte, esiste infatti una forma X a~,dx~ °~ dx~ ~,
che F trasibrma in s~. Osservazi(me 12 Se uno di questi cosifatti sistemi di quadriche ~ for-
mato di elissoidi, to spazio corrispondente si pub supporre reale. Osservazione 2. ~ Le condizioni analitiche~ che traducono queste condizioni
geometriche sono date dall'annullarsi di quella matric G che esprime essere
le (4)'" compatibili. § 7. ]~ ben evidente ora ehe i gruppi G~ transitivi sun variabili pos-
sono essere considerati come gruppi di movimenti (teorema del prof. BiA~cm)
perch~ in tal caso il sottogruppo F si riduce all'identitb~; o in altre parole~
compiutamente determinata la trasformazione the porta un punto B in un
punto A; e quindi~ dati arbitrariamente i valori delle a,.~ in A riescono sen-
z'altro, coi nostri metodi~ determinati i valori delle a,7~ nel punto B. Quest'os-
servazione rende evidente a priori il bel teorema del prof. BiAl~Cm. 54 Fubini: Sugli spazii che ammettono Ma questa stessa osservazione si pub senz'altro applicare a ogni gruppo
con trasformazioni infinitesime linearmente indipendenti e ne traggiamo il
teorema : Ogni gruppo a trasformazioni infinitesime linearmenle indipe~denti si
pub co~siderare come gruppo di movimenti. Confi'ontando questo risultato, che ~ immediato corollario del precedente
teorema generale, con quello del § 2 otteniamo il seguente teorema: Ogni gruppo G,~ generato dan trasfovmazioni linearmente indipendenti
su un numero qualsiasi n q-m di variabili ~ simile a u~ gruppo G',~ sem-
piicemente transilivo in uno spazio ctd n dimensioni~ e~ cib ch'~ lo stesso, si
pub pensare sottogruppo di un gruppo transitivo sun-[-m q--k lettere e con
n -~ m + k parametri (dove k ~ un intero qualsiasi nullo o positivo). Net precedenti paragrafi not abbiamo risoluto due questioni: la prima,
di riconoscere se un gruppo contin~to si pub considerate come gruppo di mo-
vimenti, la seconda di indicare un mezzo, col quale si possa determinare con
sole quadrature, differenziazioni e risoluzione di equazioni finite tutti questi
gruppi, e gli spazii che loro corrispondono. g
pp
g
p
p
§ 8. Not aggiungeremo ora una facile osservazlone, che ci condurrb. ossia aun notevole risultato: Nessun gruppo, chesi possa considerare come gruppo di movimenti,
pii~ di una volta transilivo. p
Infatti se G bun tale ~'ruppo ed S lo spazio corrispondente, due punti
di S hanno almeno un invariante: la loro distanza geodetica. Per la stessa ragione: Per la stessa ragione: Per la stessa ragione:
Se un gruppo G, si pub considerate come gruppo di movimenti~ quel sub Se un gruppo G, si pub considerate come gruppo di movimenti~ quel sub
soltogruppo che lascia fisso u~ punto generico ~ certo intransitivo. f
Ne deduciamo : Ne deduciamo : Nessun spazio S,~ aun numero u > 2 di dimensioni pub ammettere ur~
n ~ -~ n -- 2 parametri n~ come gruppo di movimenli, n~ come
gruppo reale a
2
sottogruppo del gruppo totale di movimenti Questo teorema per n ~ 3 fu dimostrato dal prof. BiAscm; not lo am-
metteremo vero per n ~---m-- 1 lo dimostreremo per n ~m. St% se b possi-
bil% Sm uno spazio che ammetta uu G,,,+,,_2 di movimenti. Questo gruppo
2 b certamente transitivo, perch~ se non lo fosse, esso sarebbe per il teorema
del § 1 simile aun gruppo di movimenti di uno spazio a meno dim
di- un gruppo eontinuo di movimenti. 55 assurdo, pereh~ m~ + 2m -- ~ > m (m2-- 1)
Quindi quel
mensioni: cib che b
suo sottogruppo che lascia fisso un punto generico b proprio un gruppo a m~ + m -- 2
m
2 parametri~ ciob b proprio un G(,n_l~+(,,_l)_ 2 . Questo gruppo per l'osservazione
'2
preeedente b intransitivo in G,~ e per il teorema del § 1 b simile a un
gruppo di movimenti di uno spazio a , m--1,
dimensioni. Cib che b pure
assurdo, perchb not abbiamo supposto dimostrato il nostro teorema per
~t~m~
1. parametri~ ciob b proprio un G(,n_l~+(,,_l)_ 2 . Questo gruppo per l'osservazione
'2
preeedente b intransitivo in G,~ e per il teorema del § 1 b simile a un
gruppo di movimenti di uno spazio a , m--1,
dimensioni. Cib che b pure
assurdo, perchb not abbiamo supposto dimostrato il nostro teorema per
~t~m~
1. La nostra asserzione resta cosl dimostrata in generale. Cosl pure si di-
mostra: Se un Sn ammette un G,%n_ ~ (certo immaginario sen > 2), esso ~
2 a curvalura costante~ ossia ammette anche un G~+,. ossia Anche questo teorema
2 si dimostra col metodo di induzione completa. Ecco p. es. come si dimostra
per un $4. Quel sottogruppo G~ di G9 che lascia fisso un punto generico
di S~ sar'h intransitivo e avr~t delle V~ per variet~ minime invarianti~ p. es. le ,c4 ~ cost.~ geodeticamente parallele. Esso si potr~ immaginare operante
(transitivamente) sulle x~, x~, x~ (§ 1). Questo Gs pub chiaramente portare
ogni segmento T di una di queste variet~ in ogni altro segmento della va-
riet~ stessa uguale a T. Quindi dovendo esso rieondurre tutto lo $4 in s~
senza mutare le distanze, a due segmenti uguali di una delle x, = cost. cor-
rispondono (per mezzo delle geodetiche normali alle x4 ----- cost.) due segmenti
tra di loro uguali su ogni x4 =cost. Ma una x4 ~ cost. ammettendo un Gs,
ammetterh un G~. Quel gruppo G'o che ope~:a sulle x,~ x~, x~ come questo
Gs e lascia invariato x4 trasformerh dunque in sb ogni x4 = cost., senza mutar
l'elemento lineare e percib sarh un gruppo di movimenti dello S~, ehe quindi
ammetter~ un G,o. § 9. Ritorniamo ora alle condizioni necessarie e sufficienti date al § 6
afflnehb un dato gruppo continuo si possa considerare come un gruppo di
movimenti. Queste condizioni si possono anche enunciate cost: Quel sotto-
gruppo che lascia fisso un punto A deve essere un sottogruppo di uno spazio
a curvatura costante, quando lo si pensi operante sulle rette uscenti da A~
considerate come elementi, o, in altre parole~ deve lasciare fisso un cono qua-
drico col vertice in A; di pi?t egli deve laseiare invariale le quadriche in- § 9. Ritorniamo ora alle condizioni necessarie e sufficienti date al § 6
afflnehb un dato gruppo continuo si possa considerare come un gruppo di
movimenti. Queste condizioni si possono anche enunciate cost: Quel sotto-
gruppo che lascia fisso un punto A deve essere un sottogruppo di uno spazio
a curvatura costante, quando lo si pensi operante sulle rette uscenti da A~
considerate come elementi, o, in altre parole~ deve lasciare fisso un cono qua-
drico col vertice in A; di pi?t egli deve laseiare invariale le quadriche in- Fubini: Sugli spazii che ammettono 56 finitamente vici~e ad A aventi questo cono per cono assintotico. La prima
parte di queste condizioni non sarebbe evidentemente sufficiente~ perch5 p. es. ossia anehe ii G7 delle similitudini dello spazio euc]ideo soddisfa ad esse. Esistono
dunque altri gruppi che soddisfano so]tanto a quella prima Condizione e che
non sono altro che i gruppi di trasformazioni conformi. Osserverb che]a con-
dizione affineh~ una trasformazione infinitesima generi un gruppo di trasfor-
mazioni conformi per lo spazio d s ~ ~ ~ av~ d x~ d x:, the esista una funzione k delle xl tale ehe the esista una funzione k delle xl tale ehe X (2 a+k d xl d x~) ----- k 2 alk d xi d x~. Se k ~ costante essa si pub ehiamare una trasformazione simile. Se k ~ costante essa si pub ehiamare una trasformazione simile. Un'altra osservazione ~ la seguente: Almeno per gruppi transitivi val-
gono ancora i teoremi seguenti, che si dimostrano come gli analoghi dei pa-
ragrafi precedenti per le forme quadratiehe: g
p
p
q
Si possono costruire per quadrature tutte le forme di2~erenziali di or-
dine qualunque che ammettono un gruppo continuo transitivo di operazioni,
e i gruppi corrispondenti. Condizione necessaria e sufficiente affinch~ un gruppo transitivo possa
trasformare in s~ una forma differenziale di ordine qualunque n, ~ che quel
suo sottogruppo che lascia fisso un punto A trasformi i punti infinitamente
vicini ad A in modo che esista un sistema di superficie omotetiche din ~'~°
ordine infinitamente vicine ad A~ ciascuna delle quali venga dal detto sotto-
gruppo trasformata in s~. Cosi si possono pure generalizzare molti altri dei precedenti risultati. § 10. Rieerchiamo ora effettivamente i gruppi a 1, 2~ 3~ 4 parametri
che possono essere realmente considerati come gruppi di movimenti. Osser-
viamo chese il numero delle loro trasformazioni dipendenti ~ minore o uguale
a 3, essi devono (§ 1) potersi eonsiderare come gruppi di movimenti di uno
spazio a non pi~ di tre dimensioni~ e quindi si possono prendere senz'altro
dalla Memoria del prof. BiA~cm; si potrebbe per5 anehe~ poich~ il LIE
diede tutti i gruppi su due variabili e insegnb un metodo per trovare tutti
quelli su tre variabili, esaminare l'un dopo l'altro tutti quelli dei tipi enun-
ciati da Lm che non posseggono pih di quattro parametri, e di questi rite-
nere soltanto quelli che soddisfano alle condizioni del § 6. Questo metodo
sarebbe assai rapido~ e servirebbe a trovare senz'altro direttamente i ri- un gruppo eontinuo di movimenti. 57 sullali della p#t volte eitata Memoria del prof. 8
Annali di Matematiea, Ser'ie III, tomo VIII. ossia +*.3
;
"
,P a'~
a x,
a .:c,
1
a
c q
X, ---- z, £7', + 2- (*~ -
xl) ~
-- a', a x,
a
a
3
a
II")
X,----- ax, ;
X, = a:,3 ;
X3 = ax---7 -- x, a.v, ;
a
1 ( e -'x,_
."c'~) a
n e -x,
a
X, = X, ~
-t- g ~ 1 - n"-
3 x---,'
1 --- n ~ a xa I")
X,=
a
a
a
a
ax,;
X°=--;
X, .... +*.3
;
"
,P a'~
a x,
a .:c,
1
a
c q
X, ---- z, £7', + 2- (*~ -
xl) ~
-- a', a x,
a
a
3
a
II")
X,----- ax, ;
X, = a:,3 ;
X3 = ax---7 -- x, a.v, ;
a
1 ( e -'x,_
."c'~) a
n e -x,
a
X, = X, ~
-t- g ~ 1 - n"-
3 x---,'
1 --- n ~ a xa
a
a
II[ °)
X,=ax---;;
X,=ax~;
3
n sen x,
a
Xa = cos x, -- eotg x, sen x, ~
q- -sel-l x~ ~ xa ;
X, = (X, X,). I1 tipo II °) e II[)° si potrebbero (In un punto di vista generale conside-
rate come identici. I1 tipo II °) e II[)° si potrebbero (In un punto di vista generale conside-
rate come identici. § 11. Trovinmo ora i gruppi G4 a tvasformazioni infinitesime indi-
pendenti. Questo gruppo (§ 1) si deve poter ridurre a un gruppo h'ansitivo
su 4 lettere. Per determinate questi gruppi potremmo) seguendo il procedi-
mento generale del § 5, rieorrere senz'al)ro ai metodi di LIE. ]Via perb noi
possiamo usare di metodi pitt rapidi,in quanto the noi conosciamo tu(ti i G3
transitivi. Potremo dunque prendere un sottogruppo G3 di G4, di cui cer-
cheremo la composizione. Da questa pota'emo
subito dedm're (per mezzo
della penultima tabella) una form% a eui possiamo immaginare ridotte le sue
trasfovmazioni infinitesime; la quarta trasfovmazione infinitesima di G~ si de-
termina poi in guisa che non sia combinazione linea,'e delle preeedenti, e che
il G4 abbia la eomposizione voluta. Sia ora il G, non integrabile (ell'. Lm: Vol. ossia Bix~cm. Si potrebbe anche
infine cosh-uire questi gruppi secondo il mio metodo del {} 5; ma questa
via sarebbe un po' pih lunga della preeedente. ~loi nafm'almente prenderemo
questi gruppi addiritiura dalla Memoria del prof. Br.~cm, perchh il ]ettore
pub facilmente da s~ vedere che questi altri metodi condurrebbero appunto
ai risultati del prof. BIA~em. Gruppi G, :
a
X, = a x~ Gruppi G~ a trasformazioni infinitesime naturatmente linearmeute indipendenti. a
I°)
X'=b
x,
~Xq--a x,
II °)
X, -~ e-oo .... X, = --
0 x,
a x, Gruppi Ga a tra.¢formazioni infinitesime linearmente dipendenti. a
a
a
a
------
x, = ---
x~ = x, -, .... x,
I °)
X,
a oc,
a x~
v x~
0 x.-
I[ °)
a
X, ~--- sen x,
a
a
X, =
b ~
~
+ co~g x, cos z~ ~;
X, = (X, X:)
a
Xo=
a
z, a
a
1 (e_:oo,--x~) ~
Ig °)
x, = a-x~
-
~-;, --
aT,
X, - z, ~-:}~ + -g
aT.. Gruppi Ga a h'asformazioni infinitesime linearmente indipendenti. ~
v +
-+x~
~-~x.+ 2 ~x~
I")
X,=~x~. ;
X,=a.ra
;
X~-----ax--- ~
----
I[ °)
X,--
a
a
a +xo
a
8
e.=;
X':--a.~;
*~=-a.---:
-aT;x~+hX'ax~
IIi°)
X,--
0
a
a
x~ a +(x~q_hx,)
a
0
a
a
a
a
IV °)
x,---e',
-.:e~:,
--2x, e~ ..... ;
X.=
•
X,=e~,--
a x,
a x.. a x~
"
~
'
a x,
sen.r.,
0
a __ cotg x, sen x, ~7~ -~- sen x~ a xa ;
V °)
X,=ax,;
X.=cosz,~z,
--- X, = (X, :~q) Fubi,ni: Sugli spazii che ammeltono 58 VI °)
VII °)
O
a
a
O
x,=~-;;
x:=~-7, ; x~=
o.~+.,~a.,
O
a
O
X, = o x--~ ;
X~ =
~ x--- ;
X3-----O.-
~ VI °)
O
a
a
O
x,=~-;;
x:=~-7, ; x~=
o.~+.,~a., VI °)
O
a
a
O
x,=~-;;
x:=~-7, ; x~=
o.~+.,~a., VII °)
O
a
O
X, = o x--~ ;
X~ =
~ x--- ;
X3-----O.-
~ Gruppi G4 a trasformazioni infinilesime lh, earmenle dipendenti. Gruppi G4 a trasformazioni infinilesime lh, earmenle dipendenti. I")
X,=
a
a
a
a
ax,;
X°=--;
X, .... ossia III,§ 137); il gruppo de-
rivato (X,, X2, X~) dowh avere una delle seguenti eomposizioni: (X, X~) =
X,
(X, X3) = 2 X~
(X~ X,) = .X~
oppure
(~¥, G) = G
(~; X~) = X,
(G X,) = X,. (
,
)
,
(
,
)
(
,)
oppure
(~¥, G) = G
(~; X~) = X,
(G X,) = X,. 59 un gruippo continuo di movimenti. 59 E in ambi i casi si pub poi scegliere .V4 in modo the sia permutabile
con le precedenti trasformazioni. II G3(X,, X~, X~) ~ certo a trasformazioni infinitesime linearmente in-
dipendenti e (§ 1) si pub immaginare semplicemente transitivo sutre lettere. :Nel primo di questi easi awemo: :Nel primo di questi easi awemo: O
(9
0
0
0
X,=
e-'~.----x~
-
--2x~e
-a',-;
X.=--;
X.--e
~-. Posto :
't
O
(~i~-----~I(X,, X2, X3, X.))
avremo, essendo (XI, X~)= 0 (i= 1, 2, 3)
e_,~ ~ O ~,
x~ 4 ~3 e --x' = O ;
e_~, O ~,
~ x, + 2 x: ~ e - ~,, -- "~ e-(~.,+xo) __~ x~ e-x, ~
e -x,
--~2~,e
-x~-2~3x~e
-x~==O
0 ,)7-,i
~4
.t
. ~.4
e--X~ ~.r--~l =
~xo. ~x.. D.~'~
0
&~,
~
0
Ox~ = 0 (i
1, 2, 3) O
(9
0
0
0
X,=
e-'~.----x~
-
--2x~e
-a',-;
X.=--;
X.--e
~-. O
(9
0
0
0
X,=
e-'~.----x~
-
--2x~e
-a',-;
X.=--;
X.--e
~-. Posto :
't
O
(~i~-----~I(X,, X2, X3, X.))
avremo, essendo (XI, X~)= 0 (i= 1, 2, 3) Posto : 't
O
(~i~-----~I(X,, X2, X3, X.))
avremo, essendo (XI, X~)= 0 (i= 1, 2, 3) e_,~ ~ O ~,
x~ 4 ~3 e --x' = O ;
e_~, O ~,
~ x, + 2 x: ~ e - ~,, -- "~ e-(~.,+xo) __~ x~ e-x, ~
e -x,
--~2~,e
-x~-2~3x~e
-x~==O
0 ,)7-,i
~4
.t
. ~.4
e--X~ ~.r--~l =
~xo. ~x.. D.~'~
0
&~,
~
0
Ox~ = 0 (i
1, 2, 3) donde, integrando, donde, integrando, 1
~3 = -- 2 (~ x, -[- fl)
~, =
h dove ~, /3, 7, l, sono funzioni qualunque di x,. Con procedimento analogo troviamo nel secondo caso: dove ~, /3, 7, l, sono funzioni qualunque di x,. ossia Con procedimento analogo troviamo nel secondo caso: O
~
O
sell x2
X, == ~x, ;
X~ = cos x~ ~
~ cotg x, sen x~ ~
+ sen x~ ~x~
x, ---= (x,-:~;). E, posto E, posto 4
D
i=l
0
0
¢, = ¢~=- o
.L = ~ ~-x3 + ~ 0.r, 4
D
i=l si ottiene dove ?, J/ sono funzioni di x,. 60 Fu b in i: ~uyh spazii ehe ammettono Del resto questi due easi, quando non si distingua reale da immaginario,
sono da riputarsi identiei. p
Sia era il G, integrabile e sia un G~ a h'asformazioni non permutabili
il g'ruppo derivato. Indieando con X,, _X~, X.~, X, le trasformazioni genera-
triei di G4 potremo porte (Lm~ lee. eit.) o F)
(X, .X;) = (X, ~X~) = 0
(X, .~) = X,
(G G) = c G
(.X', X,) = X~
(.\~ .xS) = (c -- 1) X, oppure pp
II °)
(x, x,) = (x, -vO = o
(x, x~) = x,
(x, xO ---- 2 x,;
(.v, ~5) =
x~;
(x~ x,) =
x: + .\:~. pp
II °)
(x, x,) = (x, -vO = o
(x, x~) = x,
(x, xO ---- 2 x,;
(.v, ~5) =
x~;
(x~ In ambi-i easi potremo fare In ambi-i easi potremo fare x,=~----;;
x,=a~;;
.~=--o--~+z~ax. E posto a
i
~Xi avremo, nel primo case, p
~, = (c -
1)x,-~- ,~,
do
~, ----- x, -t- ~,
1
h
-- -'2 -- x, % q- 2 x~. q- ~,
1
5 -= ,3 -- x, + ~3
~4 = --
zU p
~, = (c -
1)x,-~- ,~,
1
h e nel seeondo -- -'2 -- x, % q- 2 x~. q- ~,
1
~4 = --
zU ~, ----- x, -t- ~, 5 -= ,3 -- x, + ~3 dove %, D, ~, 14 sono funzioni di x~. dove %, D, ~, 14 sono funzioni di x~. Sia era il gruppo derivato di G~ un G~ e non eontenga il G4 un Ga a
trasformazioni permutabili. Allora o il G~ ha la penultima eomposizion% dove
si ponga c =
17 oppure avr~ la eomposizione: (NN)=(X,~¥~)=-(X, XO=(X~N)=O
(X, X3)=X,
(X,X~)=N. (NN)=(X,~¥~)=-(X, XO=(X~N)=O
(X, X3)=X,
(X,X~)=N. Potremo fare : Potremo fare : b
~
0
X,=~x--G;
X,=
;
X,~=-
+x,
-
0.r~
0),
0 ,:e~ 61 un gruppo continuo di movimenli. ossia (..x~ ~;) =- a s~
(X, X,) =
c X~ e quindi ~, =
x, + ~,,
oppure
I[°)
(x, x,) = c x,
e quindi :
l
(& &) =-= (1 + c) x,
( x~ x,) = x,-t-cx, ~, ~- c x, + x~ -+- ~o, ;
oppure
III°)
(X, X,) = X,
e quindi :
oppure
IV °)
e quindi :
oppure :
Vo)
~, : xa -t- ~,
G--(I -[- c) z,+ %;
( x, x,) = o
1
(&iv,) = x,
1
14
(x, &) = x, + x,
(x, x,) = x,
(x, x,) = x, + x,
(x, x,) = (G x,) -
o,
(x~ x,) = G
1
h ~, ~- c x, + x~ -+- ~o, ;
oppure
III°)
(X, X,) = X,
e quindi :
oppure
IV °)
e quindi :
~, : xa -t- ~,
G--(I -[- c) z,+ %;
( x, x,) = o
1
(&iv,) = x,
1
14
(x, &) = x, + x,
(x, x,) = x,
(x, x,) = x, + x, ~, ~- c x, + x~ -+- ~o, ;
oppure
III°)
(X, X,) = X,
e quindi :
: xa t
G--(I -[- c) z,+ %;
( x, x,) = o
1
(&iv,) = x,
1 oppure III°) e quindi : oppure
IV °)
e quindi :
~, : xa -t- ~,
1
14
(x, &) = x, + x,
(x, x,) = x,
(x, x,) = x, + x, oppure : oppure : Vo)
(x, x,) = (G x,) -
o,
(x~ x,) = G Vo) Fubiui:
Sugli spazii che ammettono 62 e quindi : e quindi : e quindi :
~, =
~, ;
5 -
x~ -4- ~-~ ;
~ -- ~,~;
~, -
oppure :
VI °)
e quindi :
~,= x,~,
(x, x~) = x,
(x, x,) = x,
~., =
x~ +
x~ + ~.~
oppure
vw)
e quindi
(x~ x,) = o
h
(x~ x,) = x, + xq
(i=1,
2 7 3)
1
h e quindi :
~, =
~, ;
5 -
x~ -4- ~-~ ;
~ -- ~,~;
~, -
oppure :
VI °)
e quindi :
(x, x~) = x,
(x, x,) = x,
h
(x~ x,) = x, + xq e quindi : q
~,= x,~,
~., =
x~ +
x~ + ~.~
oppure
vw)
e quindi
(x~ x,) = o
(i=1,
2 7 3)
1
h dove ~,, ~:, ~3, l, sono funzioni di x4. ossia 61 e, posto e, posto 4
X,
~i a xi avrelrlO
: avrelrlO
: 1 dove ~,, ~,, 7a, 14 sono funzioni di x,. dove ~,, ~,, 7a, 14 sono funzioni di x,. dove ~,, ~,, 7a, 14 sono funzioni di x,. Contenga ora il G, un Ga~(X,,
XS, 3i~) a trasformazioni a due a due
permutabili. Potremo fare x,=~-~,
x,= a-~, x~=~- G,
.,c,= ~ g,a. ~..= Si potrg poi scegliere X~ in guisa the o I °)
(x, x,) = ~. (..x~ ~;) =- a s~
e quindi
(X, X,) =
c X~ (x, x,) = ~. ossia dove ~,, ~:, ~3, l, sono funzioni di x4. {} 12. Veniamo ora a un'altra parte del presente lavoro, dove io voglio
esporre un metodo per la rieerea dei sottogruppi finiti diseontinui dei gruppi
eontinui e, in modo speeiale, dei gruppi ehe si possono considerate come
gruppi di movimenti. Io svolgerb questi metodi, applieandoli dapprima effet-
tivamente a due esempii, agli spazii eio~ del tipo ¥II[ °) e IX °) del prof. BtA~cm,
eib ehe ei permetter~ anehe di trovare delle notevolissime rappresentazioni
geometriehe di tali spazii sulla sfera e sulla pseudosfera. g
p
p
Comineeremo dagli spazii del tipo IX°), ehe ammetton% con le notazioni
del prof. BIA~em un gruppo generato dalle senx~
X~ -- ~
~
eotg x, sen x~ ~r~ + senx~ ~,r3
~:~ ;
32, = cos x~ ~r,
X~ ---- (X, X~)
eomposizione : eon la eomposizione : eon la eomposizione : (X, X~) = X, ;
(X~ X3~ = X, ;
(& X,) = X,. Cosh'uiamo il gruppo aggiunto di questo gruppo. Con le usuali notazioni
di Lm, esso si oitiene dali'integrazione del sistema" de',
),, e' --
(1)
dt
de'~ = )~3 e', -- )., e'3
(2)
dt
d e'3 -- ~., e'~ -- ?,~ e', . (3)
dt (1) (2) (3) d e'3 -- ~., e'~ -- ?,~ e', . dt un gruppo conlimw di movimenti. 63 63 Donde de',
de',
de'3
0
e', -~ -1- e', -d-i- @ e'3 ~-[ = de',
de',
de'3
0
e', -~ -1- e', -d-i- @ e'3 ~-[ = ossia e'~ -~- e'g -[- ~'~ ~ cost. (4) (4) Questo gruppo aggiunto non ~ pereib altro che il gruppo delle rotazioni
di una sfera in si~ siessa; e poich~ se T ~ una trasfi)rmazione qualunque (]el
gruppo inizMe Questo gruppo aggiunto non ~ pereib altro che il gruppo delle rotazioni
di una sfera in si~ siessa; e poich~ se T ~ una trasfi)rmazione qualunque (]el
gruppo inizMe T--'TT=T, T--'TT=T, ne traggiamo che alla trasformazione )., t, ~ t, ),3 t del gruppo iniziale corri-
sponde una rotazione della sfera attorno al diametro che ha i coseni di di-
rezioni proporzionali a ),, t, ),,. t, ).3 l; cib chesi conferma col calcolo seguente. ossia Derivando ]a (1) rispetto a t, otteniamo per le (2) e (3): d~ e ',
d t, + (~'; + )4) e:, -- ~,, ,(~,., e', + ~ e ~) ~ 0 donde : d3e ''
de',
(
de',
de'31
d t~ 4 (~,~ + ),~) -dT -- ~' ~'' -f[ + )'~ --fi/= 0 e poich~ per le (1), (2), (3) si ha 3
d e'i
si avrh 3
d e'i si avrh si avrh d 3 e't
, ~
O. '
e'
Indieando con At, Bi, Ci dello
Identiea equazione si lrova per e~,
~,
costanti avremo dunque e'i-- Ai@ Bisen Ht-}- Cicos Ht
(i=1,
2, 3)
(5) (5) dove Servendoci delle (1), (2 ~, (3) e del fatto ehe per t-----0
e'i~ei
(i=l,
2, 3) Servendoci delle (1), (2 ~, (3) e del fatto ehe per t-----0
e'i~ei
(i=l,
2, 3) Servendoci delle (1), (2 ~, (3) e del fatto ehe per t-----0
e'i~ei
(i=l,
2, 3) (1), (2 ~, (3) e del fatto ehe pe
e'i~ei
(i=l,
2, 3) potremo determinare le Ai, Bi, Ci e otterremo infin% scrivendo ?.,, )~,, ),~, H potremo determinare le Ai, Bi, Ci e otterremo infin% scrivendo ?.,, )~,, ),~, H Fubini: Sugli spazii che ammettono 64 in luogo di ),, t, L t, ),3 t, H t
e, = e, I,H, + cos [t [ -- tt"-"] nt- e.~ ~-~7- @ i[ sen H
H. ~
i
tt)
(6)
-Jr- e~ \ H~
H sen It-- H---wF, cos
/ (6) o le eguaglianze ehe se ne ottengono rotando. Queste eguaglianze rappvesen-
tano appunto una rotazione attorno alla retta, i cui eoseni di direzione sono
proporzionali a )~,, ),~, ),3. Per trovarne l'ampiezza ~ si osservi che per note
formole di Geometria analitiea ),,
).~
),3
)q
),.. ),3
).,
).~
),3
•
----- cos
e~
e~
e3
e',
e'~
e'3
e,
e~
e3 donde sostituendo e sviluppando otteniamo: a=H. Abbiamo cos~ trovato, con metodo evidentemente applicabile a casi pih
generati, un gruppo di sostituzioni lineari isomorfo al gruppo dato. ossia E il no-
stro risultato si pub esporre cos]: I1 gruppo in discorso ~ oloedricamente isomorfo al gruppo dei movimenli
di una sfera in s~ : l'isomorfismo si stabilisce~ facendo corrispondere alla
trasformazione )~, X, + L X~ + ~3 323 una rolazione di ampiezza ~ X~-~- ),~ + ?,~ attorno a quel punto di una sfera
(col centro nell'origine) le cui coordhmte sono proporzionali a ),,, ;~, 2~. (
g
)
p
p
),,, ; ,
I gruppi non sono perb simili, perehb mentre uno opera su tre variabili,
l'altro opera in realt'X soltanto su due. I gruppi non sono perb simili, perehb mentre uno opera su tre variabili,
l'altro opera in realt'X soltanto su due. Ma ora osserviamo ehe dati due gruppi oloedrieamente isomorfi, non
sempre dall'esistenza di sottogruppi finiti diseontinui per l'uno si pub eon-
chiudere l'esistenza di sottogruppi finiti diseontinui per l'altro. Cosl p. es. il
gruppo detle rotazioni attorno a un asse e il gruppo delle traslazioni in una
direzione sono oloedrieamente isomorfi, mentre perb per l'uno esistono sotto-
gruppi diseontinui finiti e per l'altro non ne esistono affatto. Cib ehe si deve alle polidromie, eee., ehe si possono presentare am-
pliando il campo in eui opera un dato gruppo. 1~ ben ehiaro perb ehe a un
sottogruppo finito discontinuo di un gruppo, eorrisponderh un sottogruppo un gruppo continue di movimentL ~5 isomorfo nel gruppo aggiunto, in generale. E quindi dovremo prima ricercare
i sottogruppi discontinui finiti del gruppo aggiunto. Questo problema ~ sen-
z'altro risoluto nel nostro case: i sottogruppi cercati net gruppo aggiunto non
sono che i gruppi dei poliedri regolari. Eil nosh~o calcolo preeedente deft-
nisce senza ambig, dt~ i sottogruppi corrispondenti del gruppo iniziale, perch~
d~ nel mode pih perspicuo quale trasformazione del nostro gruppo si deve
far corrispondere a una trasformazione del gruppo aggiunto. Ma per peter
affermare che le trasformazioni del gruppo inizia]e, corrispondenti alle tra-
sformazioni del gruppo di un po]iedro regolare contenuto nel gruppo aggiunt0,
formino effettivamente un gruppo, bisogna, come risu]ta chiaramente dalle
considerazioni precedenti, fissare bene il significato delle coordinate. A tal
fine serve ]'importante osservazione che due gruppi oloedricamente isomorfi
non simili si possono spesso rendere simili, mutando le variabili su cui essi
operano. ossia Nel case nostro il gruppo aggiunto, invece di essere considerate come
un gruppo operante sui punti della sfera, sar~t pensato come gruppo operante
sugli elementi della sfera, cio~ sulla sfera immaginata come luogo degli in-
finiti enti che si definiscono dando un punto della sfera stessa e una direzione
uscente da esso tangente alla sfera. I due gruppi risultano altora senz'altro
simili, e con opportuna see]ta delle coordinate di un elemento della sfera, essi
riescono identici. Si prendano come coordinate di un elemento della sfera la
colatitudine e la longitadine ~, ~o del punto corrispondente e la derivata
d--~' dove d s ~ la lunghezza dell'elemento, d ~ l'incremento di ~ muo-
vendoei lunge di esso. Le trasformazioni infinilesime del gruppo aggiunto sono e~-~--e.~;
e,~es--e3~-~e;
e~3-~--e~-~e ~. e3 = cos ~,
e~ ~ sen ~ sen $,
e, ~ sen ~ cos ? avremo ehe l'ultima, rappresentando una rotazione attorno all'asse delle ,, e3 ,,
sark eguale a avremo ehe l'ultima, rappresentando una rotazione attorno all'asse delle ,, e3 ,,
sark eguale a X•=--. La soconda, essendo una rotazione attorno
sen ~ sen ~0 e quindi sar'~ della forma
all'asse e2, lascia invariato e~--e.~=k
-- cos ~ sen ~ ~
-{- sen6cos~
.' 9 9 Annali di Matematica, Serie IH, tome VIII. Fubini: Sugli spazii che ammettono 66 dove // ~ una funzione da determinarsi. Ma: poichb dove // ~ una funzione da determinarsi. Ma: poichb 0
D
O~------d sen 0 sen ~ ~e~ q- sen ~ cos ? ~e, 0
D
O~------d sen 0 sen ~ ~e~ q- sen ~ cos ? ossia ~e,
0--0 = -- sen 0 ~e~ -}- cos 0 sen 50 Uee~ +" cos ~ c 0--0 = -- sen 0 ~e~ -}- cos 0 sen 50 Uee~ +" cos ~ cos ~ si trova senz'altro
e quindi
Analogamente :
1
sen 0
a
X~ = sen 50 cotg 0 ~- ~ cos 50 ~-0"
0
0
X, ----- sen 50 ~- A- cos 50 cotg 0 ~
• si trova senz'altro 1
sen 0
a
X~ = sen 50 cotg 0 ~- ~ cos 50 ~-0"
0
0
X, ----- sen 50 ~- A- cos 50 cotg 0 ~
• e quindi a
X~ = sen 50 cotg 0 ~- ~ cos 50 ~-0"
0
0
X, ----- sen 50 ~- A- cos 50 cotg 0 ~
• a
X~ = sen 50 cotg 0 ~- ~ cos 50 ~-0" Analogamente : Ampliamo ora il gruppo X,~ X~, X~ immaginandolo
su ~(')
dO
~(,)
O~. Avremo:
operante anche X~--O? - -- ;
X~ =_ sen • cotg 0 ~
-- cos 50 ~
-J- 50(') sen ~f ~
q-
[~(o cos 50 cotg 0 -- 0(') sen ? ]
+
sen ~/jj ~ ~(1/
L
x
(x
x 3 x, = (x, x.3. O
Laseiando era il termine in 0,~'
perchb noi vogliamo soltanto oceu-
parci di come il gruppo trasforma ~o), e osservando che ds'= dO'q-sen~d~ 2,
donde ~(')
x,'i -- 0 ~:~
(
d 0(,)
--
sen 0
, sostltuendo alla ~(') la nuova variabile ~k =d x/1 -~-~',)'
il gruppo assume l'aspetto il gruppo assume l'aspetto 0
sen ~ 0
X2 = sen 50 cotg 0
-- cos ~ 0-~ q- sen 0 0
endo dunque
x, =
xo. X3 ~ Ponendo dunque questo gruppo assume proprio la forma data dal prof. BIANcnI. questo gruppo assume proprio la forma data dal prof. BIANcnI. un gruppo continuo di movlmenti. 67 Abbiamo dunque : Le /ormule precedenti stabiliscono una rappresentazione del nostro spazio
sulla sfera euclidea pensata come luogo di elementi~ tale che ai movimenti
dello spazio in s~ corrispondono rnovlmenti della sfera in s~ stessa. p
p
f
Se noi vogliamo che]a rappresentazione sia biunivoca su tutta la sfera
senza eceezion% resta senz'altro fissata la corrispondenza ira i punti dello
spazio e ]e ]oro coordinate. ossia E possiamo dire: I gruppi finiti discontinui degli spazii in discovso sono quelli~ che, ~zella
corrispondenza precedentemente fissata~ corrispondono ai gruppi dei poliedri
re qolari. I1 fatto che qui vediamo, ciog ehe i gruppi finiti discontinui dei nostri
spazii sono oloedricamenle isomorfi a gruppi finiti discontinui di movimenti
di uno spazio ellittico ~ un fatto generale, che si ripele sempre appena il
gruppo totale dei movimeJ~ti non contenga trasformazioni infinitesime ecce-
zionali. Infatti a un gruppo finito discontinuo di mo,dmenti corrisponde un
gruppo finito discontinuo nel gruppo aggiunto, che g in tal easo oloedrica-
mente isomorfo al gruppo primitivo. E questo gruppo~ essendo un gruppo
finito di operazioni lineari laseia sempre invariata una forma quadratica de-
finita ehe ~ p. es. la somma delia forma e~-1-e~ + el e delle sue trasformate. ~el easo precedente questa forma ~ proprio ,, e} + e~.-1-el ,. p
q
p
p
}
Da queste osservazioni scaturisce senz'altro un metodo per ricercare in
ogni caso cosiffatto se esistono nel gruppo considerato sottogruppi finiti di-
scontinui. I1 nostro metodo consiste nel formare il !lruppo aggiunto, e di de-
terminare quali delle sue trasformazioni lascino fissa una forma quadratica
definita, cib che si compie con operazioni algebriche : considerata poi questa
forma quadratica posta uguale a zero come assoluto di uno spazio ellittico,
vedere ~uali sottogruppi discontinui finiti di questo spazio appartengono pure
al gruppo aggiunto. In conclusione anche nel case pitt generale la ricerea
si riduee alla determinazione dei gruppi finiti diseontinui di uno spazio el-
littico. Applieheremo ora questo proeedimento, un po' modifieato, al tipo VIII °
del prof. B~A~cnl. Comincieremo dal costruirne il gruppo aggiunto. Esso viene
definito dalle de', 4- ).,e' --
d t
'
~
).2 e', = O
d e'~
e' --
9~ ' ~--
dt
~'2),i
3
2
3ei
0
de'3
dt de 3
dt 68 Fubini:
Sugli spazii che ammetlono Yediamo se qualche sua trasformazione lascia fissa una conica. ossia E, poich~
pet" le prccedenti equazioni, q
ale's
e' de't
1
de'2 -- 0
e'l --d- [- -{-
3 d t
2 e'2 d t
e'
"
4 e't 3 -- e ~ = cost., ale's
e' de't
1
de'2 -- 0
e'l --d- [- -{-
3 d t
2 e'2 d t e'
"
4 e't 3 -- e ~ = cost., ossia ossia tutte le trasformazioni del gruppo lasciano fissa la conica rcale
e ~
_
'~
4e'i 3
e~=O. Allora senz'altro deduciamo che il nostro gruppo ~ oloedricamente iso-
morfo ai movimenti di una pseudosfera in s~ perch~ questa conica ~ reale, e~
analogamente a quanta s'~ ratio prima noi avremmo: g
q
p
II noslro spazio si pub riferire biunivocameste alla pseudosfera pensala
come luogo dei suoi o~ ~ elehzenti in modo che ai movimenti della ioseudosfera
in s~ stessa corrispondano movimesti dello spazio is s}. Questa corrispon-
denza definisce per noi senz'alti'o la corrispondenza tra un punto di S~ e le
sue coordinate. E allora senz'altro poieh5 eccetto casi bana!i, la pseudosfera
non ammette gruppi finiti di movimenfi in s~, ma ammette bensi gruppi in-
finiti discontinui, avremo: Gli spazii in discorso non ammetlono (tranne qualche caso banale) gruppi
finiti discontinui di movimenti : essi ammettono perb i~finiti gruppi infiniti
discontinui di moviment% isomorfi oloedricamente ai gruppi di PoI:~cAR~. Per gli altri tipi di S~, che ammettono un G3 transitivo di movimenti
5 ben facile riconoscere l'impossibilith di gruppi discontinui finiti. Basra in-
fatti riconoscere l'aperiodicit~ di una trasformazione qualsiasi del gruppo ag-
o'Junto. P. es. nel tipo Vl ° il gruppo aggiunto ~ definito da d e~i
t
1
dt -]- ?. e3-- ;% e ~ = O
de'o,
e'
dt
d e'3
dr ----0. dond% se ?,3 =[= O, dond% se ?,3 =[= O, )~4
~Lt )
e~ t
e't -- ~3 @3 - e3
n t. e,
. -- ~" e3 (1 -- d ~Jt) -{- e2 e h~t
e%-
~3
t
ea~es un gruppo continuo di movimenti. 69 ehe non pub essere periodica nel campo reale, poich~, essendo ~=',=0,
e~ ' ='i= 1. ossia 8e invece ),~ = O, si avr'a: e'~ ----- -- ~.l e3 t +
e~
e', =
-- h X~ ea t +
e,
e"3 ~
ea pure aperiodica se
e'~ ----- -- ~.l e3 t +
e~
e', =
-- h X~ ea t +
e,
e"3 ~
ea
z,=]=0,
o x,=',=0. pure aperiodica se pure aperiodica se z,=]=0,
o x,=',=0. z,=]=0,
o x,=',=0. La discussione ~ analoga per gli altri tipi. Invece ora di studiare quegli &che ammettono un G~, not osserveremo
che questi G4 contengono una trasformazione infinitesima eccezionale e di. remo poehe parole sui gruppi G,. che eontengono k trasformazioni Y,... Y~
infinitesime eccezionali. Per trovarne i sottogruppi finiti discontinui si trovino
prima tutti i cosiffatti sottogruppi eventtia]i del gruppo aggiunto. Si deter-
minano in un modo qua]unque le trasformazioni eorrispondenti del gruppo
iniziale, cercando pot, se ~ possibile, di aggiungervi delle trasformazioni di
quel sottogruppo di G,. che viene generato dalle Y,... Yk~ che siano perio-
diehe e che insieme a]le preeedenti generino effettivamente un gruppo della
specie voluta. p
§ 13. Daremo ora un'app]icazione dei nostri metodi alla determina-
zione degli spazii che ammettono un Gt oppure un G~ o un Ga o un G4,
oppure un gruppo a pih di 4 parametri con sole 4 trasformazioni infinite-
sime indipendenti. :Per i primi quattro cast il problema si risolve facilmente: per il quinto
easo inveee si vedr~ ehe oeeorrono nuo~¢i e partieolari artifici, senza i quali
i ealeoli diverrebbero estremamente lunghi e ben diffieilmente condurrebbero
in rondo. A questo caso, ~ riservata una prossima Memoria. Per trovare gli spazii the ammettono un G, basta (cir. Bl~t~cm (A)
pag. 8) supporre i eoefficienti dell'elemento lineare indipendenti da x,. Per trovare gli spazii che ammettono un G,, basta, nel primo caso,
ammettere i coetficienti dell'elemento lineare indipendenti da x,, x~. lgel secondo caso posto ds~-~aikdxidxh
(i~1,
2,... n) Fubini:
Sugli s pazii che ammettono 70 le formule di KILLI~ ei danno subito ehe le formule di KILLI~ ei danno subito ehe a~ ----- x, a,~ + c,~
(k ='= 2)
a,~ = x~ ai, + 2 c,~ x, + c., dove le c;k sono arbitrarie, puretlb non dipendano da xi, x~. Spazii cite ammettono un G3 a trasformazioni
infinitesime indipendenti. Qui ricorderemo i risultati generali del § 4. ossia Per trovare gli spazii cor-
rispondenti a uno di questi tipi noi dovremo scindere il caleolo in due patti:
uno relativo alle aik per
i, k~-1,
2~ 3 e l'altro relativo alle air, per
i> 3, k ~ 3. Quanto alle alh dove i> 3, k> 3 noi sappiamo gib. ehe esse
posseno essere funzioni arbitrarie indipendenti dalle x,~ x,, ix3. Esamineremo
ora uno dopo l'altro i varii tipi di G~, transitivi helle x,, x,, x~ gi~t trovati. p
p
,
,,
,,
g
1. ° tipo}. Per ]e aia dove i~ k-= 1, 2, 3 otteniamo, iniegrando le equa-
zioni di KILT.~% .T, dove le ci~ sono indipendenti da x,, x,, x~. Se lo spazio in diseorso ~ un S~,
potremo supporre aa, = 1, a, = a~ = a~ ~
0 e le cik funzioni di x4. In ge-
nerale avremo per le aiR, dove i> 3, k ~ 3 Ctit
0 a,~ __ 1
0 a,3
1
O x,
~ x3
0 x,
0 x, equazioni~ che si integrano senz'altro. 2. ° tipo). Per le aik dove i, k = 1~ 2~ 3 aw'emo per le equazioni di
KmLis% cite subito si integrano, 2. ° tipo). Per le aik dove i, k = 1~ 2~ 3 aw'emo per le equazioni di
KmLis% cite subito si integrano, a,, = c,,
a,~ = c,, e x'
a,z = cis e hx'
a~ = c~ e :x, ;
a,a =
C~3 eO+h)x,
as3 -~ ca3 e ~hx' a,, = c,,
a,~ = c,, e x'
a,z = cis e hx'
a~ = c~ e :x, ;
a,a =
C~3 eO+h)x,
as3 -~ ca3 e ~hx' as3 -~ ca3 e ~hx' X
x3
dove le cik sono indipendenti da x,,. ~,
• X
x3
dove le cik sono indipendenti da x,,. ~,
• X
x3
dove le cik sono indipendenti da x,,. ~,
•
Se lo spazio ~ un $4 si pub fare a4, =-1 ai4 =0
(i- 1~ 2, 3). In generale per le a~n (i> 3~ k ~ 3) avremo: p
Se lo spazio ~ un $4 si pub fare a4, =-1 ai4 =0
(i- 1~ 2, 3). In generale per le a~n (i> 3~ k ~ 3) avremo: 0 x~
~ x~
~x~°i~ ~ ~ ai~ = 0
~x~a;~ + a~ ~
--[- a~ h ~
= 0 the subito si integrano. the subito si integrano. ossia un gruppo continuo di movimenti. 71 71 3. 0 tipo), kvremo per le aik (i, k == 1, 2, 3)
0 aik
0 aik
. Ox~ = ~-~ = O, 3. 0 tipo), kvremo per le aik (i, k == 1, 2, 3)
0 aik
0 aik
. Ox~ = ~-~ = O, at3
x~
~ x~
0 x~
a22
6923
0 a83
-- ~ x~
a~ +
h a~3 + a~ =
O x'--T -- 2 a~3 + 2 h a~ . Derivando la seconda di queste ultime equazioni rispetto a x, e portan-
dovi il valore di ~al3 dato dalla terza e tra la seconda e la terza formula
x~
cost ottenuta eliminando al3, si ottiene: 0 ~ al2
~ ~t2
0x~+h-yE-x
+a,, =0. E, se = +-+-i/3 sono le radiei di z'+ h z-{-1------0 si avrh:
a,, :
¢/e ~'~, cos fl x, -]- Z e:X' sen fl x, E, se = +-+-i/3 sono le radiei di z'+ h z-{-1------0 si avrh: E, se = +-+-i/3 sono le radiei di z'+ h z-{-1------0 si avrh:
a,, :
¢/e ~'~, cos fl x, -]- Z e:X' sen fl x, a,, :
¢/e ~'~, cos fl x, -]- Z e:X' sen fl x, dove q,, Z non dipendono da x,, x~, x3. Si ha quindi: dove q,, Z non dipendono da x,, x~, x3. Si ha quindi: q,,
p
Si ha quindi: ai~
~ a2~
~a~
a,3 ----- -- "0 ,~
0 x, ~
0 ,z~ Sostituendo nell'ultima per le 0 a~2
D a23
0---~[ ' ~
i loro valori, si ha: Sostituendo nell'ultima per le 0 a~2
D a23
0---~[ ' ~
i loro valori, si ha: Sostituendo nell'ultima per le 0 a~2
D a23
0---~[ ' ~
i loro valori, si ha: ~ a~3
Da23 =
2 h a33
x' ~ ÷ 4 a~3 ÷ h ~ x---7 Derivando ed eliminando tra l'equazione cosl ot/enuta e la
la a33 si trova:
precedente Derivando ed eliminando tra l'equazione cosl ot/enuta e la
la a33 si trova:
precedente ~a a~a
0 ~ a~a
0 a2a
~ x------~ -k- a h O x----~ + ( 4 + 2 h') -U-~ -[- 4 h a..~ = O. ossia analoga equazione si troverebbe per a2~ e si avrebbe:
a~2 ---- ~ e -hx' + ~ e --hx' cos 2fl x, 27 z e-~X' sen 2/3 x,
1 ~ a2~
a~3 =
2
~xi
a33--------- ~x, ~+~vx~
1+
• Un'analoga equazione si troverebbe per a2~ e si avrebbe: n'analoga equazione si troverebbe per a2~ e si avrebbe:
a~2 ---- ~ e -hx' + ~ e --hx' cos 2fl x, 27 z e-~X' sen 2/3 x,
1 ~ a2~
a~3 =
2
~xi
a33--------- ~x, ~+~vx~
1+
• a~2 ---- ~ e -hx' + ~ e --hx' cos 2fl x, 27 z e-~X' sen 2/3 x, 1 ~ a2~
a~3 =
2
~xi
a33--------- ~x, ~+~vx~
1+
• 72 Fubini:
Sugli spazii che ammettono Si ha poi a~,----- Z, dove ~,, ~, Z sono funzioni indipendenti da x,, x~, x~. Se 1o spazio ~ un S,, potremo fare a, = 1, ai, = 0 (i= 1, 2, 3). In generale per le ai~ (i> 3, k-1 3) otteniamo : Si ha poi a~,----- Z, dove ~,, ~, Z sono funzioni indipendenti da x,, x~, x~. Se 1o spazio ~ un S,, potremo fare a, = 1, ai, = 0 (i= 1, 2, 3). In generale per le ai~ (i> 3, k-1 3) otteniamo : ai~
~ alh
~ alk
ai, ~ xa
~ (x, -~ h xa) __~ 0
Dah ~
0
~ ai,
~aia
~ x---[
~[
+ ai~ -= 0
~ x----[ + h a~ ----- O ossia ossia ehe si integrano senza diffieolth. ehe si integrano senza diffieolth. g
4. 0 tipo). Per le alk (i, k---- 1, 2, 3) le equazioni e la loro integrazione
si trova gi~ nelle pag. 67-68 di (A). Baster~ immaginarvi le a, b, c, d fun-
zioni indipendenti da x,, x~, x3. ossia p
Per le ai~ (i> 3, k~ 3) troviamo le: p
Per le ai~ (i> 3, k~ 3) troviamo le: aik
~ aii
~ ai,
D aia
x---7 ----- 0
~ x-----[ =
~ x----[ ~
~ x----'7 + a i, ----- 0
0_o0 3 ~ aik .3 V ah ~ (e-x')
¢ti, ~ (x~ e -~)
~ (2 x, e -x.)
0
x~
~ xh
~ x~
ai~
~ xk
= 0_o0 3 ~ aik .3 V ah ~ (e-x')
¢ti, ~ (x~ e -~)
~ (2 x, e -x.)
0
x~
~ xh
~ x~
ai~
~ xk
= che si integrano senz'altro. che si integrano senz'altro. che si integrano senz'altro. g
5. ° tipo). Yalgono per questo considerazioni perfettamente analoghe a
quelle del tip() preeedente. 5. ° tipo). Yalgono per questo considerazioni perfettamente analoghe a
quelle del tip() preeedente. 6. 0 tipo). Per le a~ (i, k = 1, 2, 3) si trova, integrando le equazioni
di KmLI~% e indieando con Z~ delle funzioni indipendenti da x,, x~, x3 ehe: a,,~---Z,,;
a~=Z~..;
a,z-----Z,3;
a,~-----x~z~-t- Z,~;
a~3-----x~z,,d-Z,~
Per le aik (i> 3, k L 3) si ha:
aik
~ aik
~ ah
~ ai2
D ala a,,~---Z,,;
a~=Z~..;
a,z-----Z,3;
a,~-----x~z~-t- Z,~;
a~3-----x~z,,d-Z,~ Per le aik (i> 3, k L 3) si ha: Per le aik (i> 3, k L 3) si ha: aik
~ aik
~ ah
~ ai2
D ala ehe tosto si integrano. g
7. o tipo). Basta supporre le ai~ indipendenti da x,, x~ x~, x,. g
7. o tipo). Basta supporre le ai~ indipendenti da x,, x~ x~, x,. Spazii che ammettono un G3 a trasformazioni infinitesime linearmente
dipendenti. In questo caso si dovrh scindere lo studio per le ai~ in due altri
casi, uno relativo alle aih (i, k~--2), l'altro per le ai~, (i> 2, k~ 2). Spazii che ammettono un G3 a trasformazioni infinitesime linearmente
dipendenti. In questo caso si dovrh scindere lo studio per le ai~ in due altri
casi, uno relativo alle aih (i, k~--2), l'altro per le ai~, (i> 2, k~ 2). un gruppo continuo di movimenti. 73 1. ° tipo). Tutte le aik sono indipendenti da x,, x,. Per le air, (i, k= 1, 2)
si trova dalle formole di KmH~a the: dove c,, ~ indipendente da xl, x~. ossia ,,
p
,
Per le alh (i~>2, k-1 2) si ha: p
Per le alh (i~>2, k-1 2) si ha: aik
? aik --0
0 x,
D x... X~
Xt che si integrano senz'altro. che si integrano senz'altro. g
2. ° tipo). Per le a~, (i, k-----1, 2) si hanno le: g
2. ° tipo). Per le a~, (i, k-----1, 2) si hanno le: m, --0
a,~(;
~a~ .__2a~cotgx,
a~=a,,sen-'x~ the si integrano tosto. g
Per le aik (i:~ 2, k L 2) le formule di KILLI~e danno, poieh~ le ao~ sono
indipendenti da x~, che: ai~ = ai~ = O. 3. 0 tip()). La discussione ~ perfettamente analoga alla precedente. 3. 0 tip()). La discussione ~ perfettamente analoga alla precedente Spazii che ammettono un gruppo G4 a trasformazioni infinitesime di-
Tendenti. La ricerca di questi spazii in generale si fa come preeedente-
mente : noi, col solo fine di una maggiore brevith, studieremo soltanto gli S,,
accontentandoci di aver fatto rilevare dagli esempii precedenti il metodo
generale. 1. ° tipo). Potremo chiaramente porte, quando ci si restringa agli $4~ d s ~ = d xI + ~, aik cl xi d xk
i,k
(i, k = 1, 2, 3). Integrando le equazioni di KILLIN% si trova a.-----c,t;
a~=c~;
a23=x, c22; a33=x]c~+c.;
a,~+a,3=0 a.-----c,t;
a~=c~;
a23=x, c22; a33=x]c~+c.;
a,~+a,3=0 dove le ci~ sono indipendenti da x,, x., x~. dove le ci~ sono indipendenti da x,, x., x~. 2. ° tipo). Posto, come sopra d s ~ = d x~ -t- Z a~k d z, d x~
%k
(i, k=l~
2~ 3) 10 10 Annali di 2¢Iatematica. Serie III, tomo VIII. Annali di 2¢Iatematica. Serie III, tomo VIII. 74 Fubini: Sugli spazii che ammettono otteniamo dalle e(~uazioni di KXLLI~O, otteniamo dalle e(~uazioni di KXLLI~O, otteniamo dalle e(~uazioni di KXLLI~O, otteniamo dalle e(~uazioni di KXLLI~O, Cttl =
Cjj ~
(tas ~
Cs~ ~
~,~3 ----- n e x, c3a ~
at.. =
aja == 0
a,, = e'~, [(1 -
n9 c,, +
n' c3~] dove ]e elk sono indipendenti da x,, ",c,, x,. 3. ° tipo). Posto ds*~dx]+~aikdxidxk
(i, k=],
2, 3)
i,k si trova, con ii solito procediment% che: si trova, con ii solito procediment% che: al~ ~
ct~ ;
aa3 =: Caa ;
aj~ ~-- a~a =
0 ;
a~a =
n C~3 COS Xt
a~ : n ~ c3a cos' ~ + c,, sen ~ x~. ossia Spazii che ummettono un gruppo G, non integrabile transitivo a trasfor-
.mazioni infinitesime.linearmente indipendenti. Prima di trattare questo caso particolar% faremo alcune consideraz]oni
generali sugli spazii che ammettono un Ga, a trasformazioni infinitesime in-
dipendenti. Osserveremo cio~ che in ognuno dei gruppi G4 cosiffatti, prece-
dentemente determinati~ entrano degli elementi arbitrarii. E not potremmo
va]ercene per condurre in due maniere i caleoli. Il primo modo consiste nel
dare a questi elementi arbitrarii valori opportuni della massima semplicith~
ricordando il teorema di Lm che due gruppi semplicemente transitivi sullo
stesso numero di variabili e isomorfi sono anche simili. I1 secondo metodo
consisterebbe nel prefissare all'elemento lineare da determinarsi una forma
saputa , a priori , possibil% laseiando quindi agli elementi arbitrarii the en-
trano nel gruppo tutta l'arbitrariet~ compatibile con questa forma. I1 primo
metodo ~ pih opportuno per 1o studio degli spazii a un numero qua]unque
di dimensioni, il seeondo per lo studio degli $4. Diamo ora un esempio del
primo metodo, p
Prendiamo p. es. il gruppo G4 generato dalle diamo p. es. il gruppo G4 generato dalle
X,=b--~;
X~--O-~v; X~=--~x--~+X~x,;
O
X,_[(~_ 1)x, + ,.,,] U £ + [--x,~+ cz, + ~,] ~x--- +
+ [z~ + ~] ~ x~ -- 1-7 ~x," O
X,_[(~_ 1)x, + ,.,,] U £ + [--x,~+ cz, + ~,] ~x--- +
+ [z~ + ~] ~ x~ -- 1-7 ~x," un gruppo continuo di movimenti. 75 Secondo i principii test~ esposti potremo porre ~,=7,=?~=¢),
l~=:l
col che si ha X~ = (c-- 1) x, ~
-[- c x~ ~
-Jr x~ ~ x~ ~
~x~ " Per le ai~ (i> 4, k> 4) basra supporre le funzioni indipendenti dalle
x,, x~, x~, x~. Per le a~ (i, k= l, 2, 3, 4) troviamo per le formule di
K~LL~O, d as3
~aih
?Crib
---
--
0
x, -= 2 a~ ;
~ x~
~ x~
a(3
0=',=3)
D x,
a~_2c;
a~;
--=0;
~a'L--2(cx4 -- 1) a,,;
~x, --
~x4
~x,
--- (x --~1) a,, -4- c a,2, ecc., ecc.,
0 x4 --- (x --~1) a,, -4- c a,2, ecc., ecc.,
0 x4 the si integrano senz'altro. Per le a;k (i> 4, k ~-~ 4) si hanno le: DCti___._~k --~ Oaik
0 ;
--- ~ aia. ossia @ ai, 0 x3 _
0
(c -- 1)x, ~aih
~x~
~ X a
~
2c c a i~ ~
-j- a i,3 -~w k = 0 DCti___._~k --~ Oaik
0 ;
--- ~ aia. @ ai, 0 x3 _
0 (c -- 1)x, ~aih
~x~
~ X a
~
2c c a i~ ~
-j- a i,3 -~w k = 0 the pure si integrano senza difflcoltk. Con questo stesso metodo si possono
chiaramente studiare tutti gli altri tipi di G4. Noi, col solo scopo di non
allungare questa )lemoria coll formule, che poi non utilizzeremmo, ci restrin-
geremo agli $4 e useremo naturalmente del secondo metodo. Cominciamo ora
a trovare gli $4 con un G~ transitivo non integrabile del primo tip% avver-
tendo che la prima parte dell() studio ci servir~ anche per i casi ulteriori. Sic(.ome il sottogruppo formato dalle X,, X.,, X3 ha le x4 == cost. come va-
riet~ invarianfi potremo porte: ds~-dx~-~aikdxidxk
(i, k-l,
2, 3). 4
~ f ~ una trasf0rmazione infitfitesima che quelIo spa-
i=l 4
~ f ~ una trasf0rmazione infitfitesima che quelIo spa-
i=l Fubini:
5'ucjli spazii the ammettono 76 zio ammette allora~ serivendo ]e condizioni di K~LLI~ relative ad aik =-a~,
a,:~ a~3~ a, otteniamo: ~7:~ =
0
Ox4
(~) (~) (i =
l, 2, 3). @ @ Indicando al solito con Ai~ il
il valore eertamente non nullo del
complemento algebrico di alk diviso per
determinante [alh[ otterremo dalle ([3) Indicando al solito con Ai~ il
il valore eertamente non nullo del
complemento algebrico di alk diviso per
determinante [alh[ otterremo dalle ([3) ~4
~
:~ A~k--
(i= 1 2, 3). (I)
094
k=l
~ '~l~
~
" (I) esso
Nei easi in eui~ come nel nostr% ~4 pub essere soltanto funzione di x~
;~ quindi per la (~) eostante~ e pereib per le (I) abbiamo ehe:
=
~3
p
Ox--~--~ ~x,
oX~,~----0. Dunque le ~i non possono contenere x,; eosiech~ la
nostra seelta dell'elemento Iineare fa si ehe tutte le funzioni arbitrarie
(della x,) che compariseono in X, sono semplici eostanti. Queste considera-
zioni va]gono anche per molti dei casi seguenti e noi non le ripeteremo. Intanto avremo che le a, fl~ 7 sono effettive costanti. E se noi seriviamo
]e formole di KIL~,IsG per le ai~, (i, k-~-l, 2, 3) relative a X,, X~ X, ot-
teniamo le formule pag. 67 eli B. ossia E ei basterk dall'esame di X4 ricavare le
a, b~ c, d, e, f ehe nel nostro easo sono funzioni di x4. Osserviamo intanto
che nella X4 potremo (dividendo le v., /3, 7 per una costante certo non nulla)
supporre 14-----1. Scrivendo le equazioni di KmLI~( b otteniamo dei polinomii
in x,, x~ da uguagliarsi a zero. Annullando i coefflcienti dei singoli termini
(che saranno soltanto funzioni di x 0 raggiungeremo il nostro scopo. L'equa-
zione di K~Lm~(~ relativa ad a,~ ci dh cos'l: d x4 d x4 L'equazione di KIT,Lira per a33 ci dS, indicando con un apice la deri-
vazione rispetto x4: L'equazione di KIT,Lira per a33 ci dS, indicando con un apice la deri-
vazione rispetto x4: b' =a"- 7 -k 2b~--~.(cq-2d)
e' q- 2 d' ---= 6 (bT--e.)
d'
=
2 (b 7 -
-
e ~.)
e'--(c-~- 2 d) y-- 2e[3--~ ~. un gruppo continuo di movimenti. 77 L'equazione di KILLI~o relativa ad a~2 ci d~ infine: f' ---- 4 (e 7 -
f Lea m b, c, d, e~ f si ottengono dunque integrando un sistema lineare
di equazioni differenziali ordinarie del prim'ordine. q
p
2. ° tipo).,Prefisso come sopra all'elemento lineare la forma 1~2,3
dx] + Z ai~dxidxk si trova che ~=~,
6,=~
sono costanti. Per Ie ait,(i~ k~l,
2~ 3) tro-
viamo, poich~ lo spazio deve ammettere il G3 ~(X,,
X~, X3) le equazioni
di pag. 69 (A) dove soltanto le eostanti di integrazione saranno funzioni op-
portune di x4. Se ~ ~ 0, queste funzioni saranno proprio costanti effettive ;
se ~.3~ 0~ notiamo che mutando x4 in Kx4, dove K ~ una costante oppor-
tuna~ potremo fare ~3-----~4~ mentre, sostituendo al nostro spazio uno spazio
simil% potremo ancora porre a,4-----1. Si ha cosi e le aik (i-----1~ 2~ 3) si troveranno ancora date dalle formule a pag. 69
di (A)~ dove le a, b, c... si intendano ancora effettive costanti~ ma al posto
di x~ si sostituisca x~--x4. Spazii che ammettono un G4 a trasformazioni linearmente indipendenti,
integrabile e il cui gruppo derivato ~ un G3 a trasformazioni infinitesime
non permutabili. 1. o tipo). Anehe qui potremo porre (i, k~l,
2, 3). ds'~dx~A-~aihdxidXh
(i, k~l,
2, 3). ossia Avremo per l'osservazione fatta che ~,, ,%~ y3, 14 sono costanti effeitive
che indicheremo con l,, l~, 13, l~ eosicchh si potrh porte: 1 Essendovi le X,, X~, X~ il nostro spazio sar~ del tipo: ds' = dx]--~ ~ dx~-{- 2 ~ dx, dx,-~- Z dx~ +2 (x, ~ + #) dx, d x~
-~- 2 (x, Z -}- Z) d x~ d x3 -J- (x~ Z -[- 2 z, ), --[- ~) d x; , dove le ~ ~, 7., 3, ),, u sono funzioni di x,. dove le ~ ~, 7., 3, ),, u sono funzioni di x,. 78 Fubini:
Sugli spazii the ammettono Scrivendo le formule di KILLll~(3 relative a X~ abbiamo: Scrivendo le formule di KILLll~(3 relative a X~ abbiamo: [(c -
1) x, + z,] Y-kT,
~ "~'~ +
"~' (c -- a) ~ ~ x, + ,~h, (c -- 1) U~
~ :~' +
0 (c x: -- l~ x~)
~ (c x, -- l~ x,)
"q- (/is - -
X3
~Xa=
1 ~aik Sostituendovi i valori trovati delle aih~ e integrando otteniamo: Z ~ P:~ e~cl'x~
----- e a.(~-~)x, [ ~ 2 p,~ l~ e z,x, -I--p,,-[- 1] p,~ e ~,'~, ]
)==e~,(~÷,)., I z,
1
' 1)
p,~ e z(~-~)~' ~p~3
(si noti che c =,=
----= e~t'~' I
2 l,
et,(~_,)~, +
l~
p~ e a'(~-')x' + p~3 I
------ ecZx~ I c~--lli pt~ e(C_i)Z,x4__l 3 p~3 e l'x~
li ls
t
c --
i p'~ ed'~' + p,3 dove le p;k sono costanti effettive. dove le p;k sono costanti effettive. dove le p;k sono costanti effettive. 2. ° ripe). Anehe qui si pone d s ~ = d x~ + ): aih d xi d x~. ik E se ne deduce che le li sono effettive costanti. Per l'esistenza di X,,
X~, X3 si ha che ds ~ ~ del tipo gi~ citato al 1. ° case. p
g
Scrivendo le equazioni di KmLI~G relative alla X4-
l~ X,-
13 -¥2 e in-
tegrando, troviamo : Z ~P~ ea'x'
----- e ~z,x, (l~ p~2 e '~z.~' + 2 l, p~3 e t''~' + P3.0
q~ = e~Z'x' I-- (l, + 13) p..~ e z''~' -- 1, p.~3 x4 + p,~ ]
3' = e :z,', I p,3 -- l, (l, 2 r. ossia 13) p~ e ~l.x' -- (1, -~- 13) p23 e z'x' q- l, p,~ e l,x, --
--/~ p33 x4 -- l, 14 p~3 x4 e ~x' ,! ~ e'Z'~' I (l, -q- l~)~p~ e 'Z,`~, -- 2 (1, -t- 13)p,~ e 1.~ -}- 2 p~ l~ x, (1, H- 13) e Z.È~ -1-
+ 1", p~ x ~, -- 2 l~ l). z, + p. i Z ~P~ ea'x' dove le Pik sono eostanti. un gruppo continuo di movimenti. 79 Spazii che ammeltono un G~ integrabile a trasformazioni infinitesime in-
dipendenti, di eui il gruppo derivato ~ un G~. p
g
pp
Per il primo dei due casi di questo tipo, troviamo col solito proeedi-
mento delle pagine precedenti e con notazioni analoghe :. ds'= dx~ + ?dx~ + 2kdx, dx, + z dx~ + 2(x,~-~.2) dx, dxa+
+ 2 (x, >c -ki) dx~dx~ + (xl z + 2 x, X + t,)dx]
dove dove dove = e z'~' (-- h p~, e Z~' + ~,,)
---- -
-
2p,, l~ eZ, '~, q- l~p~.~ e ~hx~ + p,,
~, =
e'-Z. ~, (1, l, p,, x, +
p~,)
=
e<~, 12 l, l, p,, x~ + l~ l] zlp. + p,, I
=
e~'°°' I P,, +
l, 1, p,, x, -- 1, 1~ p,, e z.°°, -- l, p,a e z,'~, +
1, I, p.~, e z,~, --
-- 1, la 1, p,, x~ e z,~, ] . = e z'~' (-- h p~, e Z~' + ~,,)
---- -
-
2p,, l~ eZ, '~, q- l~p~.~ e ~hx~ + p,,
~, =
e'-Z. ~, (1, l, p,, x, +
p~,)
=
e<~, 12 l, l, p,, x~ + l~ l] zlp. + p,, I
=
e~'°°' I P,, +
l, 1, p,, x, -- 1, 1~ p,, e z.°°, -- l, p,a e z,'~, +
1, I, p.~, e z,~, --
-- 1, la 1, p,, x~ e z,~, ] . Studiamo il secondo tip() di tali gruppi. Studiamo il secondo tip() di tali gruppi. Esso eontiene un G~ a
trasformazioni
tipo 2. ° , dove si faccia h = 0. infinitesime indipendenti del tipo 2. ° , dove si faccia h = 0. ossia Sarh percib : d s' -- d x~ + ~ d x; ~ 2 ~ e*' d x, d x~ ~ S e '*, d x~ -~ ~ d x~ .-9
+ 2 Z dx, dx3 -4 2). e'~,dx~dx3 7 + 2 Z dx, dx3 -4 2). e'~,dx~dx3 7 dove le ?, ~,... sono funzioni di x4. dove le ?, ~,... sono funzioni di x4. Nell'espressione trovata per ]a 2/4 f saranno al solito ]e ~i vere costanti~
% come si vede percib tost% potremo porr% indicando con a~ ~ l, costanti 7 0
0
~
1
X, = ~ U~ + [~ e-~. ~
-t- x ~
. . . . ~x~
~x3
h
~x, Integrando le relative equazioni di KmLI~% e indicando con pil, delle
costanti~ abbiamo : fz ~" ]Oaa e ~l'x~ fz ~" ]Oaa e ~l'x~ Fubini:
Sugli spazii cite ammeUono 80 (
)
~------ 2~ p,~-
T Se ~ ~ 0, valgono le stesse formule, dove perb al termine--e"l'x' (ehe eom-
parisee in ~0, ~, Z) si deve sostituire il termine 14 X4 • Annali di Matematioa, Serie II[, tomo VIII. 14 X4 • Spazii che ammetlono un G, che contengono un G3 a trasformazioni in-
finitesime permutabili e le cui trasformazioni
infinitesime sono linearmente
indi2endenti. In tutti i tipi gik enumerati al § 11 i coeflleienti dell'elemento lineare
corrispondente saranno funzioni della sola x~. E noi potvemo porte: d s' = d xl -]-- ~ ai~ d xi d xt,
(i, k = 1, 2, 3). -$~P., Se ne hg. ehela %, ~, ~, l~ sono eostanti. g
Le equazioni di Kmrm~(~ diventano: g
Le equazioni di Kmrm~(~ diventano: 2
aik UY~ + ~' 0 x,
Z a,~
+ ~
az~
~
= 0
(i, l = 1, 2, 3). Sostituendovi per le ~i i loro valori 7 integrando, indieando con hia delle
costanti, troviamo : I °) a,, = h,, e a,x,
a~2 = h2~ e ~l'~x'
a33 == h~3 e ~lxx~
a,~ =
h,, e l,(i4aix,
a.~ -=- h,s e l,(~+c)x'
a,s ~ h,s e l'('+°)w'
a.~ = (l, h,, x, -1- h,~) e t,('+'c)x*
a,~--= e,h~x, (l h,, x, + h,~)
a~ = e a-*~, (l~ h,, x~ + 2 h,~ l, x, + ~) un gruppo conlinuo di movimenti. 81 III °)
IV °)
V ° )
1
a
3 l, h,,
° ÷ (h,, + h.,~) l, x, + h,~
a,, ---= l, h,~ x~ .{- 2 h,, 14 x, -[- h.,, ;
II
. a33 = -~ h,, xl --~ l~ h,~. x] -4- 14 (h,, + h.~) xi + 2 l, h,~ x, + h~
a.22 ~- h~, e?t.~, ;
a,~ = e:l. ~, [ h,..~ 1,, x, -4- h,, ] ;
a,, = e<~, (li h:~ x~ -4- 2 l, h,, x, A- h,,)
a,~ ~--e ~t,°~, h,3 --~ ~
x] -4- ~2- li h,2 x4 --}- (h,, + h23) l, x,
a~---- e *~,', h. II ¥ -t-- l~ h,, :r~ + (h,, -4- h:~) l~ z~ + 2 l, x, h,3 + h~
a,,~h,;
a~----h,~;
a,,----h,.;
a.,3=14h.~2x~Th~;
a,3 = l, h,~ X4 "-1 t- hia III °)
1
a
3 l, h,,
° ÷ (h,, + h.,~) l, x, + h,~ VI °)
]
[
,
a~3 ~
e ~,x, [ l, h,.. x, -[- h,s [
a23 ---- e ~t':e~ [ 14 h..a x, -[- h23 [ 11
|
https://openalex.org/W2801734876
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https://www.pure.ed.ac.uk/ws/files/58553714/1_s2.0_S0034425718301421_main.pdf
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Heterogeneous and rapid ice loss over the Patagonian Ice Fields revealed by CryoSat-2 swath radar altimetry
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Remote Sensing of Environment Edinburgh Research Explorer
Heterogeneous and rapid ice loss over the Patagonian Ice Fields
revealed by CryoSat-2 swath radar altimetry Edinburgh Research Explorer Citation for published version:
Foresta, L, Gourmelen, N, Weissgerber, F, Nienow, P, Williams, J, Shepherd, A, Drinkwater, MR &
Plummer, S 2018, 'Heterogeneous and rapid ice loss over the Patagonian Ice Fields revealed by CryoSat-2
swath radar altimetry', Remote Sensing of Environment. https://doi.org/10.1016/j.rse.2018.03.041 Citation for published version: Digital Object Identifier (DOI):
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Remote Sensing of Environment Heterogeneous and rapid ice loss over the Patagonian Ice Fields revealed by
CryoSat-2 swath radar altimetry L. Forestaa,⁎, N. Gourmelena,b, F. Weissgerbera, P. Nienowa, J.J. Williamsa, A. Shepherdc,
M.R. Drinkwaterd, S. Plummerd a School of GeoSciences, University of Edinburgh, UK
b Université de Strasbourg, CNRS, IPGS UMR 7516, F-67000 Strasbourg, France
c Centre for Polar Observation and Modelling, School of Earth and Environment, University of Leeds, UK
d European Space Agency, ESA-ESTEC, Keplerlaan 1, 2201 AZ Noordwijk, Netherlands A R T I C L E I N F O The Northern and Southern Patagonian Ice Fields (NPI and SPI) in South America are the largest bodies of ice in
the Southern hemisphere outside of Antarctica and the largest contributors to eustatic sea level rise (SLR) in the
world, per unit area. Here we exploit swath processed CryoSat-2 interferometric data to produce maps of surface
elevation change at sub-kilometer spatial resolution over the Ice Fields for six glaciological years between April
2011 and March 2017. Mass balance is calculated independently for nine sub-regions, including six individual
glaciers larger than 300 km2. Overall, between 2011 and 2017 the Patagonian Ice Fields have lost mass at a
combined rate of 21.29 ± 1.98 Gt a−1, contributing 0.059 ± 0.005 mm a−1 to SLR. We observe widespread
thinning on the Ice Fields, particularly north of 49° S. However the pattern of surface elevation change is highly
heterogeneous, partly reflecting the importance of dynamic processes on the Ice Fields. The Jorge Montt glacier
(SPI), whose tidewater terminus is approaching floatation, retreated ~2.5 km during our study period and lost
mass at the rate of 2.20 ± 0.38 Gt a−1 (4.64 ± 0.80 mwe a−1). In contrast with the general pattern of retreat
and mass loss, Pio XI, the largest glacier in South America, is advancing and gaining mass at 0.67 ± 0.29 Gt a−1
rate. Keywords:
CryoSat-2
Radar altimetry
Swath processing
Glacier elevation change
Patagonian ice fields https://doi.org/10.1016/j.rse.2018.03.041
Received 24 November 2017; Received in revised form 22 March 2018; Accepted 29 March 2018
0034-4257/ ©
2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecom
m
ons.org/licenses/BY/4.0/). ⁎ Corresponding author.
E-mail address: luca.foresta@ed.ac.uk (L. Foresta). Take down policy Take down policy
The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer
content complies with UK legislation. If you believe that the public display of this file breaches copyright please
contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and
investigate your claim. Download date: 24. Oct. 2024 1. Introduction comparatively high amounts of shortwave energy (Lenaerts et al.,
2014). Thus, the glaciers west and east of the ice divide are thought to
be more sensitive to changes in precipitation and air temperature re-
spectively (Rivera and Casassa, 1999; Warren and Sugden, 1993). The Northern and Southern Patagonian Ice Fields are the two largest
ice bodies in the Southern Hemisphere excluding Antarctica, with areas
of about 4200 and 13,000 km2 and volumes of about 1200 and
4300 km3 (Carrivick et al., 2016), respectively, and elevation ranging
from sea level to about 3900 m. They experience relatively warm and
wet climatic conditions (Sagredo and Lowell, 2012) and lie on top of
the narrow Andean mountain range, which forms an efficient barrier to
the predominantly westerly winds and moisture rich air transported
inland from the Pacific Ocean (e.g. Garreaud et al., 2013). The moun-
tain and associated ice divide separates areas with contrasting climatic
conditions. On the western side, orographic uplift of moist air produces
extreme annual precipitation of up to 10 m a−1 (e.g. Lenaerts et al.,
2014; Sakakibara and Sugiyama, 2014 and references within) as well as
extensive and persistent cloud coverage with associated low shortwave
and high longwave energy fluxes (Lenaerts et al., 2014). To the east of
the divide, the Ice Fields are in the rain shadow and receive The two Ice Fields have been experiencing long-term thinning and
retreat. Between the end of the Little Ice Age (LIA, ~1870) and 2011,
their area shrank by ~12.5% on average (Davies and Glasser, 2012),
associated with a combined mass loss of about 1.70 ± 0.25 Gt a−1
(Glasser et al., 2011). However, geodetic studies based on gravimetry
(Chen et al., 2007; Ivins et al., 2011; Jacob et al., 2012; Gardner et al.,
2013) and comparisons of Digital Elevation Models (DEM; Rignot et al.,
2003; Willis et al., 2012a, 2012b; Jaber, 2016) estimated that between
1975 and 2012 the rate of mass loss of the Ice Fields has been in the
range of 15 to 35 Gt a−1, one order of magnitude more compared to the
long term trend. 1. Introduction L2swath data are then used to compute rates of surface
elevation change for six glaciological years between April 2011 and
April 2017 using a plane-fit algorithm (e.g. McMillan et al., 2014b). One glaciological year is defined as the period between 1st April in year
n and 31st March in year n + 1. CS2's acquisition dates vary spatially
for different pixels due to the satellite's orbital path as well as to the
local topography, so that the temporal resolution at the pixel scale is
non-uniform and longer than monthly. However, seasonality biases are
avoided given the regular flight path followed by CS2, which ensures
that data within each pixel are acquired at the same epochs (within a
few days) in each glaciological year. L2swath data are gridded at
500 m × 500 m spatial resolution and for each pixel, we model eleva-
tion z(x,y,t) using a linear relationship in space and time: (Glasser et al., 2011 and references within), increasing to 15.4% in the
first decade of the 21st century (Jacob et al., 2012; Gardner et al.,
2013), second only to glaciers in Alaska and the Canadian Arctic, and
larger than high mountain Asia (Brun et al., 2017) which all extend
over areas ~5–8 times larger. Currently, the Patagonian Ice Fields are
the largest contributor to sea level rise per unit area in the world
(Gardner et al., 2013; Carrivick et al., 2016). Velocities of glaciers draining the Ice Fields (up to 10 km a−1) are
amongst the fastest in the world (Sakakibara and Sugiyama, 2014;
Mouginot and Rignot, 2015) and substantial ice flow acceleration has
been observed, coincident with rapid frontal retreat, for a number of
tidewater and lacustrine glaciers (Sakakibara and Sugiyama, 2014). These observations implicate the importance of the role of dynamics
and tidewater glacier calving in the rapid wastage of the Ice Fields. In
fact, > 80% of them terminate in proglacial lakes (mostly across the
NPI and east of the SPI) or fjords (western side of the SPI) (Sugiyama
et al., 2016 and references within). Since 2010, the European Space Agency (ESA) radar altimetry
mission CryoSat-2 (CS2) (Drinkwater et al., 2005; Wingham et al.,
2006) has been acquiring topography data over land ice. Radar in-
struments are particularly suited to land ice applications since they can
penetrate through clouds and do not depend on sunlight. 1. Introduction We iteratively fit the model to the data using 3σ clipping until
there are no more outliers. The formal uncertainty εḣ on each pixel's
rate of elevation change ḣ is extracted from the model covariance
matrix M: where x, y and t are measured easting, northing, and acquisition time,
respectively, and c0, c1, ḣ, c2 are the model coefficients. The time-de-
pendent coefficient ḣ retrieved from the model fit is the linear rate of
surface elevation change for each given pixel. Each observation is as-
signed a weight according to the sample power as in Gourmelen et al. (2017a). We iteratively fit the model to the data using 3σ clipping until
there are no more outliers. The formal uncertainty εḣ on each pixel's
rate of elevation change ḣ is extracted from the model covariance
matrix M: =
=
−
−
P
cov p
G
cov z
G
( )
( )[
]T
1
1 Despite their important contribution to ice mass loss and global SLR,
studies quantifying mass changes of the Patagonian Ice Fields are lim-
ited in number and do not cover the most recent period. This paper
focuses on quantifying the mass balance of the NPI and SPI during six
glaciological years between April 2011 and March 2017. To this aim,
we exploit swath processed CS2 SARin data to generate maps of surface
elevation change rates at sub-kilometer spatial resolution, and convert
them into estimates of glacier volume and mass change. For a number
of large catchments on the SPI, such estimates are derived at the basin
scale. Additionally, the dense L2swath elevation field enables the pro-
duction of time series of elevation change for different sub-regions of
the Ice Fields exhibiting contrasting patterns of change. where p is the vector of coefficients [c0 c1 ḣ c2] of the model para-
meters, z are the input elevations and G = [x y t 1] is the model matrix. We simplify the data covariance matrix cov(z) to a variance matrix
whose diagonal values are the squared elevation differences between
the observed and modelled estimates (z-z′)2. The square root of the
diagonal elements of P represents the standard deviations of the model
parameters p. Due to the complex topography (see Discussion), the ḣ maps do not
have complete coverage. 1. Introduction We use the relation between elevation and
elevation change to model estimates for the gaps in the maps of surface
elevation change rates (i.e. hypsometric averaging, see e.g. Moholdt
et al., 2010; Nilsson et al., 2015a; Foresta et al., 2016), using the SRTM
DEM for the elevation field. Polynomial models of order 1 to 3 are fitted
to the data and used to generate elevation change rates for the in-
dividual pixels without an estimate. In order to avoid over-fitting the 1. Introduction During the last 50 years, the Patagonian Ice Fields contributed an
estimated 10% to the total mass loss from glaciers and ice caps, ex-
cluding those at the periphery of the Greenland and Antarctic ice sheets https://doi.org/10.1016/j.rse.2018.03.041
Received 24 November 2017; Received in revised form 22 March 2018; Accepted 29 March 2018
0034-4257/ ©
2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecom
m
ons.org/licenses/BY/4.0/). Please cite this article as: Foresta, L., Rem
ote Sensing of Environm
ent (2018), https://doi.org/10.1016/j.rse.2018.03.041 e this article as: Foresta, L., Rem
ote Sensing of Environm
ent (2018), https://doi.org/10.1016/j.rse.2018.03.041 Rem
ote Sensing of Environm
ent xxx (xxxx) xxx–xxx L. Foresta et al. this technique (Hawley et al., 2009; Gray et al., 2013; Foresta et al.,
2016; Gourmelen et al., 2017a) can generate up to two orders of
magnitude more data than conventional Point-Of-Closest-Approach
(POCA) processing and, importantly, provides more homogeneous
spatial coverage over relatively small glaciated regions with consider-
able topography (Foresta et al., 2016; Gourmelen et al., 2017a). The
L2swath processing scheme is similar to Foresta et al. (2016) and
Gourmelen et al. (2017a), but we use a different procedure to discard
noisy waveform samples before performing the phase unwrapping. This
procedure, first developed for InSAR images (Weissgerber, 2016) and
updated for CS2 SARIn data (Weissgerber et al., 2018), was shown to
further increase the density of the L2swath elevation field and to im-
prove the spatial coverage of the Jakobshavn glacier, Greenland
(Weissgerber and Gourmelen, 2017) (Appendix A). The L2swath algo-
rithm makes use of an external DEM to improve the precision of ele-
vation measurements in the presence of slopes larger than 0.54°, where
an entire waveform may be affected by a phase shift. Without this
correction, observations may be wrong by several tens of meters in
elevation and a few kilometres in geo-location (Gourmelen et al.,
2017a). It is not straightforward to predict the accuracy needed for the
DEM (Gourmelen et al., 2017a). However given the magnitude of the
geolocation and elevation shifts, the DEM need not be extremely ac-
curate. We used the SRTM C band DEM (Farr et al., 2007) acquired in
2000 as a reference for elevation, after including a correction to ac-
count for the elevation change occurred between 2000 and 2011
(Appendix B). 1. Introduction Radar alti-
metry data have been previously exploited to map elevation change
over ice caps (Rinne et al., 2011a, 2011b), but the technique has not
been applied widely due to the limitation caused by the large radar
footprint. CS2's state-of-the-art radar altimeter uses Synthetic Aperture
Radar (SAR) along-track to reduce the footprint size as well as inter-
ferometry across-track to accurately locate the position of the surface
reflection (SARIn mode; Wingham et al., 2006). Additionally, its orbit
inclination of 92° and repeat cycle of 369 days provides an inter-track
spacing of ~5 km on average over the Patagonian Ice Fields. Finally,
CS2's relatively short wavelength (2.2 cm; Ku band) restricts the pe-
netration of the radar pulse in the snowpack, compared to, e.g., sensors
working in C or X bands. These characteristics make CS2 better suited
for monitoring changes in glacier areas with frequent cloud cover and
considerable slopes. CS2 SARIn data have successfully mapped topo-
graphic changes over Arctic ice caps (McMillan et al., 2014a; Gray
et al., 2015). Additionally, swath processing (Hawley et al., 2009) of
CS2 SARIn data has been applied to generate high resolution DEMs of
ice caps and selected areas of the Greenland and Antarctic ice sheets
(Gray et al., 2013; Ignéczi et al., 2016; Gourmelen et al., 2017a) and to
produce sub-kilometer maps of surface elevation change (Christie et al.,
2016; Foresta et al., 2016; Gourmelen et al., 2017a; Gourmelen et al.,
2017b), with a wide range of applications such as the identification of
supraglacial lakes in NE Greenland (Ignéczi et al., 2016), subglacial
lakes in West Antarctica and regions of subsidence in Iceland (Smith
et al., 2017; Gourmelen et al., 2017a) as well as quantifying channe-
lized basal melt under the Dotson ice shelf in West Antarctica
(Gourmelen et al., 2017b) and volume and mass change of individual
ice caps in Iceland (Foresta et al., 2016). =
+
+
+
z x y t
c x
c y
ht
c
( , , )
̇
0
1
2 where x, y and t are measured easting, northing, and acquisition time,
respectively, and c0, c1, ḣ, c2 are the model coefficients. The time-de-
pendent coefficient ḣ retrieved from the model fit is the linear rate of
surface elevation change for each given pixel. Each observation is as-
signed a weight according to the sample power as in Gourmelen et al. (2017a). 2. Data and methods We exploit swath processed CS2 SARIn baseline C data (L2swath) as 2 Rem
ote Sensing of Environm
ent xxx (xxxx) xxx–xxx L. Foresta et al. Combining data from different glaciers is needed if coverage is insufficient
to provide a representative figure of elevation change in each and is jus-
tified providing that they show a similar pattern of change. data, the F-test is used to evaluate if the improvement of the additional
model parameters on the fit is statistically significant at the 99% con-
fidence level. The median rate of elevation change is then computed in
each 50 m elevation band k and multiplied to the area Ak, extracted
from the DEM, to produce elevation dependent volume change V̇ k. The
sum of these contributions represents the total rate of volume change. Uncertainties are calculated by error propagation using the same
method as in Foresta et al. (2016), summarized in Appendix C. The observed median rates of elevation change for these nine sub-
regions are shown as a function of cumulative surface aerial extent
(10% steps; Fig.1, side panels) and as a function of elevation (50 m
steps; Fig.2, side panels). Widespread thinning is occurring in the
northern part of the SPI across all elevations, with average rates of
2 m a−1 on the plateau up to and above 1400 m elevation (SPI-G1) and
in excess of 10 m a−1 at the terminus margins of Jorge Montt (tide-
water), Viedma and Upsala (both lacustrine) glaciers. Most glaciers in
the south/southwest are close to balance (SPI-G2), with the exception of
Grey and Tyndall on the eastern side. Pio XI glacier is thickening at
rates of ~2 and 1 m a−1 below 1000 m and between 1000 and 1500 m
elevation, respectively, and thinning by about 1 m a−1 above 1500 m
altitude. Similar to the northern part of the SPI, the NPI is experiencing
widespread thinning of up to 8 m a−1 with the exception of the ice
divide close to the eastern margin of the ice field (Figs. 1 and 2). Hypsometric averaging is applied in each sub-region (Fig. 2, red lines)
to generate maps of modelled elevation change rates for the Ice Fields
(Fig. 2), from which mass change is computed (Table 1). The poly-
nomial models (Fig. 2, red lines) compare well with the observed
median rates of elevation change (Fig. 2, black lines with dots). 2. Data and methods A few
exceptions are visible over the Tyndall and Upsala glaciers (at low and
high elevation respectively) as well as over the Pio XI glacier below
1000 m and above 2500 m elevation. Model misfits have marginal im-
pact on the glacier mass change when glacier area is negligible or data
coverage is high (Fig. 2). For example the rate of mass loss of glacier
Tyndall, assuming no elevation change below 350 m elevation, is re-
duced by about 9% (or 0.054 Gt a−1), which is well within its asso-
ciated uncertainty (Table 1). Similarly for Pio XI glacier, the thinning
predicted by the model above 2500 m elevation reduces the glacier net
mass gain by only 6% (or 0.04 Gt a−1). However, at low elevation
where the area of Pio XI glacier is not negligible and where there are no
observations to constrain the elevation change (Figs. 1 and 2), the
impact of the misfit on the glacier mass balance may be significant (see
Discussion). Between April 2011 and April 2017, the NPI and SPI have
been
losing
mass
at
rates
of
−6.79 ± 1.16
and −14.50 ±
1.60 Gt a−1, respectively, contributing 0.059 ± 0.005 mm a−1 to eu-
static SLR. About 35% of the SPI mass loss is concentrated on glaciers in
the SPI-G1 group (−5.07 ± 0.79 Gt a−1), which represent ~28% of
the SPI surface. The Upsala glacier is the single largest contributor to
the mass loss (−2.68 ± 0.40 Gt a−1) and is also the glacier with the
second highest rate of loss per unit area (Table 1) after Jorge Montt. Both glaciers have retreated between 2011 and 2017, by about 0.5 and
2.5 km, respectively. Pio XI is the only glacier in the Patagonian Ice
Fields with positive mass balance (0.67 ± 0.29 Gt a−1). Its southern
tidewater and northern lacustrine termini have both advanced, re-
spectively by about 500 m and 800 m during our study period. Using a
dual density scenario, the rates of mass change in the nine sub-regions
are lower by 11–19% compared to using the density of glacial ice at all
elevations and the total rate of mass loss of the Ice Fields is
17.89 ± 2.03 Gt a−1 (Table 2). For most basins, dynamic processes are
dominating the mass loss and hence the ice density scenario is the
preferred option. 2. Data and methods However, in a few sectors the dual density scenario
may be more accurate. For example, over the Pio XI glacier (SPI), where
surface thickening is suspected (Malz et al., 2018), the mass change is
0.67 ± 0.29 Gt a−1 and 0.57 ± 0.25 Gt a−1 for the single and dual
density scenarios, respectively. Ti
i
f
b
d l
i
l
ti
h
(Fi
3) h This interpolation scheme is applied independently for the NPI and
for different sub-regions of the SPI displaying highly contrasting pat-
terns of change at similar elevations (Fig. 2). Finally, we assume that all
changes relate to the gain or loss of ice of density ρice = 900 kg m−3
when converting the rate of volume change to mass balance. This
simplification is based on the assumption that at least part of the ob-
served changes are due to dynamics (see Discussion) and ignores pos-
sible differences in snow pack density below and above the firn line. To
explore mass loss related to material with lower density, we calculate
mass balance estimates using a dual density scenario. In this case the
densities of glacial ice and firn are used when converting volumetric
changes occurring, respectively, in the ablation and accumulation
areas. We assign ρfirn = 600 kg m−3 (Malz et al., 2018 and references
within). We used Equilibrium Line Altitude (ELA) values as reported in
De Angelis (2014) and Barcaza et al. (2009), respectively, for the gla-
ciers on the SPI and NPI. For each group of glaciers (SPI-G1, SPI-G2,
NPI), we computed an average ELA from all glaciers with surface area
larger than 100 km2 in the given catchment. Glacier outlines over the Ice Fields record their extent in 2000–2001
(RGI Consortium, 2017) and the Upsala and Jorge Montt glaciers (SPI)
have retreated considerably since then and, for the latter, even during our
study period. Their front positions in 2017 were manually digitized using
Landsat8 scenes (Appendix D, Table D1) and their mass loss between 2011
and 2017 is calculated against their updated fronts. Area changes between
2011 and 2017 are not included in our estimates of mass loss. The only
exception is Jorge Montt (SPI), which retreated considerably in this time
period and for which we provide an additional estimate of mass loss due to
area change. 2. Data and methods The front outline of the Jorge Montt and Upsala, as well as of
Pio XI (SPI), was additionally digitized for a number of years between
2005 and 2017 (Appendix D, Table D1). This data is used for context in the
Discussion (subsections Jorge Montt, Upsala and Pio XI) and is not em-
ployed for calculating mass balance. Finally, we produce time series of mean observed glacier elevation
change with the same methodology as Gray et al. (2015) and Foresta
et al. (2016). The time series are generated at the catchment scale for
each of the nine sub-regions with 90 (Pio XI, SPI-G2, SPI) or 120 days
time step (Fig. 3). 3. Results Swath processing of CryoSat-2 SARIn data provides 6.7 and 26.6 mil-
lion valid observations of ice topography over the NPI and SPI, respec-
tively, with the rate of elevation change for a single pixel being constrained
by ~1700 elevations (median) over a period of 5.6 years (median) between
April 2011 and March 2017. For comparison conventional CS2 POCA de-
livers about 17,000 and 55,000 observations over the NPI and SPI re-
spectively. Fig. 1 displays the maps of observed rates of elevation change
over the Ice Fields. On the SPI, different catchments show distinct patterns
of change over the study period (Fig. 1). Given such heterogeneity, we
apply the hypsometric averaging model independently for six large glaciers
on the SPI, namely Jorge Montt, Viedma, Upsala, Pio XI, Grey and Tyndall. The spatial coverage of ḣ estimates, at 500 m posting, ranges between 61
and 73% of total catchment areas (Table 1), with the exception of Grey and
Tyndall (~52%). We combine data from the rest of the SPI in two groups of
neighbouring glaciers, labelled SPI-G1 and SPI-G2. The former includes all
glaciers north of Pio XI and Viedma excluding Jorge Montt, while the latter
is composed of all the glaciers west and south of Upsala excluding Grey and
Tyndall (Fig. 1). Over the NPI, all glaciers are analysed together. Time series of mean observed glacier elevation change (Fig. 3) show
negative trends for all sub-regions with the exception of Pio XI, which
shows increasing elevation. Most sub-regions display a seasonal oscil-
lation on the order of 1–3 m. The amplitude is highest (4 m) for the Grey
Glacier, while it is less discernible for glaciers with the strongest mass
losses per unit area (Jorge Montt and Upsala), possibly reflecting the
importance of dynamic thinning also during the accumulation period. 3 Rem
ote Sensing of Environm
ent xxx (xxxx) xxx–xxx L. Foresta et al. Maps of observed rates of surface elevation change of the Northern and Southern Patagonian Ice Fields between April 2011 and March 2017 based
h elevations. The insets show observed median rates of elevation change (black lines with dots) against cumulative glacier surface area (10% steps), to Maps of observed rates of surface elevation change of the Northern and Southern Patagonian Ice
elevations. The insets show observed median rates of elevation change (black lines with dots) aga
uncertainty envelope (grey shade). 3. Results Elevation (non-linear) is also shown for clarity. Fields between April 2011 and March 2017 base
ainst cumulative glacier surface area (10% steps) Maps of observed rates of surface elevation change of the Northern and Southern Patagonian Ice Fields between April 2011 and March
elevations. The insets show observed median rates of elevation change (black lines with dots) against cumulative glacier surface area (1
uncertainty envelope (grey shade). Elevation (non-linear) is also shown for clarity. served rates of surface elevation change of the Northern and Southern Patagonian Ice Fi
. The insets show observed median rates of elevation change (black lines with dots) agai
ty envelope (grey shade). Elevation (non-linear) is also shown for clarity. Fig. 1. Maps of observed rates of surface elevation change of the Northern and Southern Patagonian Ice Fields between April 2011 and March 2017 based on CS2
L2swath elevations. The insets show observed median rates of elevation change (black lines with dots) against cumulative glacier surface area (10% steps), together
with the uncertainty envelope (grey shade). Elevation (non-linear) is also shown for clarity. Rem
ote Sensing of Environm
ent xxx (xxxx) xxx–xxx L. Foresta et al. n change of the Northern and Southern Patagonian Ice Fields between April 2011 and March 2017 based
ian rates of elevation change (black lines with dots) against elevation (50 m bands), together with the pol
vation change (not shown for clarity). The normalized histograms of the distribution of glacier area a
inuous line and shaded patch, respectively). Maps of modelled rates of surface elevation change of the Northern and Southern Patagonian Ice Fields be
h elevations. The insets show observed median rates of elevation change (black lines with dots) against elev
red line) fitted to the original rates of elevation change (not shown for clarity). The normalized histogr
e versus elevation are shown in grey (continuous line and shaded patch, respectively). modelled rates of surface elevation change of the Northern and Southern Patagonian Ice
ons. The insets show observed median rates of elevation change (black lines with dots) ag
) fitted to the original rates of elevation change (not shown for clarity). The normalize
elevation are shown in grey (continuous line and shaded patch, respectively). Maps of modelled rates of surface elevation change of the Northern and Southern Patagonian Ice Fields between April 2011 and March 2017 based
th elevations. 4.1. Spatial coverage Furthermore, using a single sensor
and frequent repeat measurements is advantageous as it limits penetration
biases associated both with using multiple sensor types (e.g. optical vs radar
or radar with varying wavelengths) and seasonal variations in surface mass
density, thereby limiting the impact on surface elevation change estimates
(Jaber et al., 2013; Jaber, 2016; Willis et al., 2012b; Malz et al., 2018). Given
the similarity of the Patagonian Ice Fields to mountain glaciers, swath alti-
metry may also provide one additional tool for monitoring elevation change
over these complex areas (Paul et al., 2015). 4.1. Spatial coverage The Patagonian Ice Fields are a challenging region for radar altimetry. The topography is similar to mountain glaciers, with elevation ranging from
sea level to above 2000 m over distances of < 30 km. Furthermore, the flow
of most glaciers on the Ice Fields is almost perpendicular to CS2's approxi-
mately north-south flight direction so that elevations change abruptly
(> 1000 m) over short distances (400 m) along the flight track (Fig. 4), in-
creasing the occurrence of loss-of-lock in the altimetric record and leading to
gaps in the collected data (Dehecq et al., 2013). Over the Southern Pata-
gonian ice field, conventional CS2 POCA altimetry provides about 30%
spatial coverage at 500 m posting. Although swath altimetry is affected by
loss-of-lock as much as POCA, enhanced spatial coverage is achieved because
a swath of heights, rather than one single elevation, is acquired when the on
board tracker correctly sets the range window. Swath altimetry thereby
provides 61–73% surface coverage over the large (A > 400 km2) glaciers in
the northern part of the SPI and between 47% and 54% in other areas
(Table 1). The only exception is SPI-G2 (39%), where the ice field is narrower
and where there are no observations over a number of glaciers with rela-
tively small surface area (Fig. 1 and Fig. 2, inset). Despite the limited extent,
their mass loss may be non-negligible. For example HPS12 (south of Pio XI)
has an area of 165 km2 and lost 0.63 Gt a−1 between 2000 and 2011/12
(Willis et al., 2012b). In comparison, work based on high resolution radar
TanDEM-X DEMs have almost complete coverage at higher spatial resolution
(Jaber et al., 2013; Jaber, 2016; Malz et al., 2018), although this data does
not allow yet to generating time series of elevation change. Compared to
TanDEM-X DEMs, optical ASTER DEMs achieve similarly high spatial Fig. 3. Time series of cumulative mean observed elevation change for the nine
sub-regions (Table 1), including the SPI as a whole (grey), in order of in-
creasingly negative specific mass balance (top to bottom). coverage for decadal periods, decreasing to 57–73% for the entirety of the
Ice Fields over shorter time periods comparable to that in this paper (Willis
et al., 2012b). Despite CS2 L2swath's lower spatial coverage, we still capture
in detail the various patterns of change. Table 2 Estimates of mass balance Ṁ [Gt a−1] for the NPI and individual sub-regions of
the SPI based on CS2 L2swath data at 500 m spatial resolution using two dif-
ferent density scenarios (see text). ELA [m]
Single density
Ṁ [Gt a−1]
Dual density
Ṁ [Gt a−1]
Abs Diff
Ṁ [Gt a−1]
NPI
1005
−6.79 ± 1.16
−5.67 ± 1.26
1.13
Jorge Montt
930
−2.20 ± 0.38
−1.96 ± 0.41
0.25
Upsala
1170
−2.68 ± 0.40
−2.29 ± 0.46
0.39
Viedma
1260
−2.27 ± 0.36
−1.90 ± 0.40
0.38
SPI G1
1077
−5.07 ± 0.79
−4.17 ± 0.90
0.90
SPI G2
1096
−1.66 ± 1.16
−1.35 ± 1.03
0.31
Tyndall
940
−0.60 ± 0.14
−0.53 ± 0.14
0.07
Grey
980
−0.69 ± 0.23
−0.59 ± 0.20
0.09
Pio XI
930
+0.67 ± 0.29
+0.57 ± 0.25
0.10
NPI + SPI
−21.29 ± 1.98
−17.89 ± 2.03
3.40 3. Results The insets show observed median rates of elevation change (black lines with dots) against elevation (50 m bands), together with the poly
(red line) fitted to the original rates of elevation change (not shown for clarity). The normalized histograms of the distribution of glacier area an
ge versus elevation are shown in grey (continuous line and shaded patch, respectively). ge of the Northern and Southern Patagonian Ice
tes of elevation change (black lines with dots) ag
h
(
h
f
l
i
) Th
li Fig. 2. Maps of modelled rates of surface elevation change of the Northern and Southern Patagonian Ice Fields between April 2011 and March 2017 based on CS2
L2swath elevations. The insets show observed median rates of elevation change (black lines with dots) against elevation (50 m bands), together with the polynomial
model (red line) fitted to the original rates of elevation change (not shown for clarity). The normalized histograms of the distribution of glacier area and data
coverage versus elevation are shown in grey (continuous line and shaded patch, respectively). 5 Rem
ote Sensing of Environm
ent xxx (xxxx) xxx–xxx L. Foresta et al. Fig. 3. Time series of cumulative mean observed elevation change for the ni
ub-regions (Table 1), including the SPI as a whole (grey), in order of i
creasingly negative specific mass balance (top to bottom). Table 1 Estimates of mass balance Ṁ [Gt a−1] and specific mass balance [mwe a−1] as
well as area A [km2] and spatial coverage [%] of the maps of surface elevation
change rates ḣ for the NPI and individual sub-regions of the SPI based on CS2
L2swath data at 500 m spatial resolution. Frontal retreat of Jorge Montt (SPI)
amounts to an additional ~0.07 Gt a−1 (see Discussion). A [km2]
ḣ Coverage [%]
Ṁ [Gt a−1]
mḃ
[mwe a−1]
NPI
4046.4
45.7
−6.79 ± 1.16
−1.68 ± 0.29
Jorge Montt
474.4
68.0
−2.20 ± 0.38
−4.64 ± 0.80
Upsala
863.1
61.3
−2.68 ± 0.40
−3.11 ± 0.46
Viedma
992.3
72.7
−2.27 ± 0.36
−2.29 ± 0.36
SPI G1
3570.1
47.4
−5.07 ± 0.79
−1.42 ± 0.22
SPI G2
4829.5
39.1
−1.66 ± 1.16
−0.34 ± 0.24
Tyndall
332
49.9
−0.60 ± 0.14
−1.81 ± 0.42
Grey
333.3
54.0
−0.69 ± 0.23
−2.07 ± 0.69
Pio XI
1242.6
65.0
+0.67 ± 0.29
+0.54 ± 0.23
SPI total
12,637.2
49.9
−14.50 ± 1.60
−1.15 ± 0.13
NPI + SPI
16,683.6
−21.29 ± 1.98
−1.28 ± 0.12 A [km2]
ḣ Coverage [%]
Ṁ [Gt a−1]
mḃ
[mwe a−1] 4.2. Rates of mass change Spatial patterns in the rates of surface elevation change (Figs. 1 and 2)
are comparable with those observed over the period 2000–2011/16 (Willis
et al., 2012a, 2012b; Jaber et al., 2013; Jaber, 2016; Malz et al., 2018). The 6 Rem
ote Sensing of Environm
ent xxx (xxxx) xxx–xxx L. Foresta et al. NPI and the northern part of the SPI (SPI-G1, Jorge Montt, Viedma and
Upsala) are thinning very rapidly and account for 89% of the mass loss of
the Patagonian Ice Fields between 2011 and 2017 (Table 1). The rate of
mass loss of the Patagonian Ice Fields has increased in recent decades
(Fig. 5a), with our estimated mass loss rate (21.29 ± 1.98 Gt a−1) being
46% higher than between 1944 and 1996 (Aniya, 1999), 42% higher than
between 1975 and 2000 (Rignot et al., 2003) and 24% higher than between
2000 and 2012/14 (Jaber, 2016). However, for the period 2000–2011/12
Willis et al. (2012b) estimate a total rate loss of 24.39 ± 1.20 Gt a−1,
comparable to those based on gravimetry data (Chen et al., 2007; Ivins
et al., 2011; Jacob et al., 2012; Gardner et al., 2013) but 30% more negative
than that of Jaber (2016) (Fig. 5a). GRACE-based estimates rely on model
predicted corrections for postglacial rebound and land water storage, which
are a large source of uncertainty to the estimated rates of mass loss in Pa-
tagonia (e.g. Chen et al., 2007; Jacob et al., 2012). Additionally, mass loss
from glaciers and ice fields in the vicinity (Möller and Schneider, 2008;
Melkonian et al., 2013; Falaschi et al., 2017) may impact on the estimate
since gravimetry methods are always sensitive to mass leakage effects from
neighbouring areas (e.g. Sørensen et al., 2017). The disagreement between
Willis et al. (2012b) and Jaber (2016) may be related to the 2 m elevation correction applied to the SRTM data by Willis et al. (2012b) in order to
account for potential radar penetration through the glacier surface. How-
ever, analysis of the SRTM mean backscattering coefficient suggests wet
surface conditions on the Ice Fields at the time of the SRTM acquisition
(Jaber, 2016), which would be expected to prevent the radar signal from
penetrating through the surface (Nilsson et al., 2015b). Additionally,
Dussaillant et al. (2018) find that radar penetration over the NPI occurs only
above 2900 m elevation, i.e. < 0.75% of the ice field's area. 4.4. Jorge Montt glacier Jorge Montt, a tidewater glacier at the northernmost tip of the SPI, has
been retreating since 1898 when it reached its LIA maximum extent (Rivera
et al., 2012b). Its recession has been linked to fjord water depth, with periods
of stable front positions corresponding to shallow depths and underwater
pinning points (Rivera et al., 2012b). Additionally, water temperatures at
depth (> 100 m) have been shown to be as high as 8 °C in summer 2012 only
1 km from the glacier front (Moffat, 2014), which may further destabilise the
glacier through submarine melting (Straneo and Heimbach, 2013). By 2011
Jorge Montt had retreated almost 20 km w.r.t 1898, with the highest rates of
recession between 1990 and 1997 (993 m a−1) occurring when water depths
beneath the glacier increased sharply to ~300 m (Rivera et al., 2012b). The
recent retreat history of Jorge Montt's reveals a slowdown to about
100–300 m a−1 in the early 2000s, followed by increased retreat after 2009
initiated at a location where bathymetry data reveals the deepest trough in
the fjord (Rivera et al., 2012b; Moffat, 2014; Fig. 7). Between 2010 and 2011, Jorge Montt retreated almost 1 km (Rivera
et al., 2012b; Rivera et al., 2012b) and calved at a rate of 2.4 km3 a−1, when
the terminus was floating (Rivera et al., 2012a). Manual delineation of the
glacier front between 2011 and 2017 using Landsat optical data (Fig. 7)
indicates that Jorge Montt retreated by an additional ~2.5 km, likely
through enhanced calving following retreat into deeper water (Rivera et al.,
2012a). Given an average glacier freeboard height of 22 m above sea level
at the terminus (Rivera et al., 2012a, 2012b) and width of 1.15 km, the
glacier frontal retreat amounts to a mass loss rate of 0.07 Gt a−1 (~3% of
the catchment's loss due to thinning, Table 1) between 2011 and 2017. This
value is however likely underestimated since the slope of the glacier surface,
and thus upglacier thickening, has not been considered. Due to the un-
certainty associated with this calculation, and that at least part of the glacier
terminus was floating in 2010/11 (Rivera et al., 2012a) and likely during
our observational period, we report this loss separately in Table 1 and do
not include it in our total estimate of glacier contribution to sea level rise. 4.2. Rates of mass change In absolute
value, this correction has a larger impact on the SPI than on the NPI (Willis
et al., 2012b), where the estimates from Willis et al. (2012b) and Jaber
(2016) differ by only ~10%. Separating the contributions of the Ice Fields (Fig. 5b) shows the dif-
ference in the progressive increase in mass loss between the NPI and SPI. Between 2011 and 2017 the NPI's rate of loss (6.79 ± 1.16 Gt a−1) is
~70% more negative compared to the previous decade (about 4 Gt a−1,
Willis et al., 2012a, 2012b; Jaber, 2016; Dussaillant et al., 2018), which in
turn was ~37% higher than between 1975 and 2000 (Rignot et al., 2003)
(Fig. 5b). Compared to the latter, Rivera et al. (2007) record higher rates of
mass loss in a similar time period (1979–2001) (Fig. 5b), but their estimate
is based on data mostly lying in the ablation zone of the NPI. In contrast, the g. 4. (left panel) Example ascending and descending CS2 sub-satellite tracks (red) displayed over the SRTM topography for the SPI. Glacier outlines from R
otted in black. (right panel) Along-track topography (black) for the same sub-satellite tracks (red). For reference, the SPI (light blue) is shown in the ba
7 Fig. 4. (left panel) Example ascending and descending CS2 sub-satellite tracks (red) displayed over the SRTM topography for the SPI. Glacier outlines from RGI v6 are
plotted in black. (right panel) Along-track topography (black) for the same sub-satellite tracks (red). For reference, the SPI (light blue) is shown in the background. Fig. 4. (left panel) Example ascending and descending CS2 sub-satellite tracks (red) displayed over the SRTM topography for the SPI. Glacier outlines from RGI v6 are
plotted in black. (right panel) Along-track topography (black) for the same sub-satellite tracks (red). For reference, the SPI (light blue) is shown in the background. 7 mass loss over the SPI varies by just 8 10% between these three periods
4 4 Jorge Montt glacier
Fig. 5. Estimates of mass balance Ṁ [Gt a−1] for the Patagonian Ice Fields combined (a) and separate (b) as published in the literature as well as calculated in this study. Note that the time line is not linear. The estimate from Gardner et al. (2013) is for the whole of the Southern Andes. In (b) different shades are used for visual clarity. L. Foresta et al. 4.4. Jorge Montt glacier Between 2011 and 2017, Jorge Montt shows the highest mass loss per unit
area, 4 times above the average for the SPI as a whole (Table 1). Its absolute
rate of mass loss (2.20 ± 0.38 Gt a−1) is comparable to what reported by
Jaber (2016) for the period 2011–2014 (2.59 Gt a−1, uncertainty not re-
ported), which increased by 50% compared to the 1.72 Gt a−1 (uncertainty
not reported) rate of mass loss between 2000 and 2011 (Jaber, 2016). 4.3. Glacier dynamics We observe a sharp transition around 49° S (Figs. 1–2, dashed green
line) from intense thinning in the north to a large area facing limited mass
loss (SPI-G2), which spans about 4800 km2 or about 40% of the total surface
of the SPI (Table 2). This pattern is in agreement with earlier observations
between 2000 and 2012/16 (Willis et al., 2012b; Jaber et al., 2013; Jaber,
2016; Malz et al., 2018), therefore suggesting constant behaviour over
decadal time scales. The topography of the northern and southern parts of
the SPI has different characteristics, with a greater proportion of the
northerly ice field lying at lower altitudes. In fact, about 72% of SPI-G1's
surface lies below 1500 m elevation, ~12% more than SPI-G2's at the same
altitude (Fig. 1, insets SPI-G1 and SPI-G2). The Ice Field also narrows and
steepens south of 49° S and even at low elevations the southern SPI shows
only moderate thinning (Fig. 6). Glaciers Grey and Tyndall, at the south-
eastern tip, are the exception to this pattern. However the latter lies almost
entirely below 1500 m altitude (Fig. 1, inset Tyndall) and both glaciers re-
ceive scarce precipitation due to their location east of the ice divide. The NPI
has similar area-altitude distribution as SPI-G1 (Fig. 1, inset SPI-G1 and NPI)
and the two areas show comparable mass losses per unit area (Table 1). However, the northern part of the SPI contains some of the fastest
flowing glaciers on the Ice Fields, including Jorge Montt and Upsala
(Sakakibara and Sugiyama, 2014; Mouginot and Rignot, 2015) which
accelerated significantly (> 500 m a−1) in the period 1984–2000 (Jorge
Montt) and 2000–2010 (Upsala), coincident with rapid frontal retreat
(Sakakibara and Sugiyama, 2014). These observations confirm the im-
portance of dynamics in impacting the overall mass balance of the
northern part of the SPI (e.g. Sakakibara and Sugiyama, 2014; Mouginot
and Rignot, 2015), where three of the largest glaciers (Jorge Montt, Upsala
and Viedma) are thinning very rapidly (Table 1 and Fig. 1–3). 4.2. Rates of mass change Rem
ote Sensing of Environm
ent xxx (xxxx) xxx–xxx Rem
ote Sensing of Environm
ent xxx (xxxx) xxx–xxx L. Foresta et al. i
i
f
b l
̇ [
1] f
h
i
i ld
bi
d Fig. 5. Estimates of mass balance Ṁ [Gt a−1] for the Patagonian Ice Fields combined (a) and separate (b) as published in the litera
Note that the time line is not linear. The estimate from Gardner et al. (2013) is for the whole of the Southern Andes. In (b) diffe Fig. 5. Estimates of mass balance Ṁ [Gt a−1] for the Patagonian Ice Fields combined (a) and separate (b) as published in the literature as well as calculated in this study. Note that the time line is not linear. The estimate from Gardner et al. (2013) is for the whole of the Southern Andes. In (b) different shades are used for visual clarity. mass loss over the SPI varies by just 8–10% between these three periods
(Rignot et al., 2003; Jaber, 2016; Malz et al., 2018) (Fig. 5b) although its
rate of loss is still more than twice that of the NPI. Estimates from Jaber
(2016) and Malz et al. (2018), both based on comparing TanDEM-X data to
the SRTM DEM, differ by 1.29 Gt a−1 although they agree within their
uncertainties. The difference may be due to the 4 years longer time period
analysed by Malz et al. (2018), who report positive elevation changes in the
southernmost part of the SPI between 2011/12 and 2015/16. We observe
only a slightly positive trend in elevation change in this time period, fol-
lowed by a marked drop after 2015/16 (Fig. 3, SPI-G2). However, our time
series for SPI-G2 is representative of an area roughly twice as large than that
analysed by Malz et al. (2018) over multiple time periods. Finally, the es-
timated SPI's rate loss of 34.83 ± 3.96 Gt a−1 between 1995 and 2000
(Rignot et al., 2003), which is even more negative than any estimate for
both Ice Fields combined (Fig. 5a), appears out of line with other values. 4.5. Upsala glacier 20–50% (Sakakibara et al., 2013; Mouginot and Rignot, 2015) and thin-
ning at a maximum rate of ~40 m a−1 near the terminus (Sakakibara
et al., 2013). The rapid retreat may have been caused by the glacier front
reaching an area where the lake depth exceeds 560 m (Sugiyama et al.,
2016). In early 2013 the glacier's velocity at the front was 2.9 m d−1
(Moragues et al., 2018), ~33% lower compared to 2008 and more similar
to values recorded in the early 2000s (Sakakibara et al., 2013). Moragues
et al. (2018) report a doubling in maximum velocity between 2013 and
2014. However this increase is unlikely to have been sustained in time. In
fact, between 2011 and 2017, the glacier front has been comparatively
stable (Fig. 8), with a retreat rate of ~85 m a−1 similar to the period
2000–2008 (Sakakibara et al., 2013). Coincident with a more stable front
position, the average thinning rate within 16 km of the terminus decreased
by a factor two from 13.4 m a−1 between 2006 and 2010 (Sakakibara
et al., 2013) to 6.2 m a−1 between 2011 and 2017 (Fig. 8). Bertacchi
Glacier, a tributary of Upsala, shows a similar pattern with current rates of
elevation change decreasing to 8.5 m a−1 (Fig. 8) from ~15 m a−1 be-
tween 2008 and 2011 (Sakakibara et al., 2013). We observe maximum
thinning rates of ~12 m a−1 5 km from the terminus of Upsala glacier,
comparable to estimates at the front in the early 1990s (Naruse et al.,
1997). Despite these reduced thinning rates, Upsala remains the glacier
with the second most negative specific mass balance in the Patagonian Ice
Fields after Jorge Montt, and is the largest single contributor to net mass
loss amongst individual glaciers (Table 1 and Figs. 1–3). south and Lake Greve to the north. From 1945 to present, the tidewater
terminus advanced ~13 km and is currently at its Neoglacial maximum
(Wilson et al., 2016; Fig. 9 and Fig. E1). Looped supraglacial moraines
were used to identify up to six surge events since 1926 (Rivera et al.,
1997a; Wilson et al., 2016), two of which were concurrent with front
retreat, possibly due to enhanced calving at the tidewater terminus
(Wilson et al., 2016; Fig. E1). 4.5. Upsala glacier Upsala, a freshwater calving glacier located on the north-eastern side
of the SPI and draining into Argentino Lake, has also been retreating since
the late 1970s (Naruse et al., 1997). Between 2008 and 2011, retreat rates
quadrupled w.r.t the previous 8 years and the glacier retreated by almost
3 km (Sakakibara et al., 2013), while simultaneously speeding up by 8 Rem
ote Sensing of Environm
ent xxx (xxxx) xxx–xxx L. Foresta et al. Fig. 6. Median observed rates of surface elevation change for the two Ice Fields every tenth of a degree of latitude and for different elevation bands at [0-80
800-1000 m], [1000–1200 m], [1200–1500 m], [1500–1800 m] and [1800–2200 m]. The Pio XI glacier is not included in this analysis due to its anomalou
unique behaviour (see Discussion). Note that the scale on the x-axis varies to display the strong thinning at lower elevations. Fig. 6. Median observed rates of surface elevation change for the two Ice Fields every tenth of a degree of latitude and for different elevation bands at [0-800 m],
[800-1000 m], [1000–1200 m], [1200–1500 m], [1500–1800 m] and [1800–2200 m]. The Pio XI glacier is not included in this analysis due to its anomalous and
unique behaviour (see Discussion). Note that the scale on the x-axis varies to display the strong thinning at lower elevations. Fig. 7. Frontal retreat of Jorge Montt Glacier (SPI). The glacier retreated almost 2 km between 2000 and 2011 and receded a further ~2.5 km in our study period. Water depth at the glacier front was 400 m in 2013. Bathymetry data after Piret et al. (2017). Fig. 7. Frontal retreat of Jorge Montt Glacier (SPI). The glacier retreated almost 2 km between 2000 and 2011 and receded a further ~2.5 km in our study period. Water depth at the glacier front was 400 m in 2013. Bathymetry data after Piret et al. (2017). Fig. 7. Frontal retreat of Jorge Montt Glacier (SPI). The glacier retreated almost 2 km between 2000 and 2011 and receded a further ~2.5 km in our study period. Water depth at the glacier front was 400 m in 2013. Bathymetry data after Piret et al. (2017). 9 Rem
ote Sensing of Environm
ent xxx (xxxx) xxx–xxx L. Foresta et al. 4.5. Upsala glacier The glacier has been thickening in the ab-
lation area since the late 1970s, with the highest rates recorded at the
termini, while the picture is more complicated in the accumulation area
where data is sparse (Fig. E1). Between 2011 and 2017, we observe
thickening at almost all elevations, by about 2.33 m a−1 and 0.57 m a−1
(median value) in the ablation and accumulation areas respectively (Fig. 1;
Pio XI inset) with thinning at the highest elevations above 1500 m altitude;
findings which match those described in Jaber (2016). There is however
no coverage in our data close to the two termini. Assuming a thickening
trend at the two fronts, which was sustained for the last four decades
(Rignot et al., 2003; Willis et al., 2012b; Jaber et al., 2013; Jaber, 2016;
Wilson et al., 2016), we estimate that the Pio XI glacier is gaining mass at a
rate of 0.67 ± 0.29 Gt a−1 between 2011 and 2017 (Table 1 and Fig. E1). However the rate is likely underestimated since the hypsometric averaging
model for Pio XI glacier predicts less thickening compared to the ob-
servations below 1000 m elevation (Fig. 2, inset). The mass gain is likely a
result of complex dynamics associated with both surge mechanisms and
terminus calving processes, since Pio XI is the only advancing glacier
within the Patagonian Ice Fields and air temperature has increased over
the last 50 years (Rasmussen et al., 2007). 5. Conclusions The tidewater (south) and lacustrine (north) termini advanced about 500 m and 8 retreated ~2.5 km between 2011 and 2017, likely by enhanced calving in
deeper fjord waters. The only exception to the overall pattern of thinning
and retreat is the Pio XI glacier, which continues to advance at both its
tidewater and lacustrine termini. The glacier, which is currently at its
Neoglacial maximum, is estimated to have gained mass at a rate of
0.67 ± 0.29 Gt a−1 during our study period. spatial and temporal resolution are essential. CS2 swath altimetry provides
an important tool for monitoring these rapidly changing areas and quan-
tifying their ongoing mass loss. Appendix A. Filtering CS2 SARIn waveform samples Selecting waveform samples based on fixed thresholds on coherence and power is an empirical approach which has been applied successfully to infer
glacial topography and higher products based on it such as topography changes (Gray et al., 2013; Christie et al., 2016; Ignéczi et al., 2016; Foresta et al.,
2016; Gourmelen et al., 2017a). However, in this paper we use a different procedure to discard noisy waveform samples before performing the phase
unwrapping. This method relies entirely on the phase difference field and consists in identifying, within each waveform, groups of consecutive samples
which can be modelled by a straight line. The original phase difference is divided into overlapping segments, their length being set to 64 samples and the
overlap half of the length. The slope of the phase difference is then calculated independently in each segment. Instead of applying a linear regression, the
algorithm applies a Fourier transform on the normalized complex coherence e(i ðɸ), where ðɸ is the phase difference field. Compared to linear regression,
this approach is both more efficient computationally as well as independent on phase wrapping. The Fourier transform enables to testing a large number of
possible slopes and the one with the highest correlation with the input data is selected. The signal is oversampled to take into account that the slope of CS-
2's phase difference can represent non-integer frequencies. Thus, each overlapping section has two possible slopes. A correlation is applied again to the data
in each overlapping section, this time using only its two estimated slope values. Sections whose correlation is below a set threshold (for this work, 0.95) are
considered noisy and discarded. Finally, the remaining segments are used to unwrap the phase difference. With this procedure, no smoothing is applied to
the phase difference and no threshold is set on the power or coherence. Acknowledgments Between April 2011 and March 2017, the Ice Fields lost mass at a
combined
rate
of
21.29 ± 1.98 Gt a−1
(equivalent
to
0.059 ±
0.005 mm a−1 eustatic sea level rise), an increase of 24% and 42% when
compared to the periods 2000–2012/14 and 1975–2000, respectively. We
find that the NPI (−6.79 ± 1.16 Gt a−1), which is responsible for a third
of the total loss, is losing mass 70% faster compared to the first decade of
the 21st century. Given the ongoing and rapid wastage of the Patagonian
Ice Fields, and their important contribution to the global budget of mass
loss from glaciers and ice caps, continuous observations with excellent This work was performed under the European Space Agency's Support
to Science Element CryoSat+ Mountain Glacier (contract 4000114224/
15/I-SBo). L.F. acknowledges a Young Scientist Training grant from the
European Space Agency's Dragon3 program. J.J.W. acknowledges a NERC
PhD Studentship. The authors wish to thank ESA for providing free access
to CryoSat-2 full bit rate data, NASA for providing free access to the SRTM
DEM and USGS for providing free access to Landsat data. The authors are
grateful to the Editor-in-Chief, Crystal Schaaf, and to two anonymous re-
viewers, whose comments have significantly improved the manuscript. 5. Conclusions Pio XI, the largest glacier on the SPI and in South America, is the only
glacier of the Patagonian Ice Fields to have experienced a net large ad-
vance since 1926 and the only known surge-type glacier on the SPI (Rivera
et al., 1997a; Wilson et al., 2016). Published data of frontal changes, ice
flow velocity at the termini, elevation change and mass balance, sum-
marized in Fig. E1, reveal a complex history (Appendix E). Between 1951
and 1963, the glacier's westward and southward advance dammed a
proglacial river originating from Greve glacier to the north, forming the
current Lake Greve for at least the second time since 1926 (Rivera et al.,
1997a). Since then, the glacier has been terminating in Ejre Fjord to the CryoSat-2 swath radar altimetry is employed successfully to map ele-
vation change over the Patagonian Ice Fields at sub-kilometer spatial re-
solution. Despite the challenging topography, similar to that of mountain
glaciers, the technique can be used to observe changes over individual
glaciers or catchments with an area as small as 300 km2. The northern part
of the SPI displays a high degree of complexity, although most of the area
is thinning at all elevations, with Jorge Montt, Viedma and Upsala glaciers
losing mass at rates higher than 2 Gt a−1. Jorge Montt additionally Fig 8 Front location of Upsala glacier (SPI) The glacier receded about 500 m between 2011 and 2017 Thinning rates from this study are also Fig. 8. Front location of Upsala glacier (SPI). The glacier receded about 500 m between 2011 and 2017. Thinning rates from this study are also shown in the range
−10 to 0 m a−1. Fig. 8. Front location of Upsala glacier (SPI). The glacier receded about 500 m between 2011 and 2017. Thinning rates from this study are also shown in the range
−10 to 0 m a−1. 10 L. Foresta et al. Rem
ote Sensing of Environm
ent xxx (xxxx) xxx–xxx L. Foresta et al. Rem
ote Sensing of Environm
ent xxx (xxxx) xxx–xxx L. Foresta et al. Rem
ote Sensing of Environm
ent xxx (xxxx) xxx–xxx Fig. 9. Front location of Pio XI glacier (SPI). The tidewater (south) and lacustrine (north) termini advanced about 500 m and 800 m, respectively, between 2011 and 2017. Fig. 9. Front location of Pio XI glacier (SPI). Appendix B. SRTM DEM correction A number of freely available DEMs covering Southern Patagonia exist, namely: the SRTM (i) C and (ii) X band DEMs (Farr et al., 2007), the (iii)
ASTER (Advanced Spaceborne Thermal Emission and Reflection Radiometer) GDEM2 (Tachikawa et al., 2011) and (iv) the ALOS (Advanced Land 11 Rem
ote Sensing of Environm
ent xxx (xxxx) xxx–xxx L. Foresta et al. Observing Satellite) AW3D30 v1.1 (Tadono et al., 2014; Takaku et al., 2014; Tadono et al., 2016; Takaku et al., 2016). The latter is the most recent,
but has large gaps over the SPI, which are filled using the SRTM C band DEM. Version 1 of the ASTER GDEM was known to be affected by large
artefacts (Arefiand Reinartz, 2011) and, despite large overall improvements, version 2 still has high frequency noise, particularly over glacial terrain
(Meyer et al., 2011). Visual comparison between the SRTM C band and ASTER GDEM2 DEMs shows evident noise in the latter, with differences at
times on the order of tens of meters between neighbouring pixels. Finally, the SRTM X band DEM does not have complete coverage and gaps over the
Ice Fields are significant. Therefore, we used the SRTM C band DEM as a reference for elevation, which fully covers the Patagonian Ice Fields,
resampled at 300 m posting and referenced to the WGS84 vertical datum. The down-sampling of the DEM is mostly dictated by achieving a
satisfactory performance in computing time while keeping the spatial resolution somewhat comparable to that of an individual elevation based on
CryoSat-2 interferometric data (300 m in the along-track direction). We use linear interpolation when querying the DEM. The SRTM DEM is based on data acquired in February 2000, and a few areas at the margins of the SPI have thinned by at least 80 m since then
(Willis et al., 2012b; Jaber et al., 2013; Jaber, 2016). This magnitude is comparable to the elevation offset caused by a 2π shift on CS2's phase (Data
and methods, this paper; Fig. 3 in Gourmelen et al., 2017a, 2017b). Thus, over areas which experienced intense thinning rates, the swath algorithm
may select an incorrect 2π multiple which best matches current observations to the glacier topography from 2000. In order to avoid that, the SRTM
DEM needs to be registered to the beginning of our study period. Appendix B. SRTM DEM correction To this purpose, we applied a first order correction of the SRTM DEM based on a
visual inspection of results in Willis et al. (2012b) and assuming constant rates of elevation change between the SRTM and CS2 periods. This
approach was sufficient to improve the phase unwrapping, leading to further pixels meeting the quality criteria for robust rates of surface elevation
change. We refer to this corrected DEM simply as the SRTM DEM in this study. The correction of the SRTM DEM only affects the terminus areas of
Jorge Montt and Upsala glaciers (SPI) since there are no CS2 swath altimetry observations over smaller glaciers on the Ice Fields which experienced
similar thinning rates in the period 2000–2011/12 (e.g. HPS12, SPI; Willis et al., 2012b; Jaber et al., 2013; Jaber, 2016). Appendix C. Error budget The errors on the mass balance estimates are calculated as in Foresta et al. (2016). The uncertainties εḣ on the observed rates of elevation change
for the individual pixels, extracted from the model covariance matrix (see Data and methods), are propagated when applying the hypsometric
averaging method: =
∑
=
E
k
ε
m
N k
( )
(
)
( .)
h
m
N k
h
̇
1
( )
̇
2 where Eḣ(k) is the elevation change error in elevation band k and N(k) is the number of valid observations in the elevation band. A two-term
decreasing exponential is used to interpolate values for elevation bands with no observations (e.g. at low elevation for bands with limited spatial
extent). We multiply the area extent A(k) of the elevation band to the related Eḣ(k) and sum all contributions to estimate the total uncertainty on the
rate of volume change: where Eḣ(k) is the elevation change error in elevation band k and N(k) is the number of valid observations in the elevation band. A two-term
decreasing exponential is used to interpolate values for elevation bands with no observations (e.g. at low elevation for bands with limited spatial
extent). We multiply the area extent A(k) of the elevation band to the related Eḣ(k) and sum all contributions to estimate the total uncertainty on the
rate of volume change: ∑
=
∗
E
E
k
A k
( )
( ). V
k
h
̇
̇ With this method, the volume change uncertainty is only related to that area of the ice cap where there are valid rates of surface elevation
change, but does not account for incomplete data coverage. The volume change uncertainty is therefore rescaled according to the data coverage
(Table 1). This procedure generates a rather conservative (i.e. larger) error estimate since it assumes that the lack in data coverage has a direct
impact on the total error estimate, which does not hold if the sampling is sufficiently uniform. Finally, we include an error on the density: =
−
E
ρ
ρ
1
2 (
)
ρ
ice
firn when converting volume to mass change (e.g. Nilsson et al., 2015a). when converting volume to mass change (e.g. Nilsson et al., 2015a). Appendix D. Landsat scenes Appendix D. Landsat scenes Table D1
List of Landsat scenes used to manually delineate the front positions of glaciers Jorge Montt, Upsala and Pio XI (SPI). 1
andsat scenes used to manually delineate the front positions of glaciers Jorge Montt, Upsala and Pio XI (SPI). y
p
g
g
,
p
(
)
Jorge Montt
2005
LT52310942005050COA00
2009
LE72320942009156EDC00
2010
LE72310942010088COA00
2011
LE72320942011050EDC00
2013
LE72320942013087ASN00
2014
LC82310942014075LGN00
2015
LC82320942015021LGN00
2016
LC82320942016072LGN00
2017
LC82320942017106LGN00
Upsala
2011
LE72310952011123EDC00
2017
LC82310952017035LGN00
Pio XI
2011
LE07_L1TP_232094_20110219_20161210_01_T1
2014
LC08_L1TP_231094_20140401_20170424_01_T1
2017
LC08_L1TP_231094_20170204_20170216_01_T1 12 Rem
ote Sensing of Environm
ent xxx (xxxx) xxx–xxx L. Foresta et al. Appendix E. Pio XI glacier (SPI): summary of published data
Fig. E1 summarises published data of frontal changes, ice flow velocity at the fronts, elevation change and mass balance (Rivera et al., 1997a;
Rivera et al., 1997b; Rivera and Casassa, 1999; Rignot et al., 2003; Lopez et al., 2010; Willis et al., 2012b; Jaber et al., 2013; Sakakibara and
Sugiyama, 2014; Wilson et al., 2016). Fig. E1. Chart summarizing published data for the Pio XI glacier (SPI). (a) Cumulative front advance of the tidewater and lacustrine termini; (b) ice flow velocity; (c)
mass change. Elevation change in the (d) ablation and (e) accumulation area. Potential surges concurrent with advance (Rivera et al., 1997a) or retreat (Wilson et al.,
2016) of the tidewater front are highlighted in light and dark grey, respectively. All relevant references are listed in the inset. Appendix E. Pio XI glacier (SPI): summary of published data
Fig. E1 summarises published data of frontal changes, ice flow velocity at the fronts, elevation change and mass balance (Rivera et al., 1997
Rivera et al., 1997b; Rivera and Casassa, 1999; Rignot et al., 2003; Lopez et al., 2010; Willis et al., 2012b; Jaber et al., 2013; Sakakibara an
Sugiyama, 2014; Wilson et al., 2016). Fig. E1. Chart summarizing published data for the Pio XI glacier (SPI). (a) Cumulative front advance of the tidewater and lacustrine termini; (b) ice flow velocity; (
mass change. Elevation change in the (d) ablation and (e) accumulation area. Potential surges concurrent with advance (Rivera et al., 1997a) or retreat (Wilson et a
2016) of the tidewater front are highlighted in light and dark grey, respectively. All relevant references are listed in the inset. Appendix E. Pio XI glacier (SPI): summary of published data Appendix E. Pio XI glacier (SPI): summary of published data Appendix E. Appendix D. Landsat scenes Pio XI glacier (SPI): summary of published data References Lett. 43 (18), 9729–9738. http://dx.doi.org/10.1002/2016GL070338. Ivins, E.R., Watkins, M.M., Yuan, D.N., Dietrich, R., Casassa, G., Rülke, A., 2011. On-land
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RGI Consortium, 2017. Appendix E. Pio XI glacier (SPI): summary of published data Fig. E1 summarises published data of frontal changes, ice flow velocity at the fronts, elevation change and mass balance (Rivera et al., 1997a;
Rivera et al., 1997b; Rivera and Casassa, 1999; Rignot et al., 2003; Lopez et al., 2010; Willis et al., 2012b; Jaber et al., 2013; Sakakibara and
Sugiyama, 2014; Wilson et al., 2016). Fig. E1. Chart summarizing published data for the Pio XI glacier (SPI). (a) Cumulative front advance of the tidewater and lacustrine termini; (b) ice flow velocity; (c
mass change. Elevation change in the (d) ablation and (e) accumulation area. Potential surges concurrent with advance (Rivera et al., 1997a) or retreat (Wilson et al
2016) of the tidewater front are highlighted in light and dark grey, respectively. All relevant references are listed in the inset. Fig. E1. Chart summarizing published data for the Pio XI glacier (SPI). (a) Cumulative front advance of the tidewater and lacustrine termini; (b) ice flow velocity; (c)
mass change. Elevation change in the (d) ablation and (e) accumulation area. Potential surges concurrent with advance (Rivera et al., 1997a) or retreat (Wilson et al.,
2016) of the tidewater front are highlighted in light and dark grey, respectively. All relevant references are listed in the inset. 13 13 L. Foresta et al. Rem
ote Sensing of Environm
ent xxx (xxxx) xxx–xxx References Rem
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English
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Frequency of osteoradionecrosis of the lower jaw after radiotherapy of oral cancer patients correlated with dosimetric parameters and other risk factors
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Head & face medicine
| 2,022
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cc-by
| 5,562
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RESEARCH Open Access Lang et al. Head & Face Medicine (2022) 18:7
https://doi.org/10.1186/s13005-022-00311-8 Lang et al. Head & Face Medicine (2022) 18:7
https://doi.org/10.1186/s13005-022-00311-8 * Correspondence: kristin.lang@med.uni-heidelberg.de Co espo de ce:
st . a g@
ed.u
e de be g.de
1Department of Radiation Oncology, University Hospital of Heidelberg, Im
Neuenheimer Feld 400, 69120 Heidelberg, Germany
2Heidelberg Institute of Radiation Oncology (HIRO), 69120 Heidelberg,
Germany y
Full list of author information is available at the end of the article Abstract Objectives: Osteoradionecrosis (ORN) of the lower jaw is a serious late complication after radiotherapy in patients
with oral cavity cancer. The aim of this study is to generate more insight into which patient- and treatment-related
factors are associated with the development of ORN in oral cavity cancer patients undergoing postoperative
radiotherapy. Material and methods: Retrospective evaluation and comparison of 44 patients with ORN (event group 1)
matched according to 45 patients without ORN (control group 2) who received postoperative radiotherapy of oral
cavity squamous cell carcinoma at our institution between 2012 and 2020. Dosimetric factors that favor the
occurrence of ORN should be detected. The cumulative occurrence rate of ORN was calculated according to the
Kaplan–Meier method and analyzed by Cox regression and log-rank test. Results: The median time to develop ORN was 18 months (3–93 months) after radiotherapy. Dental status before
radiotherapy (RT) treatment (HR 4.5; 1.8–11.5) and dosimetric parameters including Dmean > 45 Gy (HR 2.4; 1.0–5.7),
Dmax > 60 Gy (HR 1.3; 1.1–2.8) and planning target volume (PTV) proportion > 40% intersection with the lower jaw
(HR 1.1; 1.0–1.1) were significantly associated with ORN. Conclusion: The results of this retrospective study reveal that oral cavity cancer patients who underwent pre-RT
dental surgery as well as dosimetric parameters using Dmax > 60 Gy, higher mean doses > 45 Gy and more than
40% PTV intersection with the lower jaw bone are independent risk factors for ORN. These findings can assist in the
management of patients undergoing RT for head and neck cancer regarding ORN prevention. Frequency of osteoradionecrosis of the
lower jaw after radiotherapy of oral cancer
patients correlated with dosimetric
parameters and other risk factors Kristin Lang1,2*, Thomas Held1,2, Eva Meixner1,2, Eric Tonndorf-Martini1,2, Oliver Ristow3, Julius Moratin3,
Nina Bougatf1,2, Christian Freudlsperger3, Jürgen Debus1,2,4,5 and Sebastian Adeberg1,2,4,5 © 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://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 7 (2022) 18:7 Lang et al. Head & Face Medicine Lang et al. Head & Face Medicine (2022) 18:7 Clinical relevance: Poor oral hygiene and desolate dental status as well as high radiation doses to the mandibular
bone significantly increase the risk of developing osteoradionecrosis. Before irradiating a patient with oral cavity
cancer, an appointment with the dentist should be made and teeth sanitized if necessary. Likewise, maximum
radiation doses to the lower jaw should be minimized. Keywords: Desolate dental status, Osteoradionecrosis, Radiotherapy, Mandibular bone To ensure comparability, and in particular to be able to
detect risk factors, patients were divided into two equal
groups: those who developed ORN (44 patients, group
1) and those who did not develop ORN (45 patients,
group 2). We collected basic patient and treatment data
from the National Tumor Center Heidelberg Cancer
Registry and imported them into our HIRO Research
Database for this study [16]. Inclusion criteria were as
follows: histologically squamous cell carcinoma, follow-
up duration of at least 3 months after completion of RT,
no interruptions during RT, regular follow-up examina-
tions including computed tomography (CT) examina-
tions every 3 months in the first 2 years after RT as
standard clinical practice for all head and neck cancer
patients, every 6 months in years 3 and 4 after RT and
once a year in years 5 and 6, as well as regular head and
neck examinations at the Department of Oral and Max-
illofacial Surgery and the availability of sufficient RT
treatment plan data to evaluate the dose to the lower
jaw bone. Exclusion criteria were patients with follow-up
of less than 3 months as well as patients with other hist-
ology and patients with cancer outside the oral cavity. Treatment features
f Before treatment, patients generally underwent pre-RT
dental evaluation and pre-RT dental treatment (from at-
tempts to preserve teeth to extraction of teeth not worth
preserving), as deemed appropriate by the oral and max-
illofacial surgeons, based on risk assessment. The pre-
RT dental treatment because of poor dental status was
done 2 weeks before the start of radiation. A desolate
dental status was defined in our sample as follows: peri-
odontal disease, carious changes in more than 5 teeth,
attacked gums or exposed tooth necks. In our sample,
patients with caries deep into tooth pulpa were involved. There was no special grading system used for degree of
carious changes. For periodontal status the oral and
maxillofacial surgeons used periodontal risk assessment
(RPA), which includes number of teeth and implants,
number of missing teeth, percentage of alveolar bone
loss and number of periodontal pockets with probing
depths > 5 mm. Introduction Oral cancer is sensitive to radiation and is standard
treatment either in definitive intention or in the case of
pathologic risk factors (positive resection margin, posi-
tive lymph nodes, locally advanced disease) as postopera-
tive intention. Osteoradionecrosis (ORN) is a feared late
complication of radiotherapy (RT) of oral cancer pa-
tients which affects mostly the lower jaw between 2 and
22% [1–3]. The incidence of ORN is about 4–8% [4–6]. ORN is defined as exposed irradiated bone that fails to
heal over a period of 3 months without any evidence of
persisting or recurrent tumor [7, 8]. Radiological evi-
dence of bone necrosis within the target volume is also
important for diagnosis and classification [9]. Analysis of
epidemiological studies of ORN does not offer accurate
data about incidence and prevalence of ORN in the jaws
because of inconsistencies in the length of follow-up be-
tween studies and limited data from prospective studies. Different treatment-, tumor- and patient-related risk fac-
tors of ORN have been reported: size and site of the
tumor, age, total RT dose, treatment technique (3D-con-
formal therapy (3D-CRT) vs. intensity-modulated radio-
therapy
(IMRT)),
dose
volume
histogram
(DVH)
parameters, dose per fraction, injury or dental extrac-
tions, alcohol and tobacco abuse, tumor size or stage, as-
sociation of the tumor with bone and dental hygiene
[10–14]. The international standard radiation technique
for patients treated for oral cancer is IMRT, primarily
aimed at reducing dosage at the major salivary glands in
the oral cavity. Therefore, it is not uncommon to achieve
high-dose gradients across the lower jaw bone [15]. All
patients treated with RT in the oral cavity underwent
pre-RT dental evaluation and pre-RT care if necessary
according to current uniform policies. The aim of this
study is to generate more insight into which patient-
and treatment-related factors are associated with the de-
velopment of ORN in oral cavity cancer patients under-
going postoperative RT and to predict which patients
are at higher risk of developing ORN. Material and methods Data from 89 patients who received postoperative RT to
the oral cavity between 2012 and 2020 at the University
Hospital of Heidelberg were reviewed for this retrospect-
ive analysis. Patient data were homogenized by selecting
the same tumor stage and only patients with oral cancer. Patients were treated either with IMRT or 3D-CRT. CT simulation was performed in patients immobilized
using a thermoplastic mask. Target volume definition
was performed in accordance with current guidelines Page 3 of 7 Lang et al. Head & Face Medicine (2022) 18:7 Lang et al. Head & Face Medicine (2022) 18:7 Lang et al. Head & Face Medicine Page 3 of 7 [17, 18]. Our target definition was based on preoperative
and postoperative CT and magnetic resonance imaging
(MRI) scans and included the primary tumor region
initial gross tumor volume (iGTV) according to the
International
Commission
on
Radiation
Units
and
Measurements definition [19]. The planning target
volume (PTV) was created by expansion of the clin-
ical target volume (CTV) with a 3–5 mm uniform
margin. The lymphatic CTV encompasses the patho-
logic lymph nodes (pN+) as well as any adjacent re-
gions at risk of tumor spread. The lower jaw was
contoured for each plan, and DVHs were retrospect-
ively reviewed as RT dosimetric parameters. The max-
imum dose to the lower jaw (Dmax), mean dose to
the lower jaw (Dmean) and PTV correlation to lower
jaw were reviewed. The main treatment features are
listed in Table 1. Definit
Variou
curren
The m
fails to
persist
surger
Treatm
Toxici
focus
mon T
criteri
Huma
asymp
interv
medic
Table 1 Characteristics of 44 patients with osteoradionecrosis (group 1) an
postoperative radiotherapy
Group 1 (%)
Median age at RT in years (range)
70.5 (39–94)
Gender
Male
35 (79.5)
Female
9 (20.5)
T-stage
1–2
28 (63.6)
3–4
21 (47.7)
Desolate dental status (caries, periodontal disease)
36 (81.8)
Need for pre-RT dental treatment
31 (70.5)
Tooth protection during RT
41 (93.2)
Systemic therapy
CHT
22 (50.0)
IT
8 (18.2)
none
14 (31.8)
Smoking history
yes
29 (65.9)
no
15 (34.1)
RT technique
3D-CRT
7 (15.9)
IMRT
37 (84.1)
Dmean to mandible
< 45 Gy
25 (56.8) Definition of ORN [17, 18]. Our target definition was based on preoperative
and postoperative CT and magnetic resonance imaging
(MRI) scans and included the primary tumor region
initial gross tumor volume (iGTV) according to the
International
Commission
on
Radiation
Units
and
Measurements definition [19]. The planning target
volume (PTV) was created by expansion of the clin-
ical target volume (CTV) with a 3–5 mm uniform
margin. The lymphatic CTV encompasses the patho-
logic lymph nodes (pN+) as well as any adjacent re-
gions at risk of tumor spread. The lower jaw was
contoured for each plan, and DVHs were retrospect-
ively reviewed as RT dosimetric parameters. The max-
imum dose to the lower jaw (Dmax), mean dose to
the lower jaw (Dmean) and PTV correlation to lower
jaw were reviewed. The main treatment features are
listed in Table 1. Various definitions of ORN have been proposed, but no
current accepted standard of classification or grading exists. The most prevalent definition is exposed bone after RT that
fails to heal over a period of 6 months without evidence of
persisting or recurrent tumor and required drug therapy or
surgery with radical mandibulectomy [20]. Treatment toxicity Toxicity was evaluated in this retrospective analysis with
focus on ORN and was described according to the Com-
mon Terminology Criteria for Adverse Events (CTCAE)
criteria (version 5.0, U.S. Department of Health and
Human Services, Washington, DC, USA): grade 1:
asymptomatic; clinical or diagnostic observations only;
intervention
not
indicated;
grade
2:
symptomatic;
medical intervention indicated (e.g., topical agents); Table 1 Characteristics of 44 patients with osteoradionecrosis (group 1) and 45 patients in control group (group 2) after
postoperative radiotherapy
Group 1 (%)
Group 2 (%)
p-value
Median age at RT in years (range)
70.5 (39–94)
71 (42–88)
0.886
Gender
Male
35 (79.5)
37 (82.2)
0.124
Female
9 (20.5)
8 (17.8)
T-stage
1–2
28 (63.6)
32 (71.8)
0.086
3–4
21 (47.7)
13 (28.9)
Desolate dental status (caries, periodontal disease)
36 (81.8)
17 (37.7)
0.228
Need for pre-RT dental treatment
31 (70.5)
12 (26.7)
0.001
Tooth protection during RT
41 (93.2)
40 (88.9)
0.303
Systemic therapy
CHT
22 (50.0)
28 (62.2)
0.475
IT
8 (18.2)
1 (2.2)
none
14 (31.8)
16 (35.5)
Smoking history
yes
29 (65.9)
19 (42.2)
0.453
no
15 (34.1)
26 (57.7)
RT technique
3D-CRT
7 (15.9)
2 (4.4)
0.519
IMRT
37 (84.1)
43 (95.6)
Dmean to mandible
< 45 Gy
25 (56.8)
41 (91.1)
0.023
> 45 Gy
19 (43.2)
4 (8.9)
Dmax of mandible
< 50 Gy
2 (4.5)
10 (22.2)
0.033
51–60 Gy
22 (50.0)
19 (42.2)
> 60 Gy
20 (45.5)
16 (35.5)
PTV dimension (ccm) (range)
804 (68–4838)
796 (170–1389)
0.028 ristics of 44 patients with osteoradionecrosis (group 1) and 45 patients in control group (group 2) after
th Lang et al. Head & Face Medicine (2022) 18:7 Page 4 of 7 Lang et al. Head & Face Medicine (2022) 18:7 Lang et al. Head & Face Medicine was no correlation between dental intervention before
RT and occurrence of ORN detected. limitations in the performance of instrumental activ-
ities of daily living (ADLs); grade 3: severe symptoms;
limitations
in
the performance
of self-care
ADLs;
elective operative intervention indicated; grade 4: life-
threatening consequences; urgent intervention indi-
cated; and grade 5: death [21]. This retrospective
study included all patients who had medical interven-
tion for ORN. Group 1 p
The median patient age at the time of RT was 71 years
(range 39–94 years); 35 patients (79.5%) were male and 9
(20.5%) were female. Pre-RT dental treatment was ne-
cessary in 31 patients (70.5%), with half of them having
3 or more teeth removed. At the start of radiation treat-
ment, significantly more current smokers (53%) had den-
tal extraction compared with former smokers (28%) or
never smokers (34%) (p = 0.003). Systemic therapy was
used simultaneously with RT in 30 patients. The major-
ity of patients were treated with IMRT (84.1%), and only
7 patients received 3D-CRT. Statistical analysis Univariate and multivariate Cox proportional hazard
models were used to assess patient- and treatment-related
factors associated with the development of ORN. Factors
with statistical significance in the univariate analysis were
included in the multivariate analysis. The cumulative inci-
dence was estimated using the Kaplan–Meier method,
and the log-rank test was used to compare cumulative in-
cidence curves. The follow-up time was calculated from
the last date of RT until the most recent follow-up visit at
our institution or the date of death. The time to ORN de-
velopment was calculated from the last date of RT until
the date of ORN occurrence. The Mann–Whitney U test
was used to compare patients in terms of the presence of
ORN, RT technique and DVH parameters. Cut-off points
for DVH parameters were selected. All statistical analyses
were performed using IBM SPSS software version 24. P-
values
of
0.05
or
less
were
considered
statistically
significant. Dmean to the lower jaw was 40.5 Gy (range 21–62 Gy),
Dmax was 59 Gy (49–75 Gy), and the mean PTV dimen-
sion was 804 ccm (range 68–4838 ccm), respectively. Group 2 The median patient age at the time of RT was 71 years
(range 42–88 years); 37 patients (82.2%) were male and 8
(17.8%) were female. Pre-RT dental treatment before
radiotherapy was performed in 12 patients (26.7%). Sys-
temic therapy was used simultaneously with RT in 29
patients. The majority of patients were treated with
IMRT (95.6%), and only 1 patient received 3D-CRT. Dmean to the lower jaw was 26.0 Gy (range 12.0–67.0
Gy), Dmax was 56.0 Gy (32.0–77.0 Gy), and the mean
PTV dimension was 796 ccm (range 170–1389 ccm),
respectively. Results p
y
All tested dosimetric variables were mutually related
and associated with the risk of ORN. Patients with ORN
had a significantly higher Dmean, Dmax and PTV than the
control group: 40.5 Gy vs. 26.0 Gy (p = 0.043), 59.0 Gy vs. 56.0 Gy (p = 0.033) and 804 ccm vs. 796 ccm (p = 0.028). Statistically significant differences in univariate analyses
were noted for Dmean > 45 Gy, Dmax > 60 Gy, PTV touch-
ing > 40% lower jaw as well as the need for pre-RT den-
tal
therapy
due
to
potential
focus
teeth. In
the
multivariate analysis pre-RT dental surgery (HR 4.5;
1.8–11.5) and dosimetric parameters including Dmean >
45 Gy (HR 2.4; 1.0–5.7), Dmax > 60 Gy (HR 1.3; 1.1–2.8)
and PTV > 40% touching lower jaw (HR 1.1; 1.0–1.1)
were also significantly associated with ORN. Results of
univariable and multivariable analysis are shown in
Table 2. Results are shown according to presence of ORN (group
1: with ORN; group 2: without ORN). Patient and treat-
ment characteristics are summarized in Table 1. In both
groups there were five patients with desolate dental sta-
tus included who declined pre-RT dental treatment as
preferred. An example for development of osteoradione-
crosis is shown in Fig. 1. The follow-up time for the whole cohort was 28
months, and the median time to develop ORN was 18
months (3–93 months). In total, 78% of 44 ORN sites
were located in the body of the jaw, and 22% were lo-
cated in the angle or ramus. CT scans were evaluated
for all ORN sites, and cortical erosion was observed in
73% and loss of spongiosa trabeculation in 27%. Of the
44 patients with ORN, 20 (45.5%) had CTCAE grade 2,
17 (38.6%) had grade 3 and 7 (15.9%) had grade 4. All
patients received initial anti-infective therapy with chlor-
hexidine rinsing (0.2% chlorhexidine solution; GlaxoS-
mithKline Consumer Healthcare GmbH & Co. KG) and
antibiotic treatment, followed by surgical intervention in
the infection-free interval. In the time between comple-
tion of RT and detection of ORN, no patient had any
other dental intervention. For the whole cohort there Discussion ORN of the lower jaw is known to be a chronic late com-
plication of RT in patients with oral cavity cancer, and
many studies have reported its incidence. Incidences in
several recent studies range between 4 and 8% [4–6, 15],
and there has been a decrease over the past few decades Page 5 of 7 (2022) 18:7 Lang et al. Head & Face Medicine Fig. 1 The images a–d show the planned CT in one patient with oral cavity cancer undergoing postoperative radiotherapy, including PTV that
was planned to receive a total dose of 66.0 Gy (single dose 2.0 Gy) in 33 fractions. To the top right image b shows the ORN of the lower jaw
(colored in green) in the high-dose region with maximum dose 68.7 Gy. Therefore, we matched the follow-up CT with the planned CT and dose
distribution, and then volumetric and dosimetric evaluations were performed Fig. 1 The images a–d show the planned CT in one patient with oral cavity cancer undergoing postoperative radiotherapy, including PTV that
was planned to receive a total dose of 66.0 Gy (single dose 2.0 Gy) in 33 fractions. To the top right image b shows the ORN of the lower jaw
(colored in green) in the high-dose region with maximum dose 68.7 Gy. Therefore, we matched the follow-up CT with the planned CT and dose
distribution, and then volumetric and dosimetric evaluations were performed 24]. Nabil et al. reported that a total dose above 60 Gy
was a significant parameter for ORN [12]. since more conformal techniques (IMRT) have become
available, as demonstrated by Studer et al. [15] and Ben-
David et al. [10]. Also convincing are recent studies which
clarify dosimetric prognostic factors for the incidence and
severity of ORN. In our analysis, dosimetric parameters showed signifi-
cant correlation with the development of ORN as seen
with significant values in group 1: patients treated with
Dmax to lower jaw higher than 60 Gy (45.5% vs. 35.5%,
p = 0.033), Dmean more than 45 Gy (43.25 vs. 8.9%, p =
0.023) and PTV intersection of more than 40% of the
lower jaw bone. All patients with ORN had hot spots in
the region of ORN. Discussion Dosimetric evaluation of Dmean in
our study may reflect a general damaging mechanism in
the lower jaw, and from this study, Dmean seems an ap-
propriate parameter to consider in dose planning and be
included without a threshold. Doses for the lower jaw Lee et al. and Niewald et al. reported on the dose–ef-
fect relationship in patients with oral cavity cancer. The
frequency of ORN in their analyses was 6.6% [22] and
8.6% [23] in a median time of 2 years [22, 23]; in our
analysis the follow-up duration was significantly lower
for some patients. Higher radiation doses were signifi-
cantly associated with the development of ORN [22, 23]. Lee et al. found that a cumulative total dose of > 54 Gy
was a significant factor in the development of ORN [22, Lang et al. Head & Face Medicine (202 Page 6 of 7 (2022) 18:7 Table 2 Univariable and multivariate analysis of risk factors for ORN
HR
95% CI
p-value
Univariate analysis
Gender (male vs. female)
1.837
0.847–2.984
0.124
Age
1.002
0.974–1.031
0.886
T-stage
1.320
0.962–1.813
0.086
Poor dental status (caries, periodontal disease)
1.618
0.740–3.537
0.228
Pre-RT dental treatment
4.822
2.055–11.316
0.001
Smoking history
1.279
0.672–2.435
0.453
Dmean to mandible
1.950
1.770–2.170
0.043
Dmax to mandible
1.492
1.257–2.943
0.033
Concomitant systemic therapy
1.163
0.769–1.759
0.475
PTV dimension
1.001
1.000–1.003
0.028
Multivariate analysis
Pre-RT dental treatment
4.567
1.817–11.477
0.001
Dmean to mandible
2.421
1.023–5.728
0.044
Dmax to mandible
1.334
1.135–2.827
0.018
PTV dimension
1.001
1.000–1.003
0.046 Further prospective studies are needed to further dis-
sect the dental status and evaluate the infectivity of the
focus teeth and the relationship of dental treatment and
time interval with radiation onset. However, even if we
assume that radiation contributes to the development of
ORN at this dose constraint, there might be other con-
tributory factors. The final goal would be to optimize
radiation treatment plans with adaptive treatment plan-
ning and offer patients appropriate counseling for dental
management to decrease the risk of ORN. bone should be kept as low as possible to reduce the risk
of ORN in oral cavity cancer patients. Our general dose
constraint (Dmax) for the lower jaw is 72 Gy or as low as
achievable, although we do not compromise tumor
coverage to achieve this value. The median time to develop ORN in our study was 18
months. Discussion This is in line with other studies [6, 8, 12, 14,
25]. In a recent study of our institution there was a rate
of ORN in 5.8% of patients detected [25]. An appropriate
follow-up time was ensured in this retrospective study,
and we include cases (group 1) and controls (group 2)
for better correlation with factors leading to ORN in pa-
tients undergoing postoperative radiotherapy. Therefore,
all patients had routine follow-up at our department as
well as at the Department of Oral and Maxillofacial Sur-
gery in Heidelberg. Our retrospective study showed that
poor dental status which entails pre-RT dental treatment
was significantly correlated with the development of
ORN (70.5% vs. 26.7%). This is in line with previous
studies [26]. Acknowledgements Not applicable. Authors’ contributions Kristin Lang: First author, search on the electronic databases, triaged by title,
abstract and full text, acquisition, analysis or interpretation of data and
statistics. Thomas Held, Eva Meixner, Eric Tonndorf-Martini, Oliver Ristow,
Julius Moratin, Nina Bougatf, Christian Freudlsperger, Jürgen Debus, Sebastian
Adeberg: Critical revision of the article. All authors read and approved the
final version of this manuscript. Conclusion The results of this retrospective study reveal that oral
cavity cancer patients who underwent pre-RT dental
surgery as well as dosimetric parameters using Dmax >
60 Gy, Dmean > 45 Gy and more than 40% of the PTV
touching the lower jaw bone are independent risk factors
for the development of ORN. These findings can assist
in the management of patients undergoing RT for head
and neck cancer regarding ORN prevention. A larger
study with focus on dental status is planned. The limitation of this study is its retrospective na-
ture, which led to a shortage of necessary data on
some single cases. However, we were able to retrieve
follow-up data covering a lengthy time period for all
patients. The power of this study is that we were able
to show—in a dedicated sample of oral cavity cancer
patients undergoing radiotherapy—risk factors for de-
velopment of ORN: high risk was associated with
poor dental status before beginning RT and with
dosimetric
parameters
using
Dmax
> 60 Gy,
higher
mean doses > 45 Gy and more than 40% of the PTV
touching the lower jaw bone. Acknowledgements
Not applicable. Acknowledgements Kristin Lang: First author, search on the electronic databases, triaged by title,
abstract and full text, acquisition, analysis or interpretation of data and
statistics. Thomas Held, Eva Meixner, Eric Tonndorf-Martini, Oliver Ristow,
Julius Moratin, Nina Bougatf, Christian Freudlsperger, Jürgen Debus, Sebastian
Adeberg: Critical revision of the article. All authors read and approved the
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4. https://doi.org/10.1016/j.ijrobp.2007.04.030. Received: 21 October 2021 Accepted: 15 February 2022 19. Bernier J, Cooper JS, Pajak TF, van Glabbeke M, Bourhis J, Forastiere A, et al. Defining risk levels in locally advanced head and neck cancers: a
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Not applicable. Page 7 of 7 Page 7 of 7 Lang et al. Head & Face Medicine (2022) 18:7 Lang et al. Head & Face Medicine (2022) 18:7 Lang et al. Head & Face Medicine ( Ethics approval and consent to participate This study was performed following institutional guidelines and the
Declaration of Helsinki of 1975 in its most recent version. Ethical approval for
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Heidelberg (S-329/2015). For this type of study, formal consent is not
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This research did not receive any specific grant from funding agencies in the
public, commercial, or not-for-profit sectors. Open Access funding enabled
and organized by Projekt DEAL. Author details
1 1Department of Radiation Oncology, University Hospital of Heidelberg, Im
Neuenheimer Feld 400, 69120 Heidelberg, Germany. 2Heidelberg Institute of
Radiation Oncology (HIRO), 69120 Heidelberg, Germany. 3Department of Oral
and Maxillofacial Surgery, University Hospital Heidelberg, Im Neuenheimer
Feld 400, 69120 Heidelberg, Germany. 4Heidelberg Ion Therapy Center (HIT),
Im Neuenheimer Feld 450, 69120 Heidelberg, Germany. 5Clinical Cooperation
Unit Radiation Oncology, German Cancer Research Center (DKFZ), Im
Neuenheimer Feld 280, 69120 Heidelberg, Germany. 1Department of Radiation Oncology, University Hospital of Heidelberg, Im
Neuenheimer Feld 400, 69120 Heidelberg, Germany. 2Heidelberg Institute of
Radiation Oncology (HIRO), 69120 Heidelberg, Germany. 3Department of Oral
and Maxillofacial Surgery, University Hospital Heidelberg, Im Neuenheimer
Feld 400, 69120 Heidelberg, Germany. 4Heidelberg Ion Therapy Center (HIT), 17. Gregoire V, et al. Delineation of the primary tumour clinical target volumes
(CTV-P) in laryngeal, hypopharyngeal, oropharyngeal and oral cavity squamous
cell carcinoma: AIRO, CACA, DAHANCA, EORTC, GEORCC, GORTEC, HKNPCSG,
HNCIG, IAG-KHT, LPRHHT, NCIC CTG, NCRI, NRG oncology, PHNS, SBRT,
SOMERA, SRO, SSHNO, TROG consensus guidelines. Radiother Oncol. 2018;
126(1):3–24. https://doi.org/10.1016/j.radonc.2017.10.016. Im Neuenheimer Feld 450, 69120 Heidelberg, Germany. 5Clinical Cooperation
Unit Radiation Oncology, German Cancer Research Center (DKFZ), Im
Neuenheimer Feld 280, 69120 Heidelberg, Germany. 18. Lapeyre M, Toledano I, Bourry N, Bailly C, Cachin F. Target volume delineation
for head and neck cancer intensity-modulated radiotherapy. Cancer Radiother. 2011;15(6–7):466–72. https://doi.org/10.1016/j.canrad.2011.07.239. Received: 21 October 2021 Accepted: 15 February 2022 Received: 21 October 2021 Accepted: 15 February 2022 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.
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Single Bout Exercise in Children with Juvenile Idiopathic Arthritis: Impact on Inflammatory Markers
|
Mediators of inflammation
| 2,018
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cc-by
| 4,885
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Rochette Emmanuelle
,1,2,3,4 Duché Pascale,3,4 Hourdé Christophe,5 Evrard Bertrand,6,7
Pereira Bruno,8 Echaubard Stéphane,1 and Merlin Etienne1,2,6 1Pédiatrie Générale Multidisciplinaire, Hôpital Estaing, CHU Clermont-Ferrand, 63000 Clermont-Ferrand, France
2Université Clermont Auvergne, INSERM, CIC 1405, Unité CRECHE, 63000 Clermont-Ferrand, France
3 3Laboratoire des Adaptations Métaboliques en conditions Physiologiques et Physiopathologiques (AME2P), Université Clermont
Auvergne, EA 3533, Clermont-Ferrand, France 5Laboratoire Interuniversitaire de Biologie de la Motricité (E A7424), Université Savoie Mont Blanc,
73376 Le Bourget-du-Lac, France Laboratoire Interuniversitaire de Biologie de la Motricité (E A7424), Université Savoie Mont Blanc,
73376 Le Bourget-du-Lac, France 5Laboratoire Interuniversitaire de Biologie de la Motricité (E A7424), Université Savoie Mont Blanc,
73376 Le Bourget-du-Lac, France 6Université Clermont Auvergne, INRA, UMR 1019 UNH, ECREIN, 63000 Clermont-Ferrand, France
7 6Université Clermont Auvergne, INRA, UMR 1019 UNH, ECREIN, 63000 Clermont-Ferrand, France
7 7Service d’Immunologie, CHU Clermont-Ferrand, 63000 Clermont-Ferrand, France CHU Clermont-Ferrand, Délégation de la Recherche Clinique et Innovations, 63000 Clermont-Ferrand, France Correspondence should be addressed to Rochette Emmanuelle; e_rochette@chu-clermontferrand.fr Received 28 February 2018; Accepted 27 May 2018; Published 13 June 2018 Received 28 February 2018; Accepted 27 May 2018; Published 13 June 2018 Academic Editor: Elena Dozio Academic Editor: Elena Dozio Copyright © 2018 Rochette Emmanuelle 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. Objective. In a context of inflammatory disease such as juvenile idiopathic arthritis (JIA), we do not know what impact physical
activity may have on a deregulated immune system. The objective is to measure the impact of a single bout of exercise on
plasma inflammatory markers such as calprotectin, IL-6, sIL-6R, sgp130, and the hypothalamic-pituitary-adrenal axis in
children with juvenile idiopathic arthritis. Methods. Twelve children with JIA performed a nonexercise control day and a
consecutive day that included a 20 min exercise bout at 70% of max-HR at 08:30 am. Venous blood samples were taken at 08:30,
08:50, 09:30, 10:30 am, and 12:00 pm to measure plasma concentrations of calprotectin, IL-6, sIL-6R, sgp130, cortisol, and
ACTH. Pain was evaluated at 08:30, 08:50 am, and 06:00 pm. Results. There was a transient twofold increase in postexercise self-
evaluated pain (p = 0 03) that disappeared in the evening. A single bout of exercise resulted in a 1.7-fold increase in plasma
calprotectin (p < 0 001) but not IL-6 and its soluble receptors. Calprotectin levels returned to baseline within 3 hours after
cessation of exercise. Conclusion. Hindawi
Mediators of Inflammation
Volume 2018, Article ID 9365745, 6 pages
https://doi.org/10.1155/2018/9365745 Hindawi
Mediators of Inflammation
Volume 2018, Article ID 9365745, 6 pages
https://doi.org/10.1155/2018/9365745 Rochette Emmanuelle
,1,2,3,4 Duché Pascale,3,4 Hourdé Christophe,5 Evrard Bertrand,6,7
Pereira Bruno,8 Echaubard Stéphane,1 and Merlin Etienne1,2,6 Acute exercise in children with JIA induced slightly musculoskeletal leg pain and transient
increased plasma calprotectin levels but not IL-6 levels. Trial registration in ClinicalTrials.gov, reference number NCT
02502539, registered on 29 May 2015. 1. Introduction secreted by activated monocytes and phagocytes and
induce the expression of proinflammatory cytokines (IL-
1β, IL-6, or TNF) [2]. In JIA, the MRP8/14 complex, also
called calprotectin, is a reliable marker of subclinical disease
activity [3, 4]. Juvenile idiopathic arthritis (JIA), the most common
inflammatory pathology in children, is a chronic disease
characterized by persistent joint inflammation due to
immune system disruption [1]. Some of the proteins that per-
petuate inflammatory mechanisms in JIA are calgranulins
(myeloid-related protein (MRP)-8 and MRP-14). They are It has emerged that exercise may have direct effects not
only on metabolic and fitness parameters but also on the
pathogenesis of autoimmune diseases as it attenuates chronic Mediators of Inflammation 2 10 min at 4°C and stored at −80°C until analysis, while all
other blood samples were analyzed immediately. low-grade systemic inflammation [5]. After acute exercise of
varying intensities and lengths, plasma calprotectin levels in
healthy adults increase at a rate of between 3.4-fold (VO2
max test) and 96.3-fold (marathon) [6]. Although IL-6 is
known as a potent proinflammatory signal (involved in the
pathology of JIA), the transient rise in circulating IL-6 during
exercise appears to have anti-inflammatory effects [7]. In
addition, IL-6 released from contracting muscle acts in a
hormone-like manner to mobilize extracellular substrates
or inhibits low-level TNF-α production. Likewise, physical
activity is known to impact the hypothalamic-pituitary-
adrenal (HPA) axis in healthy children [8-10] and modulate
the anti-inflammatory effect of cortisol. However, the dis-
turbed HPA axis in JIA leads to impaired secretion of adre-
nocorticotropic hormone (ACTH) and cortisol [11, 12]. Even though physical activity is increasingly recognized as a
useful intervention in patients with rheumatisms [13], to
our knowledge, there is no data on the effect of acute bouts
of physical activity on IL-6 and its receptors, calprotectin
and HPA axis in children with inflammatory disease. p
y
y
Plasma ACTH levels were measured by chemilumi-
nescent immunometric assay (Immulite 2000, Siemens
Healthcare Diagnostics;
analytical
sensitivity = 5 pg/mL). Serum cortisol levels were measured by chemiluminescence
on an ADVIA Centaur XP Analyzer (Siemens Healthcare
Diagnostics; analytical sensitivity= 27.6 nmol/L). Plasma
MRP8/14 levels were measured using commercially avail-
able ELISA kits (Bühlmann Laboratories, Schönenbuch,
Switzerland;
analytical
sensitivity =400 ng/mL). Plasma
highly sensitive C-reactive protein (hs-CRP) levels were
measured by nephelometry on a Dimension Vista® ana-
lyzer (Siemens Healthcare Diagnostics; analytical sensitivi-
ty = 0.16mg/L). 1. Introduction Plasma IL-6 levels were measured using
commercially available ELISA kits (EH2IL65, Thermo
Fisher Scientific,
USA;
analytical sensitivity < 1 pg/mL). Plasma sgp130 levels were measured using commercially
available ELISA kits (EHIL6ST, Thermo Fisher Scientific,
USA; analytical sensitivity = 4 pg/mL). Plasma sIL6-R levels
were measured using commercially available ELISA kits
(KHR0061, Thermo Fisher Scientific, USA; analytical sensiti-
vity =<8 pg/mL). All analyses were made on duplicates. Here, we set out to explore the impact of an acute bout
exercise on pain and on the secretion of molecules involved
in the inflammatory process in JIA. y
pg
y
p
Statistical analyses were performed using Stata software
version 13 (StataCorp, College Station, TX, USA). Tests
were two-sided, with a type I error set at α = 0 05. Contin-
uous data were expressed as mean ±standard deviation
(SD) or median (interquartile range) according to statistical
distribution (assumption of normality assessed by using the
Shapiro-Wilk test). To take into account between- and
within-patient variability due to several measures for the
same subject, random effects models for correlated data were
performed rather than the usual statistical tests which would
be inappropriate due to unverified assumption of indepen-
dence. Time-point evaluations, sessions, and their interac-
tions were considered fixed effects whereas a patient was a
random effect (slope and intercept). The normality of resid-
uals from these models was studied as described above. When appropriate, the data were log-transformed to achieve
normality of the dependent endpoint. 2. Methods Twelve children aged 8 to 16 years with nonsystemic JIA
according to the criteria of the International League of Asso-
ciations for Rheumatology were enrolled in the study and
were followed at the pediatric unit of Clermont-Ferrand Uni-
versity Hospital in France. Patients were excluded if they had a physician-diagnosed
infection, had been treated by IL-6 blockade in the last 6
months, and had received oral corticosteroids within the last
3 months. Treatments were continued during the study. The study was approved by the governing ethics commit-
tee (Comité de Protection des Personnes Sud-Est VI, refer-
ence number: AU1190). All participants and their parents
have given consent. Experimental design consisted of two consecutive days
(day 1: control, day 2: exercise day). 3. Results Pain was self-evaluated by the patient on a visual analog
scale at 08:30, 08:50 am, and 06:00 pm during the 2 days. Baseline patient characteristics are reported in Table 1. Mean
age was 12.3± 2.8 years, BMI was 19.7 ±2.9 kg/m2, and dis-
ease duration was 29.4 ± 18.4 months. Mean fasting hs-CRP
was 0.33 ± 0.4 mg/L. The exercise test consisted of a 20 min bout, between
08:30 am and 08:50 am, at 70% of the maximal theoretical
heart rate (max-HR) performed on a cycle ergometer
(Ergo-metrics 800S, Ergoline, Bitz, Germany) during day 2. Theoretical max-HR was defined as 220-age. Exercise bout induced a transient but significant increase
in pain postexercise that resolved in few hours (Figure 1). This musculoskeletal pain involved muscles and joints,
mainly in the legs. Venous blood samples were taken via an indwelling cath-
eter at 08:30, 08:50, 09:30, 10:30 am, and 12:00 pm, into
EDTA K2 Vacutainer® tubes (Becton Dickinson, Franklin
Lakes, NJ, USA) for analysis of ACTH, IL-6, sIL-6R,
sgp130, and calprotectin, into Vacutainer Lithium Heparin
Tubes (Becton Dickinson, Franklin Lakes, NJ, USA) for anal-
ysis of hs-CRP and into Vacutainer SST II Advance Tubes
(Becton Dickinson, Franklin Lakes, NJ, USA) for analysis of
cortisol. Samples collected for calprotectin, IL-6, sIL-6R,
and sgp130 were immediately centrifuged at 3000g for Exercise induced a 1.7-fold increase in calprotectin level
immediately after the exercise (p < 0 001) that returned to
normal within three hours after the end of the exercise bout
(Figure 2). However, there was no effect of exercise on IL-6,
sIL-6R, and sgp130 levels (Figure 3). Area under the curve (AUC) for plasmatic ACTH and
serum cortisol decreased, respectively, from 11.8% (from
3228 ± 128 to 2848 ± 124; p = 0 58) to 17.4% (from 43,455 Mediators of Inflammation 3 Table 1: Baseline demographics, clinical characteristics, and baseline laboratory findings. 3. Results Age (years) Sex BMI (kg/m2) Z score JIA subtype Disease duration
(months)
Concomitant
DMARDs
hs-CRP
(mg/L)ǂ
Heart rate during exercise:
min–max (bpm)
11
F
14.8
−1.4
Psoriatic
16.8
MTX
0.24
141–149
10
M
18.1
+0.6
ERA
14.1
Sulfasalazine
<0.16
144–150
8
F
21.7
+1.8
pJIA RF−
4.9
MTX
1.4
142–152
9
M
15.5
−0.4
ERA
35.6
MTX
<0.16
143–152
14
F
19.7
+0.2
pJIA RF−
9.0
MTX
<0.16
140–149
14
M
20.1
+0.3
Psoriatic
40.1
Infliximab + leflunomide
0.33
141–149
16
F
21.1
+0.2
ERA
25.8
NSAIDs
0.53
138–147
9
M
21.3
+1.7
oJIA
55.2
NSAIDs
<0.16
140–151
15
F
23.1
+0.9
ERA
20.7
NSAIDs
<0.16
140–149
13
F
19.4
+0.2
pJIA RF+
23.0
MTX + etanercept
<0.16
138–150
12
F
16.9
−0.5
Undif
67
None
<0.16
139–151
16
F
24.4
+1.0
Undif
20.5
Etanercept
0.22
138–147
JIA: juvenile idiopathic arthritis; oJIA: oligoarticular JIA; pJIA RF−: rheumatoid factor-negative (RF−) polyarticular JIA; pJIA RF+: rheumatoid factor-positive
(RF+) polyarticular JIA; ERA: enthesitis-related arthritis; Psoriatic: psoriatic JIA; Undif: undifferentiated; DMARDs: disease-modifying antirheumatic drugs;
MTX: methotrexate; NSAIDs: nonsteroidal anti-inflammatory drugs; hs-CRP: high-sensitivity C-reactive protein; bpm: beats per minute. ǂNormal
value < 3.0 mg/L. Values of <0.16 are below the minimum detectable limit of the kit. Table 1: Baseline demographics, clinical characteristics, and baseline laboratory findings. JIA: juvenile idiopathic arthritis; oJIA: oligoarticular JIA; pJIA RF−: rheumatoid factor-negative (RF−) polyarticular JIA; pJIA RF+: rheumatoid factor-positive
(RF+) polyarticular JIA; ERA: enthesitis-related arthritis; Psoriatic: psoriatic JIA; Undif: undifferentiated; DMARDs: disease-modifying antirheumatic drugs;
MTX: methotrexate; NSAIDs: nonsteroidal anti-inflammatory drugs; hs-CRP: high-sensitivity C-reactive protein; bpm: beats per minute. ǂNormal
value < 3.0 mg/L. Values of <0.16 are below the minimum detectable limit of the kit. 8:30 8:50
Ex
⁎⁎
9:30
10:30
Calprotein
12:00
⁎⁎
⁎
⁎
(hours:minutes)
500
1000
1500
2000
2500
3000
3500
(ng/mL)
Figure 2: Plasma levels of calprotectin during the control day
(solid line) and in response to 20 min of exercise (Ex) between
08:30 and 08:50 am (dotted line). Data are means ± 95% CI. ∗Significantly different to baseline at p < 0 05. ∗∗Significantly
different to baseline at p < 0 001. 10
Pain
9
8
7
6
5
4
3
2
1
0
08:30 am
p = 0.035
p = 0.031
08:50 am
06:00 pm
Figure 1: Pain evaluations by visual analog scale during the
control day (black bar) and exercise day (white bar). Data are
means ± 95% CI. 3. Results 8:30 8:50
Ex
⁎⁎
9:30
10:30
Calprotein
12:00
⁎⁎
⁎
⁎
(hours:minutes)
500
1000
1500
2000
2500
3000
3500
(ng/mL) 10
Pain
9
8
7
6
5
4
3
2
1
0
08:30 am
p = 0.035
p = 0.031
08:50 am
06:00 pm
Figure 1: Pain evaluations by visual analog scale during the
control day (black bar) and exercise day (white bar). Data are
means ± 95% CI. Figure 1: Pain evaluations by visual analog scale during the
control day (black bar) and exercise day (white bar). Data are
means ± 95% CI. Figure 2: Plasma levels of calprotectin during the control day
(solid line) and in response to 20 min of exercise (Ex) between
08:30 and 08:50 am (dotted line). Data are means ± 95% CI. ∗Significantly different to baseline at p < 0 05. ∗∗Significantly
different to baseline at p < 0 001. ±1294 to 35,943 ±1217; p = 0 06) between the nonexercise
control day and the exercise day (Figure 4). 4. Discussion increase of calprotectin and no effect on IL-6. The absence
of IL-6 increase could be explained by the short duration
and a low intensity of the exercise bout tested. Furthermore,
as exercise does not increase calprotectin levels in neutrophils
[6], the likely source is skeletal muscle, since S100A8 and
S100A9 mRNA rates increase in skeletal muscle during exer-
cise [16]. Therefore, as our child population has a lower mus-
cle mass and incomplete muscular recruitment [17], there is
likely a lower production of calprotectin and IL-6 by the
muscles than in adults. In addition, pubertal status, physical
exercise training, and BMI seem to be factors involved in
the impact of exercise-induced IL-6 release in children
[18, 19]. Nevertheless, in our child population with systemic
low-grade inflammation, as in studies in children with Overall, the exercise session was well tolerated in all children. There was a slight increase in pain immediately postexercise,
but the effect was transient and disappeared before the eve-
ning. In terms of pain, it is therefore conceivable to advocate
exercise in this child population, as more and more studies
are showing that physical activity improves quality of life
and symptoms in JIA patients [14, 15]. In healthy adults, acute exercise is known to induce a sig-
nificant secretion of calprotectin [6] and IL-6 [7] by muscle
in an exercise intensity-dependent manner. In fact, exercise
promotes muscle recruitment of activated neutrophils, this
phenomenon is followed by a systemic counterregulation
involving IL-10 and IL-1ra [7]. Here, we observed a slight 4 Mediators of Inflammation 25
(pg/mL)
20
15
10
5
0
1300
Ex
sgp130
IL-6 25
(pg/mL)
(ng/mL)
20
15
10
5
0
1300
1200
1100
1000
900
800
Ex
Ex
sgp130
IL-6
sIL-6R 400
(nmol/L)
(pg/mL)
350
300
250
200
150
100
50
30
25
20
15
10
5
8
9
10
(hours)
11
12
Ex
Ex
Cortisol
ACTH
⁎⁎
⁎⁎
⁎
⁎ Cortisol 400
(nmol/L)
(pg/mL)
350
300
250
200
150
100
50
30
25
20
15
10
5
8
9
10
(hours)
11
12
Ex
Ex
Cortisol
ACTH
⁎⁎
⁎⁎
⁎
⁎
Figure 4: HPA axis secretion in children with JIA. Serum cortisol
level and plasma ACTH level during the control day (solid line)
and in response to 20 min of exercise (Ex) between 08:30 and
08:50 am (dotted line). ∗Significantly different to baseline at p <
0 05. 4. Discussion 400
(nmol/L)
(pg/mL)
350
300
250
200
150
100
50
30
25
20
15
10
5
8
9
10
(hours)
11
Ex
Ex
ACTH
⁎⁎
⁎⁎
⁎
⁎
Figure 4: HPA axis secretion in children with JIA. Se
level and plasma ACTH level during the control day
and in response to 20 min of exercise (Ex) betwee
08:50 am (dotted line). ∗Significantly different to ba
0 05. ∗∗Significantly different to baseline at p < 0 0
means ± 95% CI. (nmol/L) (pg/mL)
30
25
20
15
10
5
8
9
10
(hours)
11
12
Ex
ACTH (ng/mL)
1300
1200
1100
1000
900
800
Ex
sgp130
sIL-6R (ng/mL)
(hours:minutes)
360
320
280
240
200
8:30
8:50
9:30
10:30
12:00
Ex
sIL-6R Figure 4: HPA axis secretion in children with JIA. Serum cortisol
level and plasma ACTH level during the control day (solid line)
and in response to 20 min of exercise (Ex) between 08:30 and
08:50 am (dotted line). ∗Significantly different to baseline at p <
0 05. ∗∗Significantly different to baseline at p < 0 001. Data are
means ± 95% CI. (ng/mL) outbreak may have been feared in response to exercise in a
context of proinflammatory disease, this is not the case in
response to an acute exercise of moderate intensity. IL-6 is involved not only in the activation of the immune
system but also in regenerative processes and in the regula-
tion of metabolism, the maintenance of bone homeostasis,
and many neural functions [7, 20]. IL-6 is usually concerned
as a proinflammatory cytokine. However, in the setting of
muscular release, it is known to induce anti-inflammatory
effects with inhibition of TNF-α production and may
thus prevent TNF-α-induced insulin resistance [7]. Taken
together, these observations raise questions over the different
role for IL-6 in children compared to adults, making it neces-
sary to address whether acute physical activity is followed by
systemic counterregulation in children with JIA. Figure 3: Plasma level of IL-6, sgp130, and sIL-6R during the
control day (solid line) and in response to 20 min of exercise (Ex)
between 08:30 and 08:50 am (dotted line). Area under the curve
(AUC) for plasma sIL-6R decreased 8.4% between control day
and exercise day (from 63.36 106 ± 1.06 106 to 58.03 106 ± 0.97 106;
p = 0 006). 4. Discussion ∗∗Significantly different to baseline at p < 0 001. Data are
means ± 95% CI. 25
(pg/mL)
(ng/mL)
(ng/mL)
(hours:minutes)
20
15
10
5
0
1300
360
320
280
240
200
8:30
8:50
9:30
10:30
12:00
1200
1100
1000
900
800
Ex
Ex
Ex
sgp130
IL 6
sIL-6R
Figure 3: Plasma level of IL-6, sgp130, and sIL-6R during the
control day (solid line) and in response to 20 min of exercise (Ex)
between 08:30 and 08:50 am (dotted line). Area under the curve
(AUC) for plasma sIL-6R decreased 8.4% between control day
and exercise day (from 63.36 106 ± 1.06 106 to 58.03 106 ± 0.97 106;
p = 0 006). AUC for plasma sgp130 increased 2.6% between
control day and exercise day (from 188.96 106 ± 4.81 106 to 193.91
106 ± 5.08 106; p = 0 11). Data are means ± 95% CI. outbreak may have been feared in response to e
context of proinflammatory disease, this is not
response to an acute exercise of moderate intensi
IL-6 is involved not only in the activation of t
system but also in regenerative processes and in
tion of metabolism, the maintenance of bone h
and many neural functions [7, 20]. IL-6 is usually
as a proinflammatory cytokine. However, in th
muscular release, it is known to induce anti-in
effects with inhibition of TNF-α production
thus prevent TNF-α-induced insulin resistance
together, these observations raise questions over t
role for IL-6 in children compared to adults, maki
sary to address whether acute physical activity is
25
(pg/mL)
(ng/mL)
(ng/mL)
(hours:minutes)
20
15
10
5
0
1300
360
320
280
240
200
8:30
8:50
9:30
10:30
12:00
1200
1100
1000
900
800
Ex
Ex
Ex
sgp130
sIL-6R
Figure 3: Plasma level of IL-6, sgp130, and sIL-6R during the
control day (solid line) and in response to 20 min of exercise (Ex)
between 08:30 and 08:50 am (dotted line). Area under the curve
(AUC) for plasma sIL-6R decreased 8.4% between control day
and exercise day (from 63.36 106 ± 1.06 106 to 58.03 106 ± 0.97 106;
p = 0 006). AUC for plasma sgp130 increased 2.6% between
control day and exercise day (from 188.96 106 ± 4.81 106 to 193.91
106 ± 5.08 106; p = 0 11). Data are means ± 95% CI. References Concerning the soluble receptors of IL-6, our results showed
no effect of exercise on plasma sgp130 and sIL-6R levels. These results can be explained by the small number of
patients and the fact that within-subject variation was lower
than between-subject variation for IL-6, sIL-6R, and sgp130
in response to exercise, as reported in another study [23]. This finding can also in part explain why data on the effect
of exercise on the rate of sgp130 is contradictory, with reports
citing either an increase or a decrease or no effect [23-28]. Furthermore, all IL-6 family cytokines use gp130 as a
receptor [29], and this may partly explain the high concen-
trations of this receptor and its homeostasis. Finally, our
levels of sIL-6R and sgp130 are higher than reported in
literature, although sIL-6R levels are high in the serum
and synovial fluid of JIA patients [30]; this discrepancy
could be explained by the fact that we have used plasma
or by the detection kits used. [1] Y.-T. Lin, C.-T. Wang, M. E. Gershwin, and B.-L. Chiang,
“The pathogenesis of oligoarticular/polyarticular vs systemic
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no. 8, pp. 482–489, 2011. [2] C. Kessel, D. Holzinger, and D. Foell, “Phagocyte-derived
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HPA axis except for cortisol. The decrease in cortisol level
observed on the second day was probably due to lower stress
at this session, as the children were aware of the study envi-
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the physiological response to exercise at blood level, which
may not be an accurate reflection of the muscular or articular
response in this population. However, muscle biopsies or
articular aspirations in these children would have been
complicated to perform. Nevertheless, further research is
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control day and exercise day (from 188.96 106 ± 4.81 106 to 193.91
106 ± 5.08 106; p = 0 11). Data are means ± 95% CI. y
g
IL-6 acts through a heterodimeric signaling complex
consisting of the IL-6 receptor (IL-6R) and the signal-
transducing subunit glycoprotein 130 (gp130). Trans-
signaling of IL-6 through its soluble receptors is involved in
the promotion of chronic inflammatory diseases [21, 22]. obesity or type 1 diabetes, single bout exercise is not followed
by increased IL-6 levels [19]. Finally, the fact that our patients
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Stress & Chaperones, vol. 13, no. 2, pp. 247–251, 2008. [25] M. Leggate, M. A. Nowell, S. A. Jones, and M. A. Acknowledgments This work was supported by a grant from the Clermont-
Ferrand University Hospital (AOI 2015). The authors
thank the pediatric nurse Alexandra Usclade; the data
manager Aurélie Chausset and Khaled Messaoudi, Chloé
Plantard, and Clara Martin for their help with handling
the blood sampling. [14] T. Takken, M. Van Brussel, R. H. H. Engelbert, J. Van Der Net,
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pp. 287–297, 2008. 6 Mediators of Inflammation Mediators of Inflammation Nimmo, “The
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English
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Spanish Version of the Flourishing Scale (FS) on the Parents of Children With Cancer: A Validation Through Rasch Analysis
|
Frontiers in psychology
| 2,019
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cc-by
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Spanish Version of the Flourishing
Scale (FS) on the Parents of Children
With Cancer: A Validation Through
Rasch Analysis Carmen Pozo Muñoz 1* and Blanca Bretones Nieto 2 1 Department of Psychology, University of Almería, Almería, Spain, 2 Psychosocial Intervention and Health (HUM-792)
Research Group, University of Almería, Almería, Spain The interest in the study of flourishing is due to the fact that it has been proven that
it contributes to a good adjustment to the demands of the environment, relating to
indicators of health and well-being. There are many researches that have tried to find out
what dimensions make it up. The goal of this study is to validate the Spanish Version
of The Flourishing Scale (FS), being the first time it is applied to Spanish parents of
children with cancer. A total of 138 parents of children with cancer participated in a
semi-structured interview. Through IBM SPSS and Winsteps descriptive analyzes and
the internal consistency of the FS were calculated. Rasch analysis was used to study
the dimensionality of the scale, the adjustment of each one of the items, the reliability
values for the items and for the people, the validity of the construct, the functioning of
the response categories, and the differential item functioning (DIF). The external construct
validity of the FS was examined with associated measures. Results found that parents
indicated a flourishing attitude (M = 3.85; sd =0.63). We confirm that the FS maintains
an adequate internal consistency and a unidimensional structure. We observed a good
alignment between the question and the person’s abilities as soon as a high reliability
for the items. Although the sample is large enough to corroborate the difficulty hierarchy
of the items, the construct validity could be improved by introducing items of moderate
and high difficulty. On the other hand, the answer category 3 overlaps with the 2 and
4, so we propose possible solutions. Regarding a possible DIF, this exists in relation to
“gender,” “level of education” and “by the situation with respect to treatment.” Finally,
the convergent validity of the FS is demonstrated, there being a significant correlation
with well-being, satisfaction with social support and coping. In general, the results show
adequate psychometric properties of the Spanish version of the FS, so we recommend
integrating it in psychosocial interventions aimed at parents of children with cancer, in
order to provide them with resources to deal with the disease. Keywords: validation, rasch analysis, flourishing, parents, childhood cancer Edited by:
Eduardo A. Garza-Villarreal,
Instituto Nacional de Psiquiatría
Ramón de la Fuente Muñiz (INPRFM),
Mexico Edited by:
Eduardo A. Garza-Villarreal,
Instituto Nacional de Psiquiatría
Ramón de la Fuente Muñiz (INPRFM),
Mexico Reviewed by:
Kathy Ellen Green,
University of Denver, United States
Alessandro Giuliani,
Istituto Superiore di Sanità (ISS), Italy
Corrado Caudek,
Università degli Studi di Firenze, Italy Reviewed by:
Kathy Ellen Green,
University of Denver, United States
Alessandro Giuliani,
Istituto Superiore di Sanità (ISS), Italy
Corrado Caudek,
Università degli Studi di Firenze, Italy Reviewed by:
Kathy Ellen Green,
University of Denver, United States
Alessandro Giuliani,
Istituto Superiore di Sanità (ISS), Italy
Corrado Caudek,
Università degli Studi di Firenze, Italy *Correspondence:
Carmen Pozo Muñoz
cpozo@ual.es Specialty section:
This article was submitted to
Clinical and Health Psychology,
a section of the journal
Frontiers in Psychology Received: 05 May 2018
Accepted: 08 January 2019
Published: 25 January 2019 ORIGINAL RESEARCH
published: 25 January 2019
doi: 10.3389/fpsyg.2019.00035 INTRODUCTION Recently,
flourishing has been analyzed as a positive coping mechanism
for health and well-being in the context of childhood cancer,
specifically in the parents of diagnosed children and adolescents
(Bretones et al., 2016; Bretones, 2018). Agreement has not been reached regarding the dimensions
that comprise the Flourishing Scale. The first results come from
the application of the original Flourishing Scale (FS) by Diener
et al. (2009). The FS scale was found to have a single strong factor
with an eigenvalue of 4.24, which accounted for 52% of the items’
variance, with Cronbach’s alpha of 0.87 and a test-retest reliability
of 0.71. Subsequent studies have obtained similar results. In Brazil,
Nunes et al. (2015) support the one-dimensionality of the scale in
two studies carried out, respectively with students and the general
population. Similarly, Tang et al. (2016) obtained a single factor
in a sample Chinese population (α > 0.90 and a good convergent
and discriminant validity); as did Schotanus-Dijkstra et al. (2016)
in research conducted with adults in the Netherlands (α = 0.86;
adequate global reliability and a good fit of most of the elements
in the Rasch analysis). Although the psychometric properties of FS have previously
been studied through the Classical Test Theory (CTT) (Diener
et al., 2009; Silva and Caetano, 2013; Hone et al., 2014; Sumi,
2014), on a few occasions analysis of the psychometric properties
of this instrument has been used by new models of the Item
Response Theory (IRT), such as the Rasch model (Pozo et al.,
2016; Schotanus-Dijkstra et al., 2016). The main advantages of the Rasch Model (the most
parsimonious IRT model) in comparison with CTT are the
possibility of estimating the extent to which each test or item
measures the skill of the participants, the joint estimates of
respondents’ parameters and items, and the invariance of the
parameters obtained from the different samples (Arias et al.,
2013; Garzón Umerenkova et al., 2017). In contrast to the CTT,
the Rasch model establishes the probability of a person’s response
to a stimulus, i.e., the difference between the measure of a
person’s trait and the measure of the stimulus used (Tristán,
2002). INTRODUCTION It is worth highlighting the cross-cultural research of Pozo
et al. (2016), in which Colombian and Spanish university students
participated. Satisfactory psychometric properties (α Colombian
= 0.88; α Spanish = 0.85) and good levels of reliability were found
(the Rasch person reliability was fairly acceptable -Colombia =
0.77; Spain = 0.80-, and the person separation statistic was 1.83
for Colombia and 2.02 for Spain. Rasch item reliability was 0.93
for Colombia and 0.94 for Spain, and the item separation statistic
was 3.69 for Colombia and 4.13 for Spain. In both samples, the
Cronbach alpha values were higher than 0.70). The central concept developed by Keyes (2007) defines the
flourisher as the individual who experiences a high level of
emotional, psychological and social well-being. In subsequent
studies, Diener et al. (2010) consider that the flourishing consists
of a sense of competence, self-acceptance, optimism, and to
contribute to the well-being of others. According to Huppert and
So (2009) flourishing comprises a set of central characteristics
including: positive emotions, delivery, interest, meaning and
purpose; and an additional set of competencies: self-esteem,
optimism, resilience, vitality, self-determination, and positive
relationships. Giuntoli et al. (2017) analyze the dimensionality of the
construct (they found a superior fit for a two factor model) and its
invariance in two studies (with students, unemployed and healthy
samples) through a multigroup analysis. Concurrent validity was
verified with other well-being, depression and anxiety measures. Well-being Theory is based on the Theory of Authentic
Happiness, centered on the study of the latter to increase
satisfaction with life. However, from the theory of well-being,
positive psychology focuses its interest on the analysis of it, using
flourishing for its measurement (Seligman, 2011). Flourishing
has been related to psychological well-being and has been studied
in conjunction with positive and negative emotions in order to
elaborate scales to help us understand well-being (Diener et al.,
1999). In relation to the fields of application, previous investigations
have verified the association between flourishing and less
limitations in daily life, as well as flourishing and better health
(Howell et al., 2013; Huppert and So, 2013; Gilmour, 2015). Along the same lines, the psychosocial factor in the context of
education has been studied and found to be beneficial regarding
the well-being, academic performance, and abilities of university
students (Fredrickson and Branigan, 2005; Howell, 2009; Wilson-
Strydom and Walker, 2015), as well as for occupational
health workers (Rautenbach and Rothmann, 2017). Citation: Pozo Muñoz C and Bretones Nieto B
(2019) Spanish Version of the
Flourishing Scale (FS) on the Parents
of Children With Cancer: A Validation
Through Rasch Analysis. Front. Psychol. 10:35. doi: 10.3389/fpsyg.2019.00035 January 2019 | Volume 10 | Article 35 Frontiers in Psychology | www.frontiersin.org 1 Flourishing Scale in Childhood Cancer Pozo Muñoz and Bretones Nieto January 2019 | Volume 10 | Article 35 INTRODUCTION Taking into account
these circumstances and since flourishing has previously been
related to variables related to health and well-being in the context
of illness (Bretones et al., 2016), we consider it important to verify
the effects that Flourishing causes in terms of coping with the
disease. For this, first of all we believe that it is necessary to
continue deepening the analysis of the Flourishing Scale, as a
novelty, in a sample of Spanish parents of children with cancer. that they have been considered as “invisible patients” (Hinds,
1992). On the other hand, it has been shown that the adaptation
of children and adolescents is determined by factors related to
the degree of adjustment of their parents, especially in the initial
stages of the disease (Fedele et al., 2013). During the childhood
illness the parents of children with cancer have to suffer a lot
of critical moments in relation with diagnosis, treatments, side
effects, surgeries, e.g., In this way, stress symptoms represent a
common experience in the familiar context, also after the illness
situation (Vrijmoet-Wiersma et al., 2010). Taking into account
these circumstances and since flourishing has previously been
related to variables related to health and well-being in the context
of illness (Bretones et al., 2016), we consider it important to verify
the effects that Flourishing causes in terms of coping with the
disease. For this, first of all we believe that it is necessary to
continue deepening the analysis of the Flourishing Scale, as a
novelty, in a sample of Spanish parents of children with cancer. objective and subjective health, well-being, and stressors, the
majority used coping strategies (including flourishing), as well as
support from their formal and informal network. Because of the characteristics of the study, sociodemographic
and
clinical
variables
of
interest
were
collected. Socio-
demographics included: age, gender, nationality, educational
level, marital status, and employment situation. Regarding the
children, clinical information was required. These were: the
diagnosis, date of diagnosis, type of treatment and current status
post-treatment. The adaptation of the Symptom Scale (Martos et al., 2009)
consists of a 9-point Likert scale that assesses perceived physical
and psychological symptoms. Subjective health was assessed
with the item “In general, I am in poor health.” The internal
consistency in the original scale, according to Cronbach’s alpha
statistic is 0.79 (Jou and Foucada, 1997), whereas in the present
study it is 0.85. INTRODUCTION Likewise, the Rasch model quantifies the amount of
information and error with which it is measured in each point of
the dimension and allows the selection of those items that make
it possible to increase the information in previously specified
regions of the construct (Jiménez and Montero, 2013). Recent studies in Spain have applied the Spanish version of
the FS. In a study by Ramírez et al. (2017), of university students
and patients with chronic back pain, the main axis factor, and
the simultaneous component analysis in both groups showed
a common unidimensional structure. In addition, the omega
coefficient showed high reliability. The principal conclusion
was that flourishing had a mediating effect between personality
variables and positive and negative affect, which explains the
association between anxiety, optimism, pessimism, and positive
affect. De la Fuente et al. (2017) have also supported the
Spanish version of the FS as a suitable measure. Specifically,
they applied it with university students from different fields
of knowledge. They obtained a single factor structure of
the FS, with good internal consistency. The convergent
validity of the scale was demonstrated with another measure
of
psychological
well-being
and
its
discriminant
validity
with the scale of symptoms of depression, anxiety, and
stress. In light of the above, it has already been supported that a
flourishing attitude is related to well-being indicators. Childhood
cancer is understood as a “family disease” because it affects the
health of all its members and determines its ongoing functioning
(Bretones, 2018). The importance of analyzing the psychosocial
repercussion of this pathology in the parents is due to the fact January 2019 | Volume 10 | Article 35 Frontiers in Psychology | www.frontiersin.org 2 Flourishing Scale in Childhood Cancer Pozo Muñoz and Bretones Nieto that they have been considered as “invisible patients” (Hinds,
1992). On the other hand, it has been shown that the adaptation
of children and adolescents is determined by factors related to
the degree of adjustment of their parents, especially in the initial
stages of the disease (Fedele et al., 2013). During the childhood
illness the parents of children with cancer have to suffer a lot
of critical moments in relation with diagnosis, treatments, side
effects, surgeries, e.g., In this way, stress symptoms represent a
common experience in the familiar context, also after the illness
situation (Vrijmoet-Wiersma et al., 2010). Procedure The study was presented to and approved by the Research Ethics
Committee of Torrecárdenas Hospital (Almeria, Spain). Research
methodology was planned considering the design of the inclusion
and exclusion criteria, the sample selection, the interview design,
etc. The requirements for participation in the interview were
being the parent of a child diagnosed with cancer who had been
treated in that hospital. Exclusion criteria was determined as the
following: being the parent of a child who had died due to the
disease and a parent who did not speak Spanish with sufficient
command. INTRODUCTION The Spanish version of the Satisfaction with Life Scale
(Goodwin and Hernández, 2000) is a 5-item Likert scale used
to assess the level of well-being experienced by parents. Answers
range from 1 (completely disagree) to 5 (completely agree). Cronbach’s alpha in the original scale is 0.87 (Diener et al., 1985),
while it is 0.82 in this study. Objectives The purpose of this study was to validate the Spanish version
of the Flourishing Scale following administration in a sample
of parents of children with cancer. This was done using Rasch
analysis in particular, also dimensionality, the fit of the items to
the model, the functioning of the measurement scale, construct
validity, reliability, and the differential item functioning (DIF). Finally, the convergent validity of the FS was determined in
relation to other measures that study the following variables:
well-being, coping styles and social support (these variables have
been chosen because they represent indications of the state of
health and quality of life of the parents of children with cancer,
in this case). In order to analyze the coping styles in parents, the
reduced Spanish version of the Cope Inventory (Cuestionario
de Afrontamiento del Estrés, CAE) (González and Landero,
2007) was applied. This scale consists of 21 items, which
are divided into seven subscales (although the total score of
the scale was used): problem-solving coping, negative auto-
focused coping, positive reappraisal, overt emotional expression,
avoidance coping, social support seeking, and religious coping. Items are answered on a 5-point Likert scale ranging from
0 (never) to 5 (almost always). While Cronbach’s alpha for
Sadín’s and Chorot’s original questionnaire reached 0.77, in CAE’s
reduced version internal consistency was 0.74, and finally, in this
study it reached 0.62. Sample The sample consisted of 138 adults: 60 men and 78 women,
who were the parents of 94 children (aged between 1 and 14
years) diagnosed with cancer. The parents’ ages ranged from
41 to 50 years old 0.84% were of Spanish nationality and
16% of foreign origin (10% African and 6% Romanian). In
this cohort 88.4% were married or living together as a couple,
60.2% had completed secondary school and 53.9% worked
in the services sector (includes subsectors such as commerce,
communications, call center, finance, tourism, hospitality,
leisure, culture, entertainment, public administration, and so-
called public services, provided by the State or private initiative
(health, education, care dependency; among others). The
diagnoses of the children were: leukemia (44.2%) and solid
tumors (55.8%). The flourishing attitude was evaluated through the Spanish
version of the Flourishing Scale (original by Diener et al., 2009). This scale is made up of eight items. The values of the answers
range from 1 to 5 (1 being “strongly disagree”; 2 “disagree”; 3
“neither agree nor disagree”; 4 “agree” and 5 “totally agree”). Cronbach’s alpha in the original scale is 0.87 (Diener et al., 2009),
whereas it is 0.74 in the present study. Measures This study comprises a quantitative part (on which this article
focuses, with validated Likert-type scales) and a qualitative part (a
series of open-ended questions designed to investigate the main
problems in fundamental areas of the family’s daily life) of the
data gathered during the semi-structured interviews in which
they evaluated: socio-demographics and clinical characteristics, The interview was drawn up according to the research
objectives and administered individually (a single session) in a January 2019 | Volume 10 | Article 35 Frontiers in Psychology | www.frontiersin.org 3 Flourishing Scale in Childhood Cancer Pozo Muñoz and Bretones Nieto between 0.5 and 1.5 (the MNSQ values of Infit and Outfit) (see
Table 2). private room of the hospital (approximately one hour’s duration),
following detailed explanation of the study’s characteristics. Participants signed consent forms and all data was treated in
accordance with current privacy laws. These were recorded and
transcribed verbatim to a text file. Audio and text files were then
saved and processed confidentially. The PT-Measure-CORR column allows observation of the
alignment between the question and the subject’s abilities. The
results indicate that there are no inverse correlations and the
associations are moderate (the lowest value is set in item 7,
with a correlation of 0.49, and the highest item being 4, with a
value of 0.73). In addition, columns PT-Measure-EXP. show that
there are no notable differences between observed and expected
correlations. Data Analysis Pearson correlation coefficients were used to
evaluate the convergent validity between the Spanish version
of the Flourishing Scale (FS), the Spanish version of the
Satisfaction with Life Scale, the reduced Spanish version of the
Coping Stress Questionnaire (CAE) and the adaptation of the
Perceived Social Support Scale (EASP/PSS). Data Analysis The results were analyzed using IBM SPSS software (version 22
for Windows) and Winsteps (v. 3.72.3) statistical package. The
following analyses were conducted: On the other hand, the reliability for the items was 0.99 with
a separation of 8.58, with high values. The values of separation
are >3. In relation to the reliability for people, a value of 0.65 and
a separation of 1.36 was obtained. The next step was to analyse
the construct validity based on the hierarchy of the items using
the Wright map (Figure 1). Unlike the results obtained in other
studies (Pozo et al., 2016), in this case, there was a number of
subjects in the center of the figure who do not have items in front
of them. They are at a moderate level of flourishing, but the scale
does not have adequate items to measure the level of the variable. – Descriptive analyses. – Internal consistency of the items calculated using Cronbach’s
alpha. – The unidimensionality of the test was verified by “main
components.” – The goodness of fit of each of the eight FS items (infit and outfit
estimates) to the Rasch model. – Reliability values were calculated for the items and for the
subjects. – The validity of the construct was verified based on the
hierarchy of the items (Wright map). TABLE 1 | Items of the Spanish Version of the FS. Ítems
x ± sd
I am a good person and live a good life (item 6)
4.48 ± 0.81
People respect me (item 8)
4.41 ± 0.87
I am competent and capable in the activities that are important to
me (item 5)
4.28 ± 1.10
I actively contribute to the happiness and wellbeing of others
(item 4)
4.25 ± 1.03
I lead a purposeful and meaningful life (item 1)
4.11 ± 1.08
I am engaged and interested in my daily activities (item 3)
3.76 ± 1.25
I am optimistic about my future (item 7)
3.62 ± 1.24
My social relationships are supportive and rewarding (item 2)
1.91 ± 0.95 – The functioning of the response categories was verified
through the Rating Scale Model (RSM) for polytomous items. – The
possible
differential
item
functioning
(DIF)
was
contrasted. – Finally, FS construct validity was examined with a wide variety
of measures. Descriptive Analysis TABLE 2 | Infit and outfit estimations for each item. TABLE 2 | Infit and outfit estimations for each item. INFIT
OUTFIT
PT-MEASURED
Item
Measure
Model SE
MNSQ
MNSQ
CORR. EXP. Item7
0.35
0.09
1.37
1.40
0.49
0.65
Item2
2.30
0.10
1.16
1.36
0.48
0.57
Item6
−0.88
0.12
1.11
0.97
0.48
0.52
Item5
−0.50
0.11
1.04
0.79
0.66
0.57
Item1
−0.25
0.10
1.03
1.02
0.60
0.60
Item8
−0.75
0.12
0.96
0.98
0.54
0.54
Item4
−0.46
0.11
0.89
0.75
0.65
0.58
Item3
0.19
0.09
0.83
0.81
0.73
0.64
Item mean
0.00
0.11
1.05
1.01
–
–
SD
0.96
0.01
0.16
0.23
–
–
The order of the items corresponds to the adjustment of each of the items according to
the output of the table in the Rash model. Measure, Location of the latent variable; Model SE, Error estándar; INFIT-MNSQ, Infit
Mean Squared; “OUTFIT-MNSQ, Outfit Mean Squared; PT Measured, Point Measure
Correlation. The results exceed the midpoint of the scale (M = 3.85; sd
= 0.63). This also happens in specific items, such as being
considered a “good person and lead a good life,” feeling respected,
subjectively perceived as “competent and capable,” contributing
to the happiness and well-being of people close to them and
having a “useful and meaningful” life (Table 1). On the other hand, defining oneself as optimistic and being
involved in daily activities was also considered. Despite this,
the lowest scores are related to parent’s perceptions about their
supportive and rewarding social relations. The Rasch Model on the Flourishing Scale The Rasch Model on the Flourishing Scale
Cronbach’s alpha for the Spanish version of the FS was 0.74. In this sense, it is possible to conclude that it is structured
in a single dimension. The variance explained by the measure
was 59.2%, with a first contrast eigenvalues of 1.6, meeting the
recommendations for assuming the dimensionality (variance
>0.30; first contrast eigenvalue <3.0) stated by of Bond and Fox
(2007, 2012) or Smith (2012). The item fit to the Rasch Model of
each of the eight items of the scale was checked. All items were January 2019 | Volume 10 | Article 35 Frontiers in Psychology | www.frontiersin.org 4 Flourishing Scale in Childhood Cancer Pozo Muñoz and Bretones Nieto FIGURE 2 | Probability curves for the response categories. FIGURE 2 | Probability curves for the response categories. FIGURE 1 | Wright map of person and items. FIGURE 2 | Probability curves for the response categories. (2011), an item has a DIF associated with belonging to a group
when people with the same value in the measured variable, but
from different groups, have a different probability of solving
that item correctly. In this way, it was established as a criterion
for a possible DIF that the values of “DIF contrast” were >0.5
logits between the comparison groups and that this difference
in the t values was significant (p ≤0.05; Bond and Fox, 2007,
2012). )
A possible DIF by “gender” was found for item 7 (“I am
optimistic about my future”) with a contrast of 0.62 (p ≤0.05),
being a more difficult item for women than for men. There was
also a possible DIF by “level of education” for item 5 (“I am
competent and capable in the activities that are important to me”),
with a contrast of 1.41 (p ≤0.05), being a more difficult item for
participants without studies than for those with higher education. It is equally important to consider a possible DIF with “the
child’s situation with respect to treatment” for item 6 (“I am a
good person and live a good life”), with a contrast of 0.84 (p ≤
0.05). It is a more difficult item for parents whose children are
receiving treatment, compared to participants whose children
have been “out of treatment” for >5 years. The Rasch Model on the Flourishing Scale A possible DIF by “gender” was found for item 7 (“I am
optimistic about my future”) with a contrast of 0.62 (p ≤0.05),
being a more difficult item for women than for men. There was
also a possible DIF by “level of education” for item 5 (“I am
competent and capable in the activities that are important to me”),
with a contrast of 1.41 (p ≤0.05), being a more difficult item for
participants without studies than for those with higher education. It is equally important to consider a possible DIF with “the
child’s situation with respect to treatment” for item 6 (“I am a
good person and live a good life”), with a contrast of 0.84 (p ≤
0.05). It is a more difficult item for parents whose children are
receiving treatment, compared to participants whose children
have been “out of treatment” for >5 years. FIGURE 1 | Wright map of person and items. Response categories’ function was studied through the
Rating
Scale
Model
(RSM)
for
polytomous
items. The
response categories were the following: (1) means “strongly
disagree”; (2) “somewhat disagree”; (3) “neither agree nor
disagree”; (4) “somewhat in agreement,” and (5) “totally
agree.” In the probability curves of the categories it was
found that category 3 overlapped with categories 2 and 4 (see
Figure 2). Finally, regarding convergent and divergent validity, our
results largely confirmed the correlations between FS and other
measures under study with a Bonferroni-adjusted (Table 3). We
found a moderate relationship between flourishing and well-
being (r = 0.403, p ≤0.01). The FS also showed a low correlation
with the satisfaction with the social support received (r = 0.293,
p ≤0.01) and with coping strategies that parents use; specifically,
“the positive re-appraisal” (r = 0.211, p ≤0.05); with “the
problem-solving coping” (r = 0.186; p ≤0.05), the “negative
auto-focused coping” (r = −0.248; p ≤0.01) and the “over
emotional expression” (r = −0.171; p ≤0.05). The possible Differential Item Functioning (DIF) of the items
was checked by “gender,” “educational level” and “the child’s
situation with respect to treatment.” According to Prieto et al. January 2019 | Volume 10 | Article 35 Frontiers in Psychology | www.frontiersin.org 5 Flourishing Scale in Childhood Cancer Pozo Muñoz and Bretones Nieto TABLE 3 | Correlation between Flourishing and other measures. 1
2
3
4
5
6
7
1. Flourishing
1
–
–
–
–
–
–
2. DISCUSSION Originally called “item bias” (Lord, 1980), Differential Item
Functioning (DIF) has been recognized as a potential source of
bias in person measure. However, other authors indicate that the
existence of the DIF does not necessarily imply a bias in the
item (Wright and Stone, 1999). When analyzing the differential
behavior of items (DIF), the results indicate a possible DIF by
gender, by level of education and by situation with respect to
treatment. In short, the aforementioned items should continue
to be investigated in order to corroborate if there really is a bias
or not. The purpose of the present study consisted of validate the Spanish
version of the Flourishing Scale following administration in
a sample of parents of children with cancer. As a first step,
descriptive analyzes were carried out to check the tendency of
the sample to show a flourishing attitude. If were considered the
results of this study with those obtained in other investigations
in which other groups (specifically, university students) have
contributed (Pozo et al., 2016; Garzón Umerenkova et al., 2017)
it is possible to affirm that the parents who have participated in
this study have a flourishing attitude, because the scores obtained
exceed the midpoint of the scale. Good results are maintained
with regard to the internal consistency of the Spanish version of
the FS. The Rasch Model on the Flourishing Scale Wellbeing
0.403**
1
–
–
–
–
–
3. Satisfaction with social support
0.293**
0.099
1
–
–
–
–
4. Positive re–appraisal
0.211*
0.265**
0.137
1
–
–
–
5. Problem–solving coping
0.186*
0.158
0.010
0.177*
1
–
–
6. Negative auto–focused coping
−0.248**
−0.188*
−0.029
−0.328**
−0.161
1
–
7. Over emotional expression
−0.171*
−0.057
−0.111
−0.043
−0.008
0.247**
1
*p ≤0.05; **p ≤0.01. TABLE 3 | Correlation between Flourishing and other measures. CONCLUSION As a strength, the Rasch analysis performed after the application
of the Spanish version of the FS on the parents of children
with cancer, indicates that generally the test presents a good
psychometric performance. However, it would be appropriate
to continue investigating the previously mentioned aspects to
further improve its performance. The findings of the Rasch analysis have shown the one-
dimensionality of the scale and there is a good fit to the model
of each of the eight items that make up the scale. The infit and outfit mean-square (MNSQ) statistics provide
evidence of construct validity when expected values are close to
1.0, with values from 0.5 to 1.5 being useful for measure, so in
this case a good fit to the model is confirmed. Equally, there
is a good alignment between the questions and the skills of the
respondent. In relation to the convergent validity, our results are in
agreement with the postulates of well-being Theory in the
field of Positive Psychology (Seligman, 2011), since there is a
moderate correlation between flourishing and perceived well-
being. For this reason, it is important to continue researching,
using flourishing as a measure. In this connection, in principle,
we could interpret that flourishing is an effective coping strategy
for the parents of children with cancer, because it is positively
related to styles considered healthy (“satisfaction with social
support,” “positive re-appraisal” and “problem-solving coping”)
and in a negative way with others, that are not (“negative auto-
focused coping” and “over emotional expression”). Therefore,
it is possible to affirm that flourish people are more likely
to develop more favorable behavior patterns in situations that
may be problematic (such as the experience of having a child
with cancer). It would be convenient to take these results into
account in order to develop psychosocial intervention programs
(planning actions that stimulate the development of Flourishing
components) aimed at responding to the needs of the parents of
children with cancer. In accordance with Smith (2012), the Rasch model verifies that
the sample is large enough to corroborate the difficulty hierarchy
of the items, namely, the construct validity of the instrument the
reliability of people is considered a low index of separation for
people with inferior values to 2. Therefore, the instrument is not
as sensitive as one would wish, for identifying people with high
and low skill. Frontiers in Psychology | www.frontiersin.org CONCLUSION The differences in this study compared to other
studies may be due to the diversity of the characteristics of the
samples used (Pozo et al., 2016; Garzón Umerenkova et al., 2017). The construct validity of FS can be improved by adding
items of moderate and high difficulty. This would increase the
sensitivity of the instrument and the reliability for people, since
these two points are related. The overlap found between the response categories could be
solved by eliminating category 3, reducing the Likert scale from
five to four points, or the term “neither agree nor disagree” could
be rewritten, which would seem to be insufficiently differentiated
from the terms “somewhat in disagreement” (2) or “somewhat in
agreement” (4). On the other hand, regarding the advantages of an IRT
analysis with respect to the usual CCT, the use of the
characteristics of the IRT method has constituted an advance for Frontiers in Psychology | www.frontiersin.org January 2019 | Volume 10 | Article 35 6 Flourishing Scale in Childhood Cancer Pozo Muñoz and Bretones Nieto this study, allowing the improvement in the measurement of the
scale as well as its refinement. the different strategies to face the childhood illness on the part of
the mother and the father, isolating this problem from the others. On the other hand, a global approach based on dyads could be
useful in this case (even for correlation with other scales and the
Rasch analysis) and could be useful to complement the results
obtained. It is worth highlighting some methodological limitations
detected that should be taken into account in subsequent studies
(low separation, no items at a mid-range of the construct, some
problem with the response scale, moderate correlations with
validation measures). On the other hand, due to the number of
cases of childhood cancer in the study context, it has not been
possible to apply a specific sampling method. In addition, a large
volume of experimental mortality has been observed. Regarding
this issue, it has been found that research related to “childhood
cancer” is a difficult field to gather large samples (Bragado et al.,
2008). The language barrier (in the immigrant population) and
the families that declined the invitation to participate, were the
greatest difficulties. REFERENCES Diener, E., Wirtz, D., Tov, W., Kim-Prieto, C., Choi, D., Oishi, S., et al. (2010). New measures of well-being: flourishing and positive and negative feelings. Soc. Indic. Res. 39, 247–266. doi: 10.1007/978-90-481-2354-4_12 Arias, B., Arias, V., and Gómez, L. E. (2013). Calibración del índice de
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Psychology). Giuntoli, L., Ceccarini, F., Sica, C., and Caudek, C. (2017). ETHICS STATEMENT This study has been developed in accordance with the
Declaration of Helsinki and has been approved by the Research
Ethics Committee of Torrecárdenas Hospital (Almeria, Spain). There was no conflict of interest between the authors and other
institutions. CONCLUSION This is associated with the fact that, for
most of the parents, submitting to the interview took great
emotional effort, due to having to exercise awareness and delve
into circumstances of their life that were especially painful for the
child, the parents themselves, and the rest of the family. AUTHOR CONTRIBUTIONS CP was the researcher in charge of coordinating research
planning, reviewing the literature, and subsequently proposing
the objectives and research design. In addition, she has
participated in the analysis phase of the data and in the writing
of the article. Finally, the importance of these results makes it necessary to
replicate this study in a population with similar characteristics
in order to contrast the resulting conclusions and implement
methodological improvements in relation to the scale. We even
propose for a future investigation to explore through a pair of
pairs of parents of the same child, so that both can individualize BB was responsible for selecting the final sample of parents
who participated; she conducted the interviews, participated in
the analysis phase of the data and in the writing of the article. REFERENCES Validation of the
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and dimensionality of the “Flourishing Scale” in Spanish speaking population. Electr. J. Res. Ed. Psychol. 14, 175–192. doi: 10.14204/ejrep.38.15044 Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
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functioning of mini-mental test items according to disease. Neurología 26,
474–480. doi: 10.1016/j.nrl.2011.01.013 Copyright © 2019 Pozo Muñoz and Bretones Nieto. 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. Ramírez, C., Correa, M., Ricas, T., López, A. E., Serrano, E. R., and Esteve, R. (2017). Psychometric characteristics of the Flourishing Scale-Spanish Version
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psychometric validation of the Flourishing-at-Work Scale – Short Form January 2019 | Volume 10 | Article 35 Frontiers in Psychology | www.frontiersin.org 8
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Archigregarines of the English Channel revisited: New molecular data on Selenidium species including early described and new species and the uncertainties of phylogenetic relationships
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RESEARCH ARTICLE Editor: Ross Frederick Waller, University of
Cambridge, UNITED KINGDOM Editor: Ross Frederick Waller, University of
Cambridge, UNITED KINGDOM
Received: May 30, 2017
Accepted: October 19, 2017
Published: November 3, 2017 Copyright: © 2017 Rueckert, Hora´k. 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. Archigregarines of the English Channel
revisited: New molecular data on Selenidium
species including early described and new
species and the uncertainties of phylogenetic
relationships Sonja Rueckert1*, Alesˇ Hora´k2,3 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 1 School of Applied Sciences, Edinburgh Napier University, Edinburgh, United Kingdom, 2 Biology Centre of
CAS, Institute of Parasitology, Česke´ Budějovice, Czech Republic, 3 University of South Bohemia, Faculty of
Science, Česke´ Budějovice, Czech Republic * s.rueckert@napier.ac.uk OPEN ACCESS Citation: Rueckert S, Hora´k A (2017)
Archigregarines of the English Channel revisited:
New molecular data on Selenidium species
including early described and new species and the
uncertainties of phylogenetic relationships. PLoS
ONE 12(11): e0187430. https://doi.org/10.1371/
journal.pone.0187430 Background Gregarines represent an important transition step from free-living predatory (colpodellids
s.l.) and/or photosynthetic (Chromera and Vitrella) apicomplexan lineages to the most
important pathogens, obligate intracellular parasites of humans and domestic animals such
as coccidians and haemosporidians (Plasmodium, Toxoplasma, Eimeria, Babesia, etc.). While dozens of genomes of other apicomplexan groups are available, gregarines are
barely entering the molecular age. Among the gregarines, archigregarines possess a unique
mixture of ancestral (myzocytosis) and derived (lack of apicoplast, presence of subpellicular
microtubules) features. Editor: Ross Frederick Waller, University of
Cambridge, UNITED KINGDOM
Received: May 30, 2017
Accepted: October 19, 2017
Published: November 3, 2017
Copyright: © 2017 Rueckert, Hora´k. 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 Gregarine apicomplexans are obligate, unicellular parasites of freshwater, marine and terres-
trial invertebrates infecting the intestine, coelom and reproductive vesicles. As gregarines do
not generally cause any harmful diseases compared to their well-studied closely related sis-
tergroups known as notorious parasites of humans and domestic animals (e.g. Plasmodium,
Cryptosporidium and Toxoplasma), they are still an undersampled and little described group of
protists. There is a strong need to overhaul the complete gregarine taxonomy and Cavalier-
Smith [1] made an attempt in doing so. Unfortunately, his way to tackle the problem by erect-
ing 18 new taxa mainly based on a single SSU rDNA analysis is rather preliminary. The poor
support of most of the clades and branching orders in question does not allow for these taxo-
nomic conclusions to be made. At this point, we will therefore stick with the separation of the
gregarines into the three major groups (archigregarines, eugregarines and neogregarines)
based on trophozoite morphology, host affinity and life history (e.g. [2–9]) The first time a gregarine was officially described was in 1828 by Dufour, who erected the
genus Gregarina [2]. While in the early years of gregarine discovery, morphological descrip-
tions were based exclusively on light microscopic and histologic studies, the importance of
ultrastructural studies using electron microscopy became quickly apparent and shed important
light on differences e.g. between archigregarines and eugregarines (e.g. [10–15]). These data
showed that archigregarines have wide, longitudinal epicytic folds with an underlying layer of
subpellicular microtubules, while eugregarines have narrow epicytic folds with apical filaments
and rippled dense structures (see [16]). Nonetheless, available data are still quite limited. In the
last decades the molecular approach for phylogenetic analyses started to boom and several
genomes of the crown apicomplexans including Plasmodium, Toxoplasma, Crytosporidium,
Babesia, etc. are already available (e.g. [17,18]). The genomes of photosynthetic apicomplexan
lineages including Chromera velia and Vitrella brassicaformis have been published recently
[19], but gregarine apicomplexans have hardly entered the genomic age (apart from a very par-
tial genome draft of Ascogregarina taiwanensis [20]). The importance of molecular phyloge-
netic data and their value in inferring relationships between gregarine species has lead to an
increase in available sequence data in public databases such as GenBank, but these are still very
limited in numbers and mostly restricted to SSU rDNA sequences. Archigregarines of the English Channel Conclusions/Significance No. 15-17643S. The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript. Our phylogenies using the extended gregarine sampling show that the archigregarines are
indeed not monophyletic with one strongly supported clade of Selenidium sequences
around the type species S. pendula. We suggest the revision of the whole archigregarine
taxonomy with only the species within this clade remaining in the genus Selenidium, while
the other species should be moved into newly erected genera. However, the SSU rDNA
phylogenies show very clearly that the tree topology and therefore the inferred relationships
within and in between clades are unstable and such revision would be problematic without
additional sequence data. Competing interests: The authors have declared
that no competing interests exist. Competing interests: The authors have declared
that no competing interests exist. Methodology/Principal findings In this study we revisited five of the early-described species of the genus Selenidium includ-
ing the type species Selenidium pendula, with special focus on surface ultrastructure and
molecular data. We were also able to describe three new species within this genus. All spe-
cies were characterized at morphological (light and scanning electron microscopy data) and
molecular (SSU rDNA sequence data) levels. Gregarine specimens were isolated from poly-
chaete hosts collected from the English Channel near the Station Biologique de Roscoff,
France: Selenidium pendula from Scolelepis squamata, S. hollandei and S. sabellariae from
Sabellaria alveolata, S. sabellae from Sabella pavonina, Selenidium fallax from Cirriformia
tentaculata, S. spiralis sp. n. and S. antevariabilis sp. n. from Amphitritides gracilis, and S. opheliae sp. n. from Ophelia roscoffensis. Molecular phylogenetic analyses of these data
showed archigregarines clustering into five separate clades and support previous doubts
about their monophyly. Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files. Funding: This project was supported by
ASSEMBLE EU FP7 research infrastructure
initiative (EU-RI-227799) call 7 (2013) and call 9
(2014), the research infrastructure funded from the
European Union Seventh Framework Programme
(FP7/2007-2013) under grant agreement n˚
316304 as well as the Czech Grant Agency project 1 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 Collection and isolation of organisms Different polychaete species were collected by hand from rocks and sandy beaches from the
English Channel near the Station Biologique Roscoff (SBR), France, in May 2013 and in June
2014. No specific permits were required for the sampling locations as they are not privately
owned or protected. This study did not involve any endangered or protected species. The collected
polychaetes were identified based on morphological characteristics. Selenidium pendula was iso-
lated from the intestines of Scolelepis squamata (Mueller, 1806) (Spionida) collected by hand from
a beach close to the SBR (48˚43´31´´N, 3˚59´26´´W). Selenidium hollandei and S. sabellariae were
isolated from the intestines of Sabellaria alveolata (Linnaeus, 1767) (Sabellida) collected by hand
from Saint Efflam (48˚68´48´´N, 3˚61´13´´W). Selenidium sabellae was isolated from the intes-
tines of Sabella pavonina Savigny, 1822 (also Sabellida) hand-collected from Pempoul (48˚68´23
´´N, 3˚95´14´´W). Selenidium fallax was isolated from Cirriformia tentaculata (Montagu, 1808),
S. spiralis sp. n. and S. antevariabilis sp. n. were isolated from Amphitritides gracilis (Grube, 1860)
(both Terebellida) both polychaete species were collected by hand from rock assemblages close to
the SBR (48˚43’44”N, 3˚59’23”W). Selenidium opheliae sp. n. was isolated from Ophelia roscoffensis
Augener, 1910 (Opheliidae) hand-collected from Le Guillec (48˚68´56´´N, 4˚06´78´´W). The trophozoites of each species were released in seawater by teasing apart the intestines of
the respective host with fine-tipped forceps under a dissecting microscope (Zeiss Stemi 2000). The gut material was examined under an inverted microscope (Zeiss Axiovert A1) and para-
sites were isolated with a hand drawn glass pipette and washed three times in filtered seawater,
before being examined and photographed under the inverted microscope or prepared for
DNA extraction. Introduction One general problem with
molecular phylogenies is that the outcome often heavily depends on the original species dataset
utilized for the analyses [21]. Archigregarines, which are found only in marine invertebrates, especially polychaetes, are
of particular interest, as they are supposed to form the paraphyletic stem group from which all
other gregarines and maybe even all apicomplexans have evolved [2,22–26]. Archigregarines PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 2 / 24 Archigregarines of the English Channel possess a unique mixture of ancestral (myzocytosis; e.g. [27]) and derived (lack of apicoplast,
presence of subpellicular microtubules; e.g. [3, 28,29]) features. There are around ~80 archigre-
garine species described to date [11,25,26,29]. Around 65 described species belong to the genus
Selenidium. Levine [3] proposed to erect a new genus Selenidioides to accommodate those 11
species that show merogony, belonging truly to the archigregarines, while the rest of the Seleni-
dium species was shuffled into the order Eugregarinorida. Because it is difficult to prove or dis-
prove the presence or absence of merogony/schizogony (asexual reproduction of trophozoite
stage) within a lifecycle, we and many other authors do not believe it to be a good indicator for
the relationships among archigregarines [16,25,26,29–32]. Therefore, we will not follow this
split of Selenidium species and thus treat Selenidium as the main genus of archigregarines. Just
recently Schre´vel et al. [33] published for the first time molecular data for the type species Seleni-
dium pendula. The presented phylogenies show one clade of true Selenidiidae around S. pendula
and two other clades of archigregarines, all reflecting their respective host species. In this study we set out to obtain molecular data of morphologically and ultrastructurally
well-described species including the type species Selenidium pendula, S. hollandei, S. sabellar-
iae, S. sabellae and S. fallax. In addition, we were able to discover three new species. We will
present morphological characterizations of all species by light and scanning electron micros-
copy and discuss the phylogenetic relationships of archigregarines within the context of a
broad apicomplexan phylogeny based on SSU rDNA data. Archigregarines of the English Channel Between 8 and 60 specimens each of the isolated gregarine species were prepared for scan-
ning electron microscopy (SEM). Some individuals were deposited directly into the threaded
hole of separate Swinnex filter holders, containing a 10 μm polycarbonate membrane filter
(Millipore Corp., Billerica, MA), that was submerged in 10 ml of seawater within a small canis-
ter (2 cm diam. and 3.5 cm tall). A piece of Whatman No. 1 filter paper was mounted on the
inside base of a beaker (4 cm diam. and 5 cm tall) that was slightly larger than the canister. The
Whatman filter paper was saturated with 4% (w/v) OsO4 and the beaker was turned over the
canister. The parasites were fixed by OsO4 vapors for 30 min. Ten drops of 4% (w/v) OsO4
were added directly to the seawater and the parasites were fixed for an additional 30 min. A
10-ml syringe filled with distilled water was screwed to the Swinnex filter holder and the entire
apparatus was removed from the canister containing seawater and fixative. Other individuals
were deposited in hand-made baskets (top end of a 1000μl pipette tip fixed with silicon to the
same 10 μm filters) and placed in 24-well culture plates. These filters were prefixed in 2.5% (v/
v) glutaraldehyde in 0.05 M cacodylate buffer (pH = 7.4) for one hour, washed and post-fixed
with 1% (w/v) OsO4 for 45 minutes. All filters were washed with water and dehydrated with a
graded series of ethyl alcohol. Filters prepared in the Swinnex filter holders were critical point
dried with CO2, filters from the hand-made baskets were air-dried. Filters were mounted on
stubs, sputter coated with 5 nm of gold, and viewed under a scanning electron microscope
(JEOL JBM7401F or Phenom 806). Some SEM data were presented on a black background
using Adobe Photoshop CS5 (Adobe Systems Incorporated, San Jose, CA). PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 Light and scanning electron microscopy Differential interference contrast (DIC) and phase contrast (PC) light micrographs of all spe-
cies were taken with a 5 megapixel CMOS camera AxioCam ERc 5s, attached to an inverted
microscope (Zeiss Axiovert 1). 3 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 Molecular phylogenetic analysis The eight new SSU rDNA sequences were aligned with 163 other SSU rDNA sequences, repre-
senting the major lineages of apicomplexans (with an emphasis on gregarines) and relevant
outgroups (ciliates, dinozoans and chromerids and colpodellids), using local alignment with
generalized affine gap costs (E-INS-I) as implemented in MAFFT [35]. Sites comprised mostly
of gaps and ambiguously aligned regions were manually excluded from the 171-taxon align-
ment in SEAVIEW 4 [36] resulting in 1,488 unambiguously aligned sites (a NEXUS file of this
alignment is available upon request). Alternatively, we have used trimAl 1.2 [37] for automatic
ambiguous sites detection/exclusion under relaxed (parameters set as ’-gt 0.3’ and ’-st 0.001’;
dataset R, 1805 sites) as well as strict (-gappyout option engaged; dataset G, 945 sites) settings. Based on the results of a preliminary analyses and in agreement with previously published
studies, we have also created a smaller dataset (S) based on the exhaustive sampling of Seleni-
diidae s.s. (as defined by [33]), as well as other archigregarine taxa (81 taxa, 1601 sites) rooted
with marine eugregarines. Homogeneity of base-composition was tested using Tree-Puzzle 5.3
[38]. Root-to-tip distances for main clades of the dataset were measured using the TreeStat 1.2
(http://tree.bio.ed.ac.uk/software/treestat/). Because the 18S rDNA of several gregarine taxa did not pass the homogeneity test, the
Bayesian inference was carried out with exchange rates defined by the general time-reversible
model and the number of categories limited to 40 (GTR + C40), implemented in Phylobayes
4.1 [39]. This model was chosen as a compromise between the robustness of CAT admixture
model with an infinite number of rate categories, which is however mostly suitable for large
phylogenomic datasets due to its complexity, and simplicity of time-tested GTR model. Two
chains were run until they converged (i.e. the maximum observed discrepancy below 0.2 and
effective number of model parameters reached 100). Posterior probabilities of the branching
were reconstructed after burn-in of the first fifth of the generations. Alternatively, we have also
performed a maximum likelihood analysis under the gamma-corrected GTR model using the
RAxML 8.2a [40]. The highest-scoring topology was estimated using the rapid-bootstrapping
algorithm from 1000 replicates, the branching support was assessed using the non-parametric
bootstrapping and the ‘thorough’ algorithm from 1000 replicates using the same model and
software. The putative monophyly of archigregarines was tested using the approximate likeli-
hood ratio test as implemented in Consel [41]. DNA isolation, PCR, cloning, and sequencing Individual trophozoites of each species were isolated from the dissected hosts, washed three times
in filtered seawater, and deposited into a 1.5-ml microcentrifuge tube: 23 trophozoites of S. pen-
dula from S. squamata, 27 trophozoites of S. hollandei and 75 trophozoites of S. sabellariae from
Sabellaria alveolata, 8 trophozoites of S. sabellae from Sabella pavonina, 45 cells of Selenidium fal-
lax from Cirriformia tentaculata, 11 and 12 trophozoites of S. spiralis sp. n. and S. antevariabilis sp. n. from Amphitritides gracilis, and 31 trophozoites of S. opheliae sp. n. from Ophelia sp. DNA was extracted using the MasterPureTM Complete DNA and RNA Purification Kit
(Epicentre Biotechnologies, Madison, WI). Small subunit rDNA (SSU rDNA) sequences were
PCR-amplified either using a total volume of 50 μl containing 1 μl of primer, 5 μl of DNA tem-
plate, 25 μl of OneTaq Mastermix (New England Biolabs, Inc., Ipswich, USA), or using a total
volume of 25μl containing 2 μl of primer, 2.5 μl of DNA template, 20.5 μl of dH2O and one
PuReTaq Ready-to-go PCR Bead (GE Healthcare, Quebec, Canada). The SSU rDNA sequences from these species were amplified in one fragment (~1800 bp)
using universal eukaryotic PCR primers F1 5´-GCGCTACCTGGTTGATCCTGCC-3´ and R1
5´-GATCCTTCTGCAGGTTCACCTAC-3´ [34] and internal primers designed to match exist-
ing eukaryotic SSU sequences F2 5´-AAGTCTGGTGCCAGCAGCC-3´ and R2 5´-TTTAAG
TTTCAGCCTTGCG-3´.PCR was performed using MJ MiniTM Gradient Thermal Cycler (Bio-
Rad) and the following protocol: After 4 cycles of initial denaturation at 94 oC for 4.5 min, 45
oC for 1 min and 72 oC for 1.75 min, 34 cycles of 94 oC for 30 sec (denaturation), 50 oC for 1
min (annealing), 72 oC for 1.75 min (extension), followed by a final extension period at 72 oC
for 10 min. PCR products corresponding to the expected size were gel isolated using the Ultra-
CleanTM 15 DNA Purification kit (MO Bio, Carlsbad, California) and cloned into the pCR 2.1
vector using the TOPO TA cloning kit (Invitrogen, Frederick, MD). Eight cloned plasmids
were digested with EcoRI and screened for size. One or two clones for each species were se-
quenced with ABI big dye reaction mix using vector primers and internal primers oriented in
both directions. DNA isolation, PCR, cloning, and sequencing PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 4 / 24 Archigregarines of the English Channel The new SSU rDNA sequences were initially identified by BLAST analysis and subsequently
verified with molecular phylogenetic analyses (GenBank Accession numbers: Selenidium pen-
dula MF882901, Selenidium hollandei MF882899, Selenidium sabellariae MF882900, Seleni-
dium sabellae MF882906, Selenidium fallax MF882905, Selenidium spiralis sp. n. MF882902,
Selenidium antevariabilis sp. n. MF882903, Selenidium opheliae sp. n. MF882904). Molecular phylogenetic analysis Prior to the analysis, we have optimized the
topology and branch lengths of the starting tree with forced monophyly of archigregarine
sequences in RAxML. Morphological observations All given measurements are based on light micrographs from fresh material, as some of the
gregarines seemed to have shrunk during preparation for scanning electron microscopy. All
trophozoites were brownish in colour under the LM, reflecting an accumulation of amylopec-
tin granules within the cytoplasm. Selenidium pendula (Fig 1, Table 1). Trophozoites were isolated from the polychaete
Scolelepis squamata (Mueller, 1806). The morphology matched the original description of Fig 1. Differential interference contrast (DIC) light micrographs and scanning electron micrographs
(SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium pendula
isolated from the polychaete Scolelepis squamata. A-C. DIC micrographs showing a spindle-shaped
trophozoite in different positions of the pendular movement. The mucron at the anterior end is rounded
(arrowhead) and visually separated from the rest of the cell with a slight restriction. The axial canal (ac) is
conspicuous at the anterior end, but ran along the entire cell. The oval nucleus (n) is situated in the middle of the
cell. D-E. Two gamonts (G1, G2) in caudal syzygy. The mucrons (arrowhead) are visible. The double arrow
marks the junction between the two gamonts. Gamonts are changing from elongated to stumpy. F. Young
gametocyst, the junction (double arrow) is still visible. G. Gametocysts in different focal planes, packed with
round oocysts. H-K. SEM micrographs of trophozoites showing the epicytic longitudinal folds (arrow), the mucron
(arrowhead) free of folds and some transverse striations in the inner curvature of the cell. Scale bars: Fig 1A-E,
30 μm; Fig 1F, 50 μm; Fig 1G, 70 μm; Fig 1H-I, 5μm; Fig J-K, 2μm. https://doi org/10 1371/journal pone 0187430 g001 Fig 1. Differential interference contrast (DIC) light micrographs and scanning electron micrographs
(SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium pendula
isolated from the polychaete Scolelepis squamata. A-C. DIC micrographs showing a spindle-shaped
trophozoite in different positions of the pendular movement. The mucron at the anterior end is rounded
(arrowhead) and visually separated from the rest of the cell with a slight restriction. The axial canal (ac) is
conspicuous at the anterior end, but ran along the entire cell. The oval nucleus (n) is situated in the middle of the
cell. D-E. Two gamonts (G1, G2) in caudal syzygy. The mucrons (arrowhead) are visible. The double arrow
marks the junction between the two gamonts. Gamonts are changing from elongated to stumpy. F. Nomenclatural acts The electronic edition of this article conforms to the requirements of the amended International
Code of Zoological Nomenclature, and hence the new names contained herein are available
under that Code from the electronic edition of this article. This published work and the nomen-
clatural acts it contains have been registered in ZooBank, the online registration system for the
ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated infor-
mation viewed through any standard web browser by appending the LSID to the prefix "http://
zoobank.org/". The LSID for this publication is: urn:lsid:zoobank.org:pub:91B0E976-C459- 5 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 Archigregarines of the English Channel 46E5-9B93-FA96177BC9CA. The electronic edition of this work was published in a journal
with an ISSN, and has been archived and is available from the following digital repositories:
PubMed Central, Edinburgh Napier Repository (http://www.napier.ac.uk/research-and-
innovation/repository). 46E5-9B93-FA96177BC9CA. The electronic edition of this work was published in a journal
with an ISSN, and has been archived and is available from the following digital repositories:
PubMed Central, Edinburgh Napier Repository (http://www.napier.ac.uk/research-and-
innovation/repository). Archigregarines of the English Channel Table 1. Morphological comparison of Selenidium species presented in this study and other species that belong to the same clades in the phylogenetic tree (data from
this study highlighted in bold). species
Host
Locality
Host
tissue
Trophozoite
shape
Trophozoite
size (L x W,
μm)
Nucleus
shape
Nucleus
size (L x W,
μm)
Position of
nucleus
Motility
# of long. epicytic
folds
Trans. surface
folds
Shape of
mucron
SSU
rDNA
Ref. S. pendula
Nerine cirratulus
E. Atlantic
Intestine
spindle-shaped
180 x 30–40
round to
oval
18–33 x
13–32
middle
bending,
twisting,
pendulum-like
20–30
? pointed
-
[3,43]
S. pendula
Scolelepis
squamata
E. Atlantic
Intestine
spindle-shaped
147–260 x 12–
48
oval
16–28 x
7–22
middle
bending,
twisting,
pendulum-like
30–34
+
rounded
+
this
study
S. terebellae
Terebella
lapidaris
E. Pacific, E. Atlantic
Intestine
spindle-shaped
30–330 x
12–40
oval
(ellipsoidal)
35 x 15
posterior
half
Bending,
twisting
46.00
+
Pointed to
slightly round
+
[3, 26]
S. hollandei
Sabellaria
alveolata
E. Atlantic
Intestine
spindle-shaped,
elongate,
flattened
up to 500 x 20–
30
spherical or
ovoid
16 x 6–8
? bending,
twisting
about 16
? heart-shaped
-
[3]
S. hollandei
Sabellaria
alveolata
E. Atlantic
Intestine
elongated,
flattened
97–411 x
13–43
spherical
or ovoid
4–19 x
6–17
middle to
posterior
half
bending,
twisting, rapid
rolling
18
+
heart-shaped,
sometimes
rounded
+
this
study
S. sabellariae
Sabellaria
alveolata
E. Atlantic
Intestine
spindle-shaped
220–500 x 16–
25
ovoid
16 x 13
? bending,
twisting
? +
pointed, spine
present
-
[3]
S. sabellariae
Sabellaria
alveolata
E. Atlantic
Intestine
spindle shaped,
slightly flattened
68–227 x
8–23
ovoid
7–15 x
6–13
middle
quick coiling
& uncoiling
14-Dec
+
pointed
+
this
study
S. neosabellaria
Neosabellaria
cementarium
W. Pacific
Intestine
vermiform
125–350 x
9–12
spherical
10
middle
bending,
twisting,
undulated
12-Oct
+
nipple-shaped
+
[49]
S. sabellae
Sabella
pavonina
E. Atlantic
Intestine
24–120 x
8–60
spherical
? ? ? 24–40
? conical
-
[3]
S. sabellae
Sabella
pavonina
E. Atlantic
Intestine
rectangualar’,
flattened
40–85 x
10–16
oval
6–12 x
5–11
middle
bending,
contracting
20–40
-
cone-shaped
+
this
study
S. fallax
Cirriformia
tentaculata
E. Atlantic
Intestine
spindle-shaped
300–500 x
10–30
? ? ? bending,
twisting
90
? knob-like
-
[3,26]
S. fallax
Cirriformia
tentaculata
E. Atlantic
Intestine
spindle-shaped
116–264 x
10–26
ellipsoid
3–9 x
6–17
anterior
third
twisting. Quick coiling
40–45
-
flat-topped tip
+
this
study
S. idanthyrsae
Idanthyrsus
alveolata
W. Morphological observations Young
gametocyst, the junction (double arrow) is still visible. G. Gametocysts in different focal planes, packed with
round oocysts. H-K. SEM micrographs of trophozoites showing the epicytic longitudinal folds (arrow), the mucron
(arrowhead) free of folds and some transverse striations in the inner curvature of the cell. Scale bars: Fig 1A-E,
30 μm; Fig 1F, 50 μm; Fig 1G, 70 μm; Fig 1H-I, 5μm; Fig J-K, 2μm. 6 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 The two individuals in syzygy underwent continuous change from elongated
cells, which showed active movement to more stumpy cells (Fig 1E), which did show restricted
movement. Different developmental stages of the gamontocyst and gametocyst were observed
(Fig 1F and 1G). The gametocyst was oval [129 (103–146) μm x 88 (61–106) μm, n = 5]. The
junction between the two cells was still visible in the young gametocyst. (Fig 1F). At the end
of sporogony the gametocyst was packed with spherical oocysts (Fig 1G). The oocysts contain-
ing 4 sporozoites were 13.1 μm in diameter (11.8–14.5μm, n = 22). The SEM micrographs
demonstrated that there were around 30–34 longitudinal epicytic folds inscribing the surface
of the trophozoites. The anterior end was free of folds (Fig 1H, 1I and 1K), while some of the
folds terminated towards the posterior end. In bended and shortened cells, transverse folds
appeared on the longitudinal epicytic folds (Fig 1K). In the middle of the cell, the density of
longitudinal folds was 1 fold/micron (Fig 1J). Single trophozoites and two individuals in early
syzygy were capable of pendular movement, which gave this type species of Selendium its
name S. pendula [42]. Selenidium hollandei (Fig 2, Table 1). The trophozoites of this archigregarine were isolated
from the intestines of the polychaete Sabellaria alveolata (Linnaeus, 1767). The morphology
matched the original description of S. hollandei by Vivier and Schre´vel [13] and several
accounts in studies on different aspects that followed [3,10,11]. Trophozoites were mostly
elongated and extremely flattened (Fig 2A, 2B, 2H and 2I). They were 250 μm (97–411 μm,
n = 25) long and 22 μm (13–43 μm, n = 25) wide (Fig 2A–2D). The anterior and posterior end
were both heart-shaped, but the anterior end was often a lot narrower than the posterior end
(Fig 2B and 2I). The anterior end showed a slight middle ridge in some specimens (Fig 2). In
some cases the anterior end was rounded and swollen and the posterior end was blunt (Fig
2D). The nucleus was spherical or ovoid and measured 9 x 10 μm (4–19 x 6–17 μm, n = 6) (Fig
2C–2E), situated in the middle of the trophozoite or slightly shifted towards the posterior end. The SEM showed broad epicytic folds covering the cell (Fig 2F–2I). The density of the folds
was up to 1 fold/micron (Fig 2H and 2J). Pacific
Intestine
spindle-shaped,
partially flattened
450–543 x
9–11
spherical
13–16 x
9–10
middle to
posterior
half
bending,
twisting
20–22
+
pointed
+
[26]
S. melongena
Thelepus
japonicus
W. Pacific
Coelom
oval, ’eggplant-
shaped’
30–155 x
10–41
spherical
19-Jul
middle
none
30
-
neck-like,
pointed
+
[50]
S. cf. mesnili
Myxicola
infundibulum
W. Pacific
Intestine
spindle-shaped
85–157 x
18–24
ellipsoidal
7–9 x
10–11
anterior to
posterior
bending, coiling
22–24
+
pointed to
round
+
[26]
S. boccardiellae
Boccardiella
ligerica
W. Pacific
Intestine
spindle-shaped,
partially flattened
87–250 x
10–12
ellipsoidal
10–12 x
4–6
anterior half
bending,
coiling,
thrashing
12-Oct
-
pointed
+
[26]
S. sensimae
Spirobranchus
giganteus
E. Pacific
Intestine
spindle shaped
130–170 x
10–13
ellipsoidal
10 x 4–6
middle
slow bending,
twisting
16–18
-
rounded to
blunt
+
[49]
S. cf.echinatum
Dodecaceria
concharum
W. Pacific
Intestine
spindle-shaped
95–205 x
8–11
spherical
10
anterior half
bending,
twisting
12-Oct
-
nipple-shaped
+
[49]
S. spiralis sp. n. Amphitritides
gracilis
E. Atlantic
Intestine
spindle-shaped
105–194 x
21–37
ovoid
10–21 x
15–25
posterior
half
little bending
6 (12)
+
rounded
+
this
study
S. antevariabilis sp. n. Amphitritides
gracilis
E. Atlantic
Intestine
spindle-shaped
131–190 x
22–31
ovoid
13–21 x
15–20
middle
little bending,
plastic
mucron
6
? plastic
+
this
study
S. opheliae sp. n. Ophelia
roscoffensis
E. Atlantic
Intestine
sugar snap pea-
like’
123–177 x
10–16
ovoid
10–16 x
7–11
middle to
posterior
half
little bending
none
+
elongated,
rounded
+
this
study
PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 7 / 24 Archigregarines of the English Channel S. pendula by Giard [42] and several accounts in studies on different aspects that followed
[10,11,33,43]). The cells were elongated and spindle-shaped (Fig 1). Trophozoites were 200 μm
(147–260 μm, n = 15) long and 25 μm (12–48 μm, n = 15) wide. There was a slight restriction
between the anterior end and the rest of the body. The anterior end was globular and rounded,
while the posterior end was tapering into a pointed tip (Fig 1A–1C, 1H and 1I). The axial canal
was visible along the entire cell starting from the restriction at the anterior end to the tip of the
posterior end (Fig 1B). The oval nucleus [20 (16–28) μm x 13 (7–22), n = 10] was situated in
the middle of the cell (Fig 1A and 1B). Mature trophozoites (or gamonts) pair up in caudal syz-
ygy (Fig 1D). PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 None of the folds terminated on either end of the
cell. When the trophozoites were maximally contracted there were densely packed transverse
striations/folds visible (Fig 2F). Micropores were situated in the shallow grooves between
the epicytic folds (Fig 2J). Syzygy between two cells was observed showing a dorso-ventrally
overlap of the two posterior ends (Fig 2E and 2G). Single trophozoites showed rapid rolling
movements. Selenidium sabellariae (Fig 3, Table 1). Trophozoites of this archigregarine species were
also isolated from the polychaete Sabellaria alveolata. The morphology matched the original
description of S. sabellariae by Schre´vel [43] and accounts in later studies (e.g. [3]). The tro-
phozoites were spindle-shaped and slightly flattened dorso-ventrally (Fig 3A–3C and 3F). Tro-
phozoites were 137 μm (68–227 μm, n = 19) long and 16 μm (8–23 μm, n = 19) wide (Fig 3A
and 3B). The anterior and posterior end both tapered into pointed tips. The ovoid nucleus
11 x 9 μm (7–15 x 6–13 μm, n = 15) situated in the middle of the cell contained a circular
nucleolus [5 (4–6) μm in diameter, n = 10) (Fig 3B and 3C). The SEM revealed around 14 epi-
cytic longitudinal folds (Fig 3F–3H). These folds split ‘superficially’ into two folds shortly after PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 8 / 24 Archigregarines of the English Channel Fig 2. Differential interference contrast (DIC) light micrographs and scanning electron micrographs (SEM)
showing the general morphology and surface ultrastructure of the gregarine Selenidium hollandei isolated from
the polychaete Sabellaria alveolata. A-B. DIC micrographs of elongated and flattened trophozoites. The anterior end
(mucron area, arrowhead) is narrower than the posterior end of the cell. C-D. Contracted trophozoites during peristaltic
movement. The ovoid nucleus is visible (n). Broad epicytic folds (arrow) inscribe the surface of the entire cell. E-F. DIC
and SEM micrographs of two gamonts (G1, G2) in lateral syzygy with overlapping posterior ends (double arrow). G-I. Trophozoites in different stages of movement. When contracted or bended, transverse striations (double arrow) form on
the surface. The mucron (arrowhead) at the anterior end is free of folds. The broad longitudinal epicytic folds (arrows)
cover most of the body. The lateral view shows dorso-ventrally extremely flattened trophozoites. J. Longitudinal epicytic
folds (arrow) with visible pores (arrowheads) in the grooves between the folds. Scale bars: Fig 2A, C-D, G-H, 20 μm; Fig
2B, E-F, 30 μm; Fig 2I, 25 μm; Fig 2J, 5 μm. Fig 2. Differential interference contrast (DIC) light micrographs and scanning electron micrographs (SEM)
showing the general morphology and surface ultrastructure of the gregarine Selenidium hollandei isolated from
the polychaete Sabellaria alveolata. A-B. DIC micrographs of elongated and flattened trophozoites. The anterior end
(mucron area, arrowhead) is narrower than the posterior end of the cell. C-D. Contracted trophozoites during peristaltic
movement. The ovoid nucleus is visible (n). Broad epicytic folds (arrow) inscribe the surface of the entire cell. E-F. DIC
and SEM micrographs of two gamonts (G1, G2) in lateral syzygy with overlapping posterior ends (double arrow). G-I. Trophozoites in different stages of movement. When contracted or bended, transverse striations (double arrow) form on
the surface. The mucron (arrowhead) at the anterior end is free of folds. The broad longitudinal epicytic folds (arrows)
cover most of the body. The lateral view shows dorso-ventrally extremely flattened trophozoites. J. Longitudinal epicytic
folds (arrow) with visible pores (arrowheads) in the grooves between the folds. Scale bars: Fig 2A, C-D, G-H, 20 μm; Fig
2B, E-F, 30 μm; Fig 2I, 25 μm; Fig 2J, 5 μm. Fig 2. Differential interference contrast (DIC) light micrographs and scanning electron micrographs (SEM)
showing the general morphology and surface ultrastructure of the gregarine Selenidium hollandei isolated from
the polychaete Sabellaria alveolata. A-B. DIC micrographs of elongated and flattened trophozoites. The anterior end
(mucron area, arrowhead) is narrower than the posterior end of the cell. C-D. Contracted trophozoites during peristaltic
movement. The ovoid nucleus is visible (n). Broad epicytic folds (arrow) inscribe the surface of the entire cell. E-F. DIC
and SEM micrographs of two gamonts (G1, G2) in lateral syzygy with overlapping posterior ends (double arrow). G-I. Trophozoites in different stages of movement. When contracted or bended, transverse striations (double arrow) form on
the surface. The mucron (arrowhead) at the anterior end is free of folds. The broad longitudinal epicytic folds (arrows)
cover most of the body. The lateral view shows dorso-ventrally extremely flattened trophozoites. J. Longitudinal epicytic
folds (arrow) with visible pores (arrowheads) in the grooves between the folds. Scale bars: Fig 2A, C-D, G-H, 20 μm; Fig
2B, E-F, 30 μm; Fig 2I, 25 μm; Fig 2J, 5 μm. PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 https://doi.org/10.1371/journal.pone.0187430.g002 the mucron with rounded tip, which was free of folds, and merged again shortly before the
posterior end (Fig 3G and 3H). There were numerous micropores visible in the grooves of the
main folds, and none in the “superficially’ or secondary ones (Fig 3E). The density of the folds
was 1 fold/micron at the anterior end (Fig 3G and 3H). Several transverse folds formed the
base of the mucron (Fig 3G). Two cells in syzygy were laterally attached to each other with the
gamonts oriented in opposite directions (Fig 3D). Single trophozoites showed quick coiling
and uncoiling movements. Selenidium fallax (Fig 4, Table 1). Trophozoites of this archigregarine species infect the
intestines of the polychaete Cirriformia tentaculata (Montagu, 1808). The morphology
matched the original description of S. fallax by MacGregor & Thomasson [44] and accounts
on different aspects that followed (e.g. [3,28]). The trophozoites were spindle-shaped (Fig 4A–
4D). Trophozoites were 174 μm (116–264 μm, n = 23) long and 16 μm (10–26 μm, n = 23)
wide (Fig 4A–4D). The anterior end tapered into a flat-topped tip with a nipple-like structure
in the middle (Fig 4A, 4B and 4E). The posterior end tapered into a pointy tip (Fig 4A–4D and
4F). The ellipsoid nucleus 5 x 11 μm (3–9 x 6–17 μm, n = 21) situated in the anterior third of
the cell (Fig 1A–1C) contained a circular nucleolus [5 (4–5) μm in diameter, n = 7]. The SEM
revealed around 40 to 45 epicytic longitudinal folds (Fig 4D and 4G) and the mucron being
free of folds (Fig 4E). The surface right beneath the bulging rim of the mucron was inscribed PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 9 / 24 Archigregarines of the English Channel Fig 3. Differential interference contrast (DIC) light micrographs and scanning electron micrographs
(SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium
sabellariae isolated from the polychaete Sabellaria alveolata. A- C. DIC micrographs of trophozoites in
different focal planes and different stages of movement. The mucron (arrowhead) is pointed to rounded and the
ovoid nucleus (n) is situated in the middle of the cell. D. Two gamonts (G1, G2) in lateral syzygy with their
orientation in opposite directions. The anterior ends are marked with arrowheads. Both gamonts are widest in the
area of the junction (double arrow). E. Higher magnification SEM of the surface ultrastructure. There are
numerous micropores (arrowheads) along the grooves of the main folds (arrow), whereas the grooves of the
secondary folds (double arrow) do not display any micropores. F. Trophozoite, showing a rounded mucron
(arrowhead) and longitudinal epicytic folds (arrow) with secondary folds expanding over parts of the cell’s length. G. Anterior end showing the rounded mucron (arrowhead) that is free of folds, but has a basal cluster of
transverse striations (double arrowhead). Shortly after the mucron the main folds (arrow) start splitting (double
arrowhead) into secondary folds. H. Posterior end of the trophozoite, showing the merging secondary folds
(arrow), before they reach the posterior end. Scale bars: Fig 3A-D, 30 μm; Fig 3F, 20 μm; Fig 3E, G-H, 2 μm. https://doi.org/10.1371/journal.pone.0187430.g003 Fig 3. Differential interference contrast (DIC) light micrographs and scanning electron micrographs
(SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium
sabellariae isolated from the polychaete Sabellaria alveolata. A- C. DIC micrographs of trophozoites in
different focal planes and different stages of movement. The mucron (arrowhead) is pointed to rounded and the
ovoid nucleus (n) is situated in the middle of the cell. D. Two gamonts (G1, G2) in lateral syzygy with their
orientation in opposite directions. The anterior ends are marked with arrowheads. Both gamonts are widest in the
area of the junction (double arrow). E. Higher magnification SEM of the surface ultrastructure. There are
numerous micropores (arrowheads) along the grooves of the main folds (arrow), whereas the grooves of the
secondary folds (double arrow) do not display any micropores. F. Trophozoite, showing a rounded mucron
(arrowhead) and longitudinal epicytic folds (arrow) with secondary folds expanding over parts of the cell’s length. G. Anterior end showing the rounded mucron (arrowhead) that is free of folds, but has a basal cluster of
transverse striations (double arrowhead). Shortly after the mucron the main folds (arrow) start splitting (double
arrowhead) into secondary folds. H. Posterior end of the trophozoite, showing the merging secondary folds
(arrow), before they reach the posterior end. Scale bars: Fig 3A-D, 30 μm; Fig 3F, 20 μm; Fig 3E, G-H, 2 μm. https://doi.org/10.1371/journal.pone.0187430.g003 https://doi.org/10.1371/journal.pone.0187430.g003 by short, narrow, superficial, longitudinal folds (4E). The main folds emerged just after this
area, split subsequently into two folds and merged again shortly before the posterior end (4F). The main grooves also called ‘primary’ grooves were deeper and wider than the superficial
‘secondary’ grooves (Fig 4E and 4G). PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 Micropores were only visible in the ‘primary grooves’. The density of the folds was up to 2 doublefolds/micron (Fig 4G). Free trophozoites showed
quick coiling and uncoiling movements (Fig 4C). The motion also involved some helical twist-
ing. The anterior end of the cell was always the center of the formed spiral (Fig 4C). After the
trophozoites had been isolated for some time, they often remained in the coiled position. Selenidium sabellae (Fig 5, Table 1). Trophozoites of this archigregarine species were iso-
lated from the intestines of the polychaete Sabella pavonina Savigny, 1822. The morphology
matched the original description of S. sabellae by Lankester [45], its re-description by Ray [46],
and several accounts in studies that followed (e.g. [3]). The cells were short and slightly dorso-
ventrally flattened (Fig 5A, 5E and 5F). Trophozoites were 61 μm (40–85 μm, n = 22) long and
14 μm (10–16 μm, n = 23) wide. The anterior end tapered into a narrow, but long, cone-shaped
tip. The posterior end was broadly rounded or blunt (Fig 5A and 5D–5F). The ovoid nucleus 9
x 7 μm (6–12 x 5–11 μm, n = 21) situated in the middle of the cell (Fig 5A and 5B), contained a
circular nucleolus [4 (3–6) μm in diameter, n = 18]. In some cases the trophozoites were
attached to a globular body (Fig 5C). The SEM revealed around 20–40 epicytic longitudinal
folds (Fig 5E). The folds were quite narrow starting at the base of the mucron (free of folds), PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 10 / 24 Archigregarines of the English Channel Fig 4. Differential interference contrast (DIC) light micrographs and scanning electron micrographs
(SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium fallax
isolated from the polychaete Cirriformia tentaculata. A-C. DIC micrographs of trophozoites in different
stages of movement. Trophozoits can be elongated to curled. The mucron (arrowhead) is rounded and
sometimes quite flattened at the tip. The ellipsoid nucleus (n) is situated in the anterior part of the cell. D. SEM
micrograph of a trophozoite, showing a flattened mucron (arrowhead) and longitudinal epicytic folds (arrows)
with secondary folds expanding over parts of the cell’s length. The posterior end is very pointed. E. Anterior end
showing the flat-topped mucron (arrowhead) that is free of folds, but has a nipple-like structure (arrow) in the
middle. The upper rim appears to be a bit bulgy (double arrowhead). The region directly after the bulge is
inscribed by short, narrow, superficial folds. The broader main folds start splitting (asterisks) into secondary folds
shortly after. F. Posterior end of the trophozoite showing the tip free of folds, but with some indentations. G. Higher magnification SEM of the surface ultrastructure. The grooves between the secondary folds (asterisks) are
often narrower than the grooves between the main folds (arrows). Scale bars: Fig 4A-B, 25 μm; Fig 4C, 15 μm;
Fig 4D, 10 μm; Fig 4E-G, 1 μm. Fig 4. Differential interference contrast (DIC) light micrographs and scanning electron micrographs
(SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium fallax
isolated from the polychaete Cirriformia tentaculata. A-C. DIC micrographs of trophozoites in different
stages of movement. Trophozoits can be elongated to curled. The mucron (arrowhead) is rounded and
sometimes quite flattened at the tip. The ellipsoid nucleus (n) is situated in the anterior part of the cell. D. SEM
micrograph of a trophozoite, showing a flattened mucron (arrowhead) and longitudinal epicytic folds (arrows)
with secondary folds expanding over parts of the cell’s length. The posterior end is very pointed. E. Anterior end
showing the flat-topped mucron (arrowhead) that is free of folds, but has a nipple-like structure (arrow) in the
middle. The upper rim appears to be a bit bulgy (double arrowhead). The region directly after the bulge is
inscribed by short, narrow, superficial folds. The broader main folds start splitting (asterisks) into secondary folds
shortly after. F. Posterior end of the trophozoite showing the tip free of folds, but with some indentations. G. Higher magnification SEM of the surface ultrastructure. The grooves between the secondary folds (asterisks) are
often narrower than the grooves between the main folds (arrows). Scale bars: Fig 4A-B, 25 μm; Fig 4C, 15 μm;
Fig 4D, 10 μm; Fig 4E-G, 1 μm. https://doi.org/10.1371/journal.pone.0187430.g004 and enlarged towards the posterior end, where they terminated gradually (Fig 5D–5F). There
were numerous micropores scattered along the grooves of the epicytic folds (Fig 5F and 5G). The density of the folds was 1 fold/micron at the anterior end (Fig 5E). Syzygy between two
cells was observed showing a dorso-ventrally overlap of the two posterior ends. Trophozoites
were able to bend, as well as expand and contract along their length (Fig 5B). Selenidium spiralis sp. n. (Fig 6, Table 1). PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 Trophozoites were isolated from the polychaete
Amphitritides gracilis (Grube, 1860). The cells were spindle-shaped (Fig 6A–6C). Trophozoites
were 139 μm (105–194 μm, n = 18) long and 31 μm (21–37 μm, n = 18) wide. The anterior end
narrowed into a rounded mucron (Fig 6B and 6D) with a slight indentation setting it apart
from the rest of the cell. The posterior end tapered into a more pointed tip (Fig 6E). The cells
were wider in the posterior part of the trophozoite compared to the anterior part (Fig 6A–6C). The ovoid nucleus 12 x 20 μm (10–15 x 15–25 μm, n = 18) situated in the posterior half of the
cell (Fig 6A and 6B), contained a circular nucleolus [7 (5–9) μm in diameter, n = 9). The tro-
phozoites appeared to have a fishnet pattern on the surface in some of the micrographs (Fig
6A and 6B). The SEM revealed six epicytic longitudinal folds (Fig 6B and 6C). These folds split
into two folds shortly after the mucron, which was free of folds, and merged again shortly
before the posterior end (Fig 6A–6F). The main grooves also called ‘primary’ grooves were
deeper than the superficial ‘secondary’ grooves (Fig 6C). The grooves between the secondary
folds become gradually shallower towards the posterior end at which point they disappear PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 11 / 24 Archigregarines of the English Channel Fig 5. Phase contrast (PC), differential interference contrast (DIC) light micrographs and scanning
electron micrographs (SEM) showing the general morphology and surface ultrastructure of the
gregarine Selenidium sabellae isolated from the polychaete Sabella pavonina. A. DIC micrographs of
trophozoites in different focal planes. The mucron (arrowhead) is cone-shaped. The ellipsoid nucleus (n) is
situated in the middle of the cell. Longitudinal epicytic folds (arrow) are visible along the cell. The posterior part
ends in a dorso-ventrally flattened, blunt end. B. PC micrographs showing the plasticity of the cell. The nucleus
(n) keeps its position in the middle of the trophozoite. C. DIC micrograph of an attached trophozoite with visible
epicytic folds (arrow). D. High magnification SEM of the anterior end showing the cone-shaped mucron
(arrowhead) that is free of folds. The longitudinal epicytic folds (arrows) start to broaden after their emergence. E. Trophozoite, showing the cone-shaped mucron (arrowhead) and longitudinal epicytic folds (arrows) that fan out
right after the mucron. The posterior end is broadly rounded or blunt. F. Posterior end of trophozoite in lateral
view, showing that the very tip is free of folds. The cell is dorso-ventrally flattened. There are micropores
(arrowheads) visible in between the folds. G. Surface ultrastructure towards the posterior end. The epicytic folds
(arrow) enlarge towards the posterior end, and gradually terminate. There are numerous micropores
(arrowheads) along the grooves between the folds. Scale bars: Fig 5A, C, 20 μm; Fig 5B, 30 μm; Fig 5E, 10 μm;
Fig 5F, D, 3 μm; Fig 5G, 1 μm. Fig 5. Phase contrast (PC), differential interference contrast (DIC) light micrographs and scanning
electron micrographs (SEM) showing the general morphology and surface ultrastructure of the
gregarine Selenidium sabellae isolated from the polychaete Sabella pavonina. A. DIC micrographs of
trophozoites in different focal planes. The mucron (arrowhead) is cone-shaped. The ellipsoid nucleus (n) is
situated in the middle of the cell. Longitudinal epicytic folds (arrow) are visible along the cell. The posterior part
ends in a dorso-ventrally flattened, blunt end. B. PC micrographs showing the plasticity of the cell. The nucleus
(n) keeps its position in the middle of the trophozoite. C. DIC micrograph of an attached trophozoite with visible
epicytic folds (arrow). D. High magnification SEM of the anterior end showing the cone-shaped mucron
(arrowhead) that is free of folds. The longitudinal epicytic folds (arrows) start to broaden after their emergence. E. Trophozoite, showing the cone-shaped mucron (arrowhead) and longitudinal epicytic folds (arrows) that fan out
right after the mucron. The posterior end is broadly rounded or blunt. F. Posterior end of trophozoite in lateral
view, showing that the very tip is free of folds. The cell is dorso-ventrally flattened. There are micropores
(arrowheads) visible in between the folds. G. Surface ultrastructure towards the posterior end. The epicytic folds
(arrow) enlarge towards the posterior end, and gradually terminate. There are numerous micropores
(arrowheads) along the grooves between the folds. Scale bars: Fig 5A, C, 20 μm; Fig 5B, 30 μm; Fig 5E, 10 μm;
Fig 5F, D, 3 μm; Fig 5G, 1 μm. https://doi.org/10.1371/journal.pone.0187430.g005 completely (Fig 6C). The folds were more or less helically arranged along the cells body (Fig
6B and 6C). Micropores were observed in the ‘primary grooves’. The density of the folds was
less than 1 fold/micron (Fig 6F). PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 The main epicytic folds (double arrowheads) start right
after the mucron and split very early into secondary folds. The grooves (asterisks) between the secondary
folds appear quite deep from the anterior end and become gradually shallower towards the posterior end at
which point they disappear completely. The cell is adorned with transverse striations (arrows) along its whole
length apart from the anterior and posterior tip. D. The mucron (arrowhead) is free of folds. The very
prominent transverse striations (arrow) start immediately posterior to the mucron, similar to the main epicytic
folds (double arrowhead) and the split (asterisks) into secondary folds soon after. E. The transverse striations
(arrows) continue to almost the tip of the posterior end, but are less prominent compared to the anterior end. One slight indentation (asterisk) of a secondary fold is visible on one of the main epicytic folds (double
arrowhead). F. High magnification SEM of the transverse striations (arrow) and the grooves (asterisk)
between the folds. Scale bars: Fig 6A-B, 25 μm; Fig 6C, 10 μm; Fig 6D-F, 2 μm. Fig 6. Phase contrast (PC), differential interference contrast (DIC) light micrographs and scanning
electron micrographs (SEM) showing the general morphology and surface ultrastructure of the
gregarine Selenidium spiralis sp. n. isolated from the polychaete Amphitritides gracilis. A. PC
micrograph showing the general spindle-like shape of the trophozoites with spirally arranged epicytic folds
(double arrowhead) that appear to overlap each other in a crisscross pattern. The ovoid nucleus (n) with its
round nucleolus (nu) is situated in the posterior half of the cell. The mucron (arrowhead) is set apart from the
rest of the cell through a slight indentation. B. DIC micrographs of the same cell as in A in different focal
planes. The main epicytic folds (double arrowhead) and the secondary epicytic folds (asterisks) are spirally
arranged along the longitudinal axis of the cell. C. SEM micrograph showing the surface ultrastructure of the
trophozoite. The mucron (arrowhead) is free of folds. The main epicytic folds (double arrowheads) start right
after the mucron and split very early into secondary folds. The grooves (asterisks) between the secondary
folds appear quite deep from the anterior end and become gradually shallower towards the posterior end at
which point they disappear completely. The cell is adorned with transverse striations (arrows) along its whole
length apart from the anterior and posterior tip. D. The mucron (arrowhead) is free of folds. The epicytic folds were covered in thick, evenly spread trans-
verse striations (2–3 transverse striations/micron), which started right after the mucron (Fig
6C–6F). The posterior end was free of the transverse striations. The striations became less
prominent over the length of the cell (4D-F). Trophozoites were not very active and showed
some bending movements. Selenidium antevariabilis sp. n. (Fig 7, Table 1). Trophozoites were isolated from the poly-
chaete Amphitritides gracilis (Grube, 1860). The cells were spindle-shaped, sometimes with a
slight indentation at the anterior end superficially separating the mucron area from the rest of
the cell (Fig 7A). Trophozoites were 165 μm (131–190 μm, n = 7) long and 29 μm (22–31 μm,
n = 7) wide (at the widest part of the cell). The anterior end was very plastic in shape, changing
from almost rounded to flat with finger-like protrusions, while the posterior end was rounded
(Fig 7A–7C). The ovoid nucleus [17 x 19 μm (13–21 x 15–20 μm) in diameter, n = 7] was situ-
ated in the middle of the cell (Fig 7A–7C). The trophozoites were widest at the position of the
nucleus. The nucleus contained a circular nucleolus [9 (7–11) μm in diameter, n = 4]. The tro-
phozoites appeared to have a fishnet pattern on the surface in some of the micrographs (Fig PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 12 / 24 Archigregarines of the English Channel Fig 6. Phase contrast (PC), differential interference contrast (DIC) light micrographs and scanning
electron micrographs (SEM) showing the general morphology and surface ultrastructure of the
gregarine Selenidium spiralis sp. n. isolated from the polychaete Amphitritides gracilis. A. PC
micrograph showing the general spindle-like shape of the trophozoites with spirally arranged epicytic folds
(double arrowhead) that appear to overlap each other in a crisscross pattern. The ovoid nucleus (n) with its
round nucleolus (nu) is situated in the posterior half of the cell. The mucron (arrowhead) is set apart from the
rest of the cell through a slight indentation. B. DIC micrographs of the same cell as in A in different focal
planes. The main epicytic folds (double arrowhead) and the secondary epicytic folds (asterisks) are spirally
arranged along the longitudinal axis of the cell. C. SEM micrograph showing the surface ultrastructure of the
trophozoite. The mucron (arrowhead) is free of folds. PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 The very
prominent transverse striations (arrow) start immediately posterior to the mucron, similar to the main epicytic
folds (double arrowhead) and the split (asterisks) into secondary folds soon after. E. The transverse striations
(arrows) continue to almost the tip of the posterior end, but are less prominent compared to the anterior end. One slight indentation (asterisk) of a secondary fold is visible on one of the main epicytic folds (double
arrowhead). F. High magnification SEM of the transverse striations (arrow) and the grooves (asterisk)
between the folds. Scale bars: Fig 6A-B, 25 μm; Fig 6C, 10 μm; Fig 6D-F, 2 μm. https://doi.org/10.1371/journal.pone.0187430.g006 7A and 7B). Six epicytic longitudinal folds were more or less helically arranged along the cells
body (Fig 7B and 7C). This helical arrangement of the folds leads to the visible fishnet pattern,
due to a crisscross overlap of the longitudinal folds on opposite cell surfaces. The density of
folds was less than 1 fold/micron (Fig 7B). Trophozoites showed some bending movements
and very active change of the morphology of the anterior end (Fig 7A and 7C). Selenidium opheliae sp. n. (Fig 8, Table 1). Trophozoites were isolated from the polychaete
Ophelia roscoffensis Augener, 1910. The cell shape was generally elongated to slightly reminis-
cent of a pea pod (Fig 8A–8C and 8E). Trophozoites were 151 μm (123–177 μm, n = 19) long
and 13 μm (10–16 μm, n = 19) wide. The anterior end tapered into an elongated, but at the tip
mostly rounded mucron (Fig 8A–8C and 8G). The posterior end was wider with a heart-
shaped or blunt end (Fig 8C, 8E and 8H). The ovoid nucleus 13 x 9 μm (10–16 x 7–11 μm,
n = 19) situated in the middle of the cell or slightly shifted to the posterior end contained a cir-
cular nucleolus [6 (5–8) μm in diameter, n = 9] (Fig 8A–8D). The SEM revealed no epicytic
folds (Fig 8F–8H). The surface of some trophozoites appeared a bit crinkled (Fig 8E and 8H). On some of the trophozoites transverse striations were visible (Fig 8F). Syzygy of two cells was PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 13 / 24 Archigregarines of the English Channel Fig 7. Differential interference contrast (DIC) light micrographs showing the general morphology of
the gregarine Selenidium antevariabilis sp. n. isolated from the polychaete Amphitritides gracilis. A-C. DIC micrographs of spindle-shaped trophozoites with plastic anterior (arrowhead) and rounded posterior
ends. The arrowhead marks the mucron area, which can be rounded or flattened, but often showing finger-like
protrusions (double arrowhead). The ovoid nucleus (n) is situated in the middle of the cell or slightly shifted to
the anterior end. Broad longitudinal epicytic folds (asterisks) run along the cells anterior-posterior axis with a
helical turn, which appears like a criss-cross pattern on the surface (arrow). Scale bars: Fig 7A-C, 30 μm. Fig 7. Differential interference contrast (DIC) light micrographs showing the general morphology of
the gregarine Selenidium antevariabilis sp. n. isolated from the polychaete Amphitritides gracilis. A-C. DIC micrographs of spindle-shaped trophozoites with plastic anterior (arrowhead) and rounded posterior
ends. The arrowhead marks the mucron area, which can be rounded or flattened, but often showing finger-like
protrusions (double arrowhead). The ovoid nucleus (n) is situated in the middle of the cell or slightly shifted to
the anterior end. Broad longitudinal epicytic folds (asterisks) run along the cells anterior-posterior axis with a
helical turn, which appears like a criss-cross pattern on the surface (arrow). Scale bars: Fig 7A-C, 30 μm. https://doi.org/10.1371/journal.pone.0187430.g007 observed, in which the outer posterior ends were attached through overlap (Fig 8D). Tropho-
zoites were not very active and showed some bending movements. PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 Molecular phylogenetic analyses The preliminary phylogenetic analysis of the pan-apicomplexan dataset revealed an increased rate
of evolution in gregarines compared to the already highly divergent crown apicomplexan lineages. The averaged root-to-tip distance of gregarines as a whole, or archigregarines, was significantly
higher than in other lineages (S1 Fig). Together with the non-homogenous base composition
observed in several gregarine species, this makes the correct phylogenetic reconstruction of api-
complexans very challenging. Therefore, together with the widely-used Maximum likelihood
inference under the gamma-corrected GTR model, we have employed also the CAT admixture
model (or rather the ‘C40’ approximation of CAT; see relevant Methods part for details and rea-
soning), which is supposed to be more robust to phylogenetic artifacts stemming from varying
rates of evolution along the phylogenetic tree, namely long-branch attraction (LBA). The resulting phylogeny (Fig 9) shows the expected topology with monophyletic myzozo-
ans (dinozoans, chrompodellids [47] and apicomplexans) and ciliates as an outgroup. Both
dinozoans and apicomplexans are monophyletic, while chrompodellids split into two clades. One, represented by Alphamonas and Vitrella, which branch out as the basal-most myzozoan
lineage, whereas the rest (Chromera, Voromonas and Colpodella) are a sister group to the api-
complexans s.s. Apicomplexans are further split into two clades. The first contains ‘true’ coccidians, hae-
mosporidians with Nephromyces spp. and the adeleorinid coccidians (Adelina and Hepato-
zoon). The second clade is comprised of gregarines including the cryptosporidians and two
highly divergent apicomplexans Platyproteum vivax and Filipodium phascolosomae, which
show typical archigregarine features, but were classified as an independent myzozoan lineages
by a previous study of Cavalier-Smith [1]. Within the gregarine clade, we were able to identify the three main currently recognized
lineages (Archigregarinorida, Eugregarinorida, Neogregarinorida), as well as Cryptosporidium
as sister clade to the gregarines from terrestrial hosts. Bayesian inference (Fig 9A) split grega-
rines into two main clades. The first contains neogregarines, cryptosporidia, cephaloidophor-
ids and terrestrial gregarines. The second comprised of archigregarines and marine PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 14 / 24 Archigregarines of the English Channel Fig 8. Differential interference contrast (DIC) light micrographs and scanning electron micrographs
(SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium
opheliae sp. n. isolated from the polychaete Ophelia roscoffensis. A-C. DIC micrographs of different
trophozoite cells showing the general elongated cell shape that is slightly reminiscent of a pea pod. Molecular phylogenetic analyses The mucron
(arrowhead) anterior end is slightly elongated and ends in a rounded tip, while the posterior end (arrow) is wider
and either blunt or heart-shaped. The ovoid nucleus (n) is situated in the middle of the cell or shifted slightly to
the posterior end. D. Two gamonts (G1, G2) in syzygy. This species forms a caudo-lateral syzygy with the two
posterior ends overlapping (arrow). Both gamonts have a visible nucleus (n) in the middle. E. SEM micrograph
showing the general morphology and ultrastructure of the trophozoite, with an elongated mucron (arrowhead)
and a heart- shaped (arrow) posterior end. The surface seemed to be crinkled in places (double arrow). F. Some surface areas showed few transverse striations (asterisks) but in no obvious pattern. G. Anterior end
with elongated mucron (arrowhead). H. Posterior end with visible crinkles (double arrow) on the surface. Scale
bars: Fig 3A–3D, 30 μm; Fig 3E, 20 μm; Fig 3F–3H, 2 μm. htt
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/10 1371/j
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0187430 008 Fig 8. Differential interference contrast (DIC) light micrographs and scanning electron micrographs
(SEM) showing the general morphology and surface ultrastructure of the gregarine Selenidium
opheliae sp. n. isolated from the polychaete Ophelia roscoffensis. A-C. DIC micrographs of different
trophozoite cells showing the general elongated cell shape that is slightly reminiscent of a pea pod. The mucron
(arrowhead) anterior end is slightly elongated and ends in a rounded tip, while the posterior end (arrow) is wider
and either blunt or heart-shaped. The ovoid nucleus (n) is situated in the middle of the cell or shifted slightly to
the posterior end. D. Two gamonts (G1, G2) in syzygy. This species forms a caudo-lateral syzygy with the two
posterior ends overlapping (arrow). Both gamonts have a visible nucleus (n) in the middle. E. SEM micrograph
showing the general morphology and ultrastructure of the trophozoite, with an elongated mucron (arrowhead)
and a heart- shaped (arrow) posterior end. The surface seemed to be crinkled in places (double arrow). F. Some surface areas showed few transverse striations (asterisks) but in no obvious pattern. G. Anterior end
with elongated mucron (arrowhead). H. Posterior end with visible crinkles (double arrow) on the surface. Scale
bars: Fig 3A–3D, 30 μm; Fig 3E, 20 μm; Fig 3F–3H, 2 μm. https://doi.org/10.1371/journal.pone.0187430.g008 eugregarines. Finally, S. Archigregarines of the English Channel Platyproteus vivax AY196708
Filipodium phascolosomae FJ832163
Paralecudina polymorpha AY196706
Veloxidium leptosynaptae JN857966
Pterospora schizosoma DQ093793
Pterospora floridiensis DQ093794
Lithocystis sp. DQ093795
Difficilina tubulani FJ832160
Difficilina paranemertis FJ832159
Lecudina tuzetae AF457128
Lecudina longissima FJ832157
Lankesteria abbotti DQ093796
Lankesteria cystodytae EU670241
Lankesteria chelyosomae EU670240
Lecudina phyllochaetopteri FJ832156
Selenidium pisinnus FJ832162
Selenidium orientale FJ832161
Selenidium pendula LN901443
Selenidium cf. echinatum KC110874
Selenidium boccardiellae JN857969
Selenidium sp. 1 KC110866
Selenidium serpulae DQ683562
Selenidium sp. 2 KC110864
Selenidium sensimae KC110869
Selenidium cf. mesnili JN857968
Selenidium sabellariae
Selenidium idanthyrsae JN857967
Selenidium idanthyrsae JN857967
Selenidium neosabellariae KC110873
Selenidium hollandei
Polyplicarium translucidae JX535351
Polyplicarium lacrymae JX535347
Polyplicarium curvarae JX535341
Selenidium terebellae AY196709
Selenidium antevariabilis sp. n. Selenidium melongena KC890799
Cryptosporidium fragile EU162753
Cryptosporidium baileyi DQ898161
Cryptosporidium parvum
Cryptosporidium parvum S40330
Cryptosporidium andersoni AB513869
Xiphocephalus triplogemmatus FJ459763
Xiphocephalus ellisi FJ459762
Syncystis mirabilis DQ176427
Prismatospora evansi FJ459756
Monocystis agilis AF457127
Hoplorhynchus acanthatholius FJ459750
Mattesia gemina AY334568
Mattesia sp. SV-2003 AY334569
Ophriocysti elektroscirrha AF129883
Paraschneideria metamorphosa FJ459755
Ascogregarina taiwanensis DQ462455
Ascogregarina culicis DQ462456
Apicomplexan Acarus siro pathogen AY490099
Gregarina polymorpha FJ459748
Gregarina niphandrodes AF129882
Gregarina coronata FJ459743
Apicomplexa sp. FJ481523
Gregarina kingi FJ459746
Protomagalhaensia granulosae FJ459757
Leidyana migrator AF457130
Leidyana haasi FJ459753
Gregarina blattarum FJ459741
Apicomplexa sp. KC890798
Amoebogregarina nigra FJ459737
Ganymedes sp. FJ976721
Heliospora cf. longissima HQ891115
Heliospora caprellae HQ876007
Thiriotia pugettiae HQ876006
Cephaloidophora cf. communis HQ876008
0.2
ciliates (6)
dinoflagellates
incl. Perkinsus (22)
chrompodellids (7)
coccidians (28)
haemosporidians and piroplasmids
incl. Nephromyces spp. (23)
Selenidium spiralis sp. n. Selenidium sabellae
Selenidium fallax
Selenidium opheliae sp. n. Selenidium pendula
Selenidium hollandei LN901442
0.2
1/3 of actual size
cephaloidophoroids
eugregarines
neogregarines and eugregarines
cryptosporidia
A1
A3
A2
marine eugregarines
A4
cephaloidophoroids
marine eugregarines
A1
A2
A3
A4
cryptosporidia
eugregarines
neogregarines and eugregarines
EF100227
EF100292
Difficilina paranemetrtes FJ832159
Lankesteria chelyosomae EU670240
Lankesteria abbotti DQ093796
Lecudina cf. tuzetae JF264877
EU087268
Selenidium melongena KC890799
Selenidium melongena KC890800
Selenidium terebellae KC890803
Selenidium terebellae KC890805
Selenidium pisinnus FJ832162
Selenidium orientale FJ832161
Platyproteus vivax AY196708
Filipodium phascolosomae FJ832163
Selenidium fallax
JF972216 Gulf of Mexico
Selenidium pendula
Selenidium pendula LN901443
Selenidium pendula LN901444
JF972220 Gulf of Mexico
JQ243319 Papua New Guinea
Selenidium opheliae sp. n. AB275007 Honshu coast, Japan
AY046619 Baja California
AY046806 Baja California
EF100333 Greenland
EF100387 Greenland
JF972228 Gulf of Mexico
Selenidium cf. echinatum KC110874
Selenidium cf. Molecular phylogenetic analyses fallax, whose phylogenetic position is for the first time presented here,
as well as Platyproteum vivax and Filipodium phascolosomae were the most basal gregarines
and sister to the two above mentioned clades. Maximum likelihood analysis split gregarines according to the ecology: terrestrial clade
containing cryptosporidia, neogregarines and terrestrial eugregarines and the marine clade
comprised of cephaloidophorids branching with marine eugregarines and several archigregar-
ine lineages (Fig 9B). Both phylogenetic methods unequivocally and consistently placed the
newly obtained sequences of Selenidium in three separate clades (Fig 9, S2 Fig, S3 Fig). Seleni-
dium pendula branched within the well supported ‘true’ Selenidium clade with S. sabellariae, S. sabellae, S. hollandei, S. opheliae and nine other representatives, while S. pisinnus formed a
weakly supported clade with S. orientale basal to it. Another putatively archigregarine clade PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 15 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 mesnili JN857968
Selenidium sabellariae
Selenidium idanthyrsae JN857967
Selenidium idanthyrsae JN857967
Selenidium neosabellariae KC110873
Selenidium hollandei
Polyplicarium translucidae JX535351
Polyplicarium lacrymae JX535347
Polyplicarium curvarae JX535341
Selenidium terebellae AY196709
Selenidium antevariabilis sp. n. Selenidium melongena KC890799
Cryptosporidium fragile EU162753
Cryptosporidium baileyi DQ898161
Cryptosporidium parvum
Cryptosporidium parvum S40330
Cryptosporidium andersoni AB513869
Xiphocephalus triplogemmatus FJ459763
Xiphocephalus ellisi FJ459762
Syncystis mirabilis DQ176427
Prismatospora evansi FJ459756
Monocystis agilis AF457127
Hoplorhynchus acanthatholius FJ459750
Mattesia gemina AY334568
Mattesia sp. SV-2003 AY334569
Ophriocysti elektroscirrha AF129883
Paraschneideria metamorphosa FJ459755
Ascogregarina taiwanensis DQ462455
Ascogregarina culicis DQ462456
Apicomplexan Acarus siro pathogen AY490099
Gregarina polymorpha FJ459748
Gregarina niphandrodes AF129882
Gregarina coronata FJ459743
Apicomplexa sp. FJ481523
Gregarina kingi FJ459746
Protomagalhaensia granulosae FJ459757
Leidyana migrator AF457130
Leidyana haasi FJ459753
Gregarina blattarum FJ459741
Apicomplexa sp. KC890798
Amoebogregarina nigra FJ459737
Ganymedes sp. FJ976721
Heliospora cf. longissima HQ891115
Heliospora caprellae HQ876007
Thiriotia pugettiae HQ876006
Cephaloidophora cf. communis HQ876008
Selenidium spiralis sp. n. Selenidium sabellae
Selenidium fallax
Selenidium opheliae sp. n. Selenidium pendula
Selenidium hollandei LN901442
0.2
1/3 of actual size
cephaloidophoroids
eugregarines
neogregarines and eugregarines
cryptosporidia
A1
A3
A2
marine eugregarines
A4
A5
ABSOLUTE SU
A B A cephaloidophoroids marine eugregarines outgroup
(95) A3 Selenidium fallax A5 ABSOLUTE SUPPORT (BI 1.0, ML 100)
STRONG SUPPORT (BI >0.95, ML >85) haemosporidians and piroplasmids
incl. Nephromyces spp. (23) coccidians (28) dinoflagellates
incl. Perkinsus (22) Fig 9. Phylogenetic relationships of apicomplexans. A. Bayesian tree of apicomplexans inferred using the GTR + C40 model of substitution in
Phylobayes on an alignment of 171 small subunit (SSU) rDNA sequences and 1488 unambiguously aligned sites. Thickened lines at branches denote
Bayesian posterior probabilities and bootstrap support (see legend in the center of the figure for details). The eight sequences derived from this study are
highlighted in bold. A1 to A5 show the positions of the five ‘archigregarine’clades. B. Schematized Maximum likelihood phylogeny analysis of the same
dataset inferred under the GTR+G model as implemented in RAxML 8.2. The outgroup composition and topology is the same as in Fig 9A and is
simplified due to space limitations. C. Bayesian topology of the dataset S with exhaustive sampling of Selenidiidae, also enriched for environmental
sequences inferred in Phylobayes under the GTR + C40 model of substitution. The branching support follows parameters of tree A. Fig 9. Phylogenetic relationships of apicomplexans. A. echinatum KC110875
EU087194 East Sea
JQ243944 Papua New Guinea
Selenidium sabellaria
Selenidium hollandei
Selenidium hollandei LN901445
Selenidium neosabellariae KC110871
Selenidium neosabellariae KC110872
Selenidium neosabellariae KC110873
JQ244240 Papua New Guinea
Selenidium cf. mesnili JN857968
Selenidium sabellae
FJ221514 NY coast, USA
Selenidium sp. 1 KC110866
Selenidium sp. 1 KC110867
Selenidium sp. 1 KC110863
Selenidium serpulae DQ683562
Selenidium sensimae KC110869
Selenidium sensimae KC110870
Selenidium sp. 2 KC110865
Selenidium sp. 2 KC110864
Selenidium sp. 2 KC110868
0.1
Lecudina phyllochaetopteri FJ832156
Pterospora floridiensis DQ093794
Pterospora schizosoma DQ093793
Lithocystis sp. DQ093795
Lecudina longissima FJ832157
Veloxidium leptosynaptae JN857966
Selenidium spiralis sp. n. Selenidium antevariabilis sp. n. Selenidium boccardiellae JN857969
Selenidium idanthyrsae JN857967
B
C
A5
1/3 of actual size
ABSOLUTE SUPPORT (BI 1.0, ML 100)
STRONG SUPPORT (BI >0.95, ML >85)
A5
Paralecudina polymorpha AY196706
A
outgroup
(95)
Polyplicarium translucidae JX535351
Polyplicarium lacrymae JX535347
Polyplicarium curvarae JX535341
Alphamonas edax AY234843
Vitrella brassicaformis DQ174732
Fig 9. Phylogenetic relationships of apicomplexans. A. Bayesian tree of apicomplexans inferred using the GTR + C40 model of substitution in
Phylobayes on an alignment of 171 small subunit (SSU) rDNA sequences and 1488 unambiguously aligned sites. Thickened lines at branches denote
Bayesian posterior probabilities and bootstrap support (see legend in the center of the figure for details). The eight sequences derived from this study are
highlighted in bold. A1 to A5 show the positions of the five ‘archigregarine’clades. B. Schematized Maximum likelihood phylogeny analysis of the same
dataset inferred under the GTR+G model as implemented in RAxML 8.2. The outgroup composition and topology is the same as in Fig 9A and is
simplified due to space limitations. C. Bayesian topology of the dataset S with exhaustive sampling of Selenidiidae, also enriched for environmental
sequences inferred in Phylobayes under the GTR + C40 model of substitution. The branching support follows parameters of tree A. Platyproteus vivax AY196708
Filipodium phascolosomae FJ832163
Paralecudina polymorpha AY196706
Veloxidium leptosynaptae JN857966
Pterospora schizosoma DQ093793
Pterospora floridiensis DQ093794
Lithocystis sp. DQ093795
Difficilina tubulani FJ832160
Difficilina paranemertis FJ832159
Lecudina tuzetae AF457128
Lecudina longissima FJ832157
Lankesteria abbotti DQ093796
Lankesteria cystodytae EU670241
Lankesteria chelyosomae EU670240
Lecudina phyllochaetopteri FJ832156
Selenidium pisinnus FJ832162
Selenidium orientale FJ832161
Selenidium pendula LN901443
Selenidium cf. echinatum KC110874
Selenidium boccardiellae JN857969
Selenidium sp. 1 KC110866
Selenidium serpulae DQ683562
Selenidium sp. 2 KC110864
Selenidium sensimae KC110869
Selenidium cf. Formal taxonomic description Phylum Apicomplexa Levine, 1970
Subphylum Sporozoa Leuckart, 1879
Class Gregarinea J.A.O. Bu¨tschli, 1882, stat. nov. Grasse´, 1953
Order: Archgregarinorida Grasse´, 1953
Family Selenidiidae Brasil, 1907
Genus Selenidium Giard, 1884
Selenidium spiralis Rueckert and Hora´k sp. n. urn:lsid:zoobank.org:act:0687AD8C-E669-47B4-8EC2-9FC22A039B urn:lsid:zoobank.org:act:0687AD8C-E669-47B4-8EC2-9FC22A039B42 Diagnosis. Trophozoites are spindle-shaped and 105–194 μm long (mean length 147 μm) and
21–37 μm wide (mean width 30 μm). Cell tapers into a rounded mucron at the anterior and a
pointed tip at the posterior end. The ovoid nucleus (13 x 20 μm) is situated in the posterior half of
the trophozoite. Trophozoites are brownish in colour under the LM due to the accumulation of
amylopectin granules within the cytoplasm. Six longitudinally and helically oriented broad epicy-
tic folds are present on the cell surface, except the mucron and the posterior tip. They are sepa-
rated by deep, primary grooves and also show shallow secondary grooves in between. Epicytic
folds are covered by thick transverse striations. The trophozoites are capable of some bending. DNA sequence. The SSU rDNA sequence; GenBank Accession No. MF882900. Type locality. Rock assemblages close to Station Biologique Roscoff (48˚43’44”N, 3˚
59’23”W), Roscoff, France. Type habitat. Marine. Type host. Amphitritides gracilis (Grube, 1860) (Annelida, Polychaeta, Terebellidae). Type host. Amphitritides gracilis (Grube, 1860) (Annelida, Polychaeta, Terebellidae). Location in host. Intestinal lumen. Hapantotype. Trophozoites on gold sputter-coated SEM stubs have been deposited in the
Beaty Biodiversity Museum (Marine Invertebrate Collection) at the University of British
Columbia, Vancouver, Canada (collection number MI-PR135). Hapantotype. Trophozoites on gold sputter-coated SEM stubs have been deposited in the
Beaty Biodiversity Museum (Marine Invertebrate Collection) at the University of British
Columbia, Vancouver, Canada (collection number MI-PR135). Hapantotype. Trophozoites on gold sputter-coated SEM stubs have been deposited in the
Beaty Biodiversity Museum (Marine Invertebrate Collection) at the University of British
Columbia, Vancouver, Canada (collection number MI-PR135). Iconotype. Fig 6B. Iconotype. Fig 6B. Etymology. The species name spiralis refers to the helically arranged longitudinal epicytic
folds. It is
interesting that while most archigregarine species are described from temperate regions, many
of these environmental sequences originate from tropical and subtropical waters clearly show-
ing their cosmopolitan distribution and research bias. Bayesian tree of apicomplexans inferred using the GTR + C40 model of substitution in
Phylobayes on an alignment of 171 small subunit (SSU) rDNA sequences and 1488 unambiguously aligned sites. Thickened lines at branches denote
Bayesian posterior probabilities and bootstrap support (see legend in the center of the figure for details). The eight sequences derived from this study are
highlighted in bold. A1 to A5 show the positions of the five ‘archigregarine’clades. B. Schematized Maximum likelihood phylogeny analysis of the same
dataset inferred under the GTR+G model as implemented in RAxML 8.2. The outgroup composition and topology is the same as in Fig 9A and is
simplified due to space limitations. C. Bayesian topology of the dataset S with exhaustive sampling of Selenidiidae, also enriched for environmental
sequences inferred in Phylobayes under the GTR + C40 model of substitution. The branching support follows parameters of tree A. https://doi.org/10.1371/journal.pone.0187430.g009 https://doi.org/10.1371/journal.pone.0187430.g009 with sequences related to S. terebellae (including two of our new isolates from Amphitritides
gracilis) is found elsewhere among the gregarines. Selenidium fallax probably represents an
independent (archi-) gregarine lineage of uncertain position. However, analysis of auxiliary
datasets R and G (S2 Fig, S3 Fig) shows the gregarine topology is unstable and highly depen-
dent on the phylogenetic method and site selection. It is worth mentioning that the archigre-
garine monophyly was also rejected using the au-test. While all the respective clades are
robustly supported in all analyses, their mutual relationships are unclear to say the least and
the support for deeper branching is missing completely. A pairwise distance calculation based
on the Kimura two-parameter model [48] of 1656 nt (with pairwise exclusion of the indels)
resulted in sequence divergences between 2% and 35.8% between all Selenidium species. The
sequence divergence ranges were 2–15.6% (A1); 5.8–10.5% (A2) and 15.3 (A3) within clades PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 16 / 24 Archigregarines of the English Channel A1-A3 and 18.5–35.8% between species from the different clades (S1 Table). Since the
sequence of the archigregarine type species S. pendula has been recently published [33], we
have also attempted to explore the diversity and phylogenetic structure of the genus Selenidium
s.s. Our exhaustive sampling revealed that almost half of the diversity is comprised of environ-
mental sequences without any morphology and host-specificity record. Selenidium pendula is
found near the base of the Selenidium clade, which lacks a strong phylogenetic structure. PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 Selenidium antevariabilis Rueckert and Hora´k sp. n. Selenidium antevariabilis Rueckert and Hora´k sp. n. urn:lsid:zoobank.org:act:F830245F-4CF9-4925-9F20-CFFB8699DBCB posterior tip. The trophozoites are capable of some bending and very active change of the mor-
phology of the anterior end. Type habitat. Marine. Type habitat. Marine. Type host. Amphitritides gracilis (Grube, 1860) (Annelida, Polychaeta, Terebellidae). L
ti
i h
t I t ti
l l Type host. Amphitritides gracilis (Grube, 1860) (Annelida, Polychaeta, Terebellidae). Holotype. The name-bearing type of this species is the specimen shown in Fig 7B (see Ico-
notype). This is in accordance with Declaration 45 recommendations to article 73 of the
ICZN. An explanation is provided in the remarks. Iconotype. Fig 7. Etymology. The species epithet antevariabilis refers to the plastic morphology of the ante-
rior end and stems from the Latin words ‘ante’ meaning front/before and ‘variabilis’ meaning
changeable. Remarks. In accordance with Declaration 45 of the ICZN we use the specimen presented in
the light micrographs (7B) as name-bearing type material (Holotype). Here we provide reason-
ing, why no preserved specimen was used as name-bearing type for the new species. This new
gregarine species occurred in very low numbers. The few specimens we were able to isolate
were used for DNA extraction, prepared for SEM and for LM analyses. Trophozoites fixed for
SEM often do represent a gregarine’s morphology better than a fixed and flattened cell on an
object slide. Light micrographs (Iconotype) were obtained from freshly isolated cells and rep-
resent the morphology of the trophozoite and especially of the plasticity of the anterior end
better than a fixed specimen on an object slide could. We were not able to find any gregarines
on the filter prepared for SEM, therefore the only documentation of this species are the light
micrographs and the cell measurements. We do provide the SSU rDNA sequence, which sepa-
rates S. antevariabilis n. sp. clearly from S. spiralis n. sp. isolated from the same host. Therefore,
we feel that we provide enough evidence for the description of a new species. Etymology. The species name opheliae refers to the genus of the polychaete type host Ophe-
lia roscoffensis Augener, 1910. urn:lsid:zoobank.org:act:F830245F-4CF9-4925-9F20-CFFB8699DBCB Diagnosis. Trophozoites are spindle-shaped and 131–190 μm long (mean length 165 μm)
and 22–31 μm wide (mean width 29 μm). Cell tapers into a rounded posterior end. The ante-
rior end is plastic in shape, being rounded, or flat, or showing finger-like protrusions. The
ovoid nucleus (17 x 19 μm) is situated in the middle of the trophozoite and contains a circular
nucleolus (9 μm in diameter). Trophozoites are brownish in colour under the LM due to the
accumulation of amylopectin granules within the cytoplasm. Six longitudinally and helically
oriented broad epicytic folds are present on the cell surface, except the mucron and the PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 17 / 24 Archigregarines of the English Channel posterior tip. The trophozoites are capable of some bending and very active change of the mor-
phology of the anterior end. posterior tip. The trophozoites are capable of some bending and very active change of the mor-
phology of the anterior end. Selenidium opheliae Rueckert and Hora´k sp. n. Selenidium opheliae Rueckert and Hora´k sp. n. Justification for newly erected species Trophozoites of Selenidium spiralis sp. n. and S. antevariabilis sp. n. were isolated from Amphi-
tritides gracilis. No gregarines have so far been reported for this terebellid polychaete. The anal-
yses of the SSU rDNA sequences available for gregarines of the genus Selenidium placed S. spiralis sp. n. and S. antevariabilis sp. n. into a clade with other Selenidium species from terebel-
lid worms (Thelepus japonicus), namely Selenidium terebellae and Selenidium melongena. While S. terebellae is a typical archigregarine of the genus Selenidium, with few longitudinal
epicytic folds, bending/twisting and coiling movements and transverse striations, S. melongena
is quite different (see Table 1 and Wakeman et al. 2014). One of the most distinct features is
that the 30–40 epicytic folds are helically arranged along the longitudinal axis of the cell and
that no movement could be observed. The new species described here present different degrees
of helically arranged epicytic folds, but a lot less in numbers (six broad folds, in one case with
secondary grooves). Along the whole length of the epicytic folds of S. spiralis sp. n. there are
thick transverse folds at regular intervals. While S. spiralis sp. n. and S. antevariabilis sp. n. were found within the intestine, S. melongena was predominantly found attached to the outer
wall of the intestine [50]. The calculated sequence divergences ranged between 8.7–10.5% for
S. spiralis sp n., S. terebellae and S. antevirabilis sp. n. compared to S. melongena (compare S1
Table). The lowest sequence divergence (2%) was actually found between two established spe-
cies S. idanthyrsae and S. neosabellariae. Apart from their sequence divergence (6.8%), the big-
gest difference between the two new species is the morphology of the anterior end. While S. spiralis sp. n. has a rounded mucron, the anterior end of S. antevariabilis sp. n. is very plastic
and changing in form constantly. Combining all evidence we are convinced that both isolated
trophozoites represent new Selenidium species. Ophelia roscoffensis has never been described as hosting any gregarine species. There are
two Rhytidocystis species (Agamococcidiorida) described from opheliid worms, namely R. opheliae from O. bicornis [51] and R. henneguyi from O. neglecta [52], but their phylogenetic
position is not quite certain yet [9]. Rotari et al. [53] described metchnikovellids (hyperpara-
sites of gregarines) from a Selenidium species infecting Ophelia limacina, but the gregarine spe-
cies was not formally described and only a line drawing is available. Discussion In the last decade more and more information has been published about the archigregarines,
which occur exclusively in marine habitats and are most likely the stem group from which all
gregarine and maybe even all apicomplexans have derived [24–26,29,32,49,50]). Most of these
publications synergize data on morphology, ultrastructure and SSU rDNA sequences to newly
describe or re-describe species in the most comprehensive way possible to date. Almost all of
the work has been done on the trophozoite stages, the most prominent and abundant life cycle
stages. Recently work on other life-cycle stages of S. pendula has been published [33]. Molecu-
lar data of archigregarines are still very limited compared to other taxa. urn:lsid:zoobank.org:act:8316105E-9419-4F06-9463-E12553F9E2C5 urn:lsid:zoobank.org:act:8316105E-9419-4F06-9463-E12553F9E2C5 Diagnosis. Trophozoites elongated ellipsoid to pea pod shape and 123–177 μm long (mean
length 151 μm) and 10–16 μm wide (mean width 13 μm). Anterior end is elongated with
rounded mucron, posterior end is wide with heart-shaped or blunt end. The ovoid nucleus
measures13 x 9 μm (10–16 x 7–11 μm, n = 19) and is situated in the middle of the cell or
slightly shifted to the posterior end. Trophozoites are brownish in colour reflecting an accu-
mulation of amylopectin granules within the cytoplasm. Epicytic folds are lacking. Surface of
trophozoites can appear crinkled and some show transverse striations. Trophozoites are capa-
ble of bending movements. DNA sequence. The SSU rDNA sequence; GenBank Accession No. MF882904. Type locality. Le Guillec (48˚68´56´´N, 4˚06´78´´W), France. Type habitat. Marine. Type habitat. Marine. Type host. Ophelia roscoffensis Augener, 1910 (Annelida, Polychaeta, Opheliidae). Location in host. Intestinal lumen. Hapantotype. Trophozoites on gold sputter-coated SEM stubs have been deposited in the
Beaty Biodiversity Museum (Marine Invertebrate Collection) at the University of British
Columbia, Vancouver, Canada (collection number MI-PR136). Etymology. The species name opheliae refers to the genus of the polychaete type host Ophe-
lia roscoffensis Augener, 1910. Etymology. The species name opheliae refers to the genus of the polychaete type host Ophe-
lia roscoffensis Augener, 1910. Etymology. The species name opheliae refers to the genus of the polychaete type host Ophe-
lia roscoffensis Augener, 1910. 18 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 Archigregarines of the English Channel PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 True Selenidiidae vs. Selenidioides Levine [3] split the genus Selenidium and its species into two: 1) Selenidioides within the family
Selenidioididae in the order Archigregarinorida encompassing species with merogony and 2)
Selenidium within the family Selenidiidae in the order Eugregarinorida without merogony. There have been many discussions before this split and ever since the split [16,25,26,29,30,32]
about the taxonomy of gregarine species described as Selenidium, as it is difficult to prove or
disprove the existence of merogony within a gregarine life-cycle. No sequence data were avail-
able back then and still the available DNA sequences are quite limited. Our phylogenetic trees
based on SSU rDNA sequence data (Fig 9) show that S. hollandei, which after Levine [3] should
belong to the genus Selenidioides, clusters within the big clade around the type species of the
genus Selenidium, S. pendula, which was moved to the Eugregarinorida due to the ‘absence’ of
merogony. Therefore, S. pendula as well as S. hollandei belong to the true Selendiidae, which
has also been shown by Schre´vel et al. [33], inferring that the split of the genus Selenidium was
premature, because it is not backed up by molecular sequence data and should therefore be
ignored from hereon. While S. sabellariae and S. sabellae also cluster within the clade A1
together with S. pendula, the species S. fallax that should belong into this genus as well clusters
out with any of the other ‘Selenidium’ sequences, which supports our suggestion to revise this
genus at some point, when better/more sequence data and morphological descriptions of
other species within this genus become available. Justification for newly erected species Compared to the other
known Selenidium species our newly described S. opheliae sp. n. from O. roscoffensis has very
different morphological features, as this is the only species that does not show any longitudinal
epicytic folds (compare Table 1). The crinkled surface and the few transverse striations could
be the result of the SEM fixation process. The sequence divergence showed values between
6.9% and 15.4% when comparing S. opheliae sp. n. with all other Selenidium species in clade
A1. The distances between all Selenidium species in clade A1 ranged from 2% to 15.6% (S1
Table). The phylogenetic tree based on gregarine SSU rDNA sequences alone (Fig 9) shows
clearly that this species has a distinct position from the other Selenidium species within the S. pendula clade of true Selenidium species, therefore validating the erection of the new species. PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 19 / 24 Archigregarines of the English Channel Archigregarine phylogeny The so called archigregarines (i.e. apicomplexans mostly with vermiform or spindle-shaped
actively moving trophozoits), represented here by all species with available SSU rDNA
sequences, apparently comprise of five independent lineages: 1) a clade around the Selenidium
type-species S. pendula, 2) a clade containing S. pisinnus and S. orientale, 3) a clade with para-
sites isolated from polychaetes Amphitritides gracilis and Thelepus japonicus and 4) a clade
made up of two very divergent species Platyproteum vivax and Filipodium phascolosomae, and
5) the single sequence of S. fallax at the base of all other gregarines. Although all these lineages
share similar morphology (see Table 1) and possibly (according to the limited insight available)
also general biology, phylogenetic analysis of SSU rDNA clearly reveals well defined and sepa-
rated lineages. However, the deeper branching reflecting their mutual relationship is again
very unstable and poorly supported. All recent studies [1,25,26,33,49,50], as well as the phylog-
eny of our main dataset presented here (Fig 9) suggest their paraphyly. Archigregarine mono-
phyly was also rejected by the approximately-unbiased test. However, short internal branches
suggest rapid archigregarine diversification, which is always hard to capture using phyloge-
netic reconstruction. In our opinion, while improbable, the archigregarine monophyly cannot
be reliably rejected based on current taxon sampling and SSU rDNA. One obvious outcome is
that only the archigregarines of the clade around S. pendula should retain the genus name Sele-
nidium and thorough revision of the whole archigregarine concept is required. Suitability of SSU rDNA for gregarine phylogeny Molecular diversity is almost exclusively represented by SSU rDNA sequences for gregarine
apicomplexans. Yet, even the most elaborate phylogenetic models designed for phylogenies of
highly divergent datasets [54,55], failed to cope with the extreme diversity of gregarine SSU
rDNA. The topology is unstable and is highly dependent on the particular model used (GTR
+ C40 vs. GTR) as well as taxon and site sampling. Therefore, we intentionally refrain from
any taxonomic revisions of gregarines s.l. as recently proposed by Cavalier-Smith [1], as we do
not find it justified by the available data. It is our opinion that we have to leave the question of PLOS ONE | https://doi.org/10.1371/journal.pone.0187430
November 3, 2017 20 / 24 Archigregarines of the English Channel relationships among the key gregarine lineages open until new sets of markers of sufficient
sampling will be available. Supporting information S1 Fig. Boxplots showing averaged root-to-tip distances of main clades used in phyloge-
netic analyses. Three groups of gregarines (representatives of Selenidium s.s., archigregarines
and gregarines as a whole) show significantly longer branch lengths and therefore also
increased rates of evolution as revealed by pairwise t-test comparison to other clades presented
in the dataset. (EPS) S2 Fig. Phylogenetic topology of auxiliary dataset G (945 sites) as revealed by Bayesian
inference under the GTR + C40 model as implemented in Phylobayes. Thickened lines
show branching supported by Bayesian posterior probabilities (dark, p.p. 1.0, grey p.p. above
0.94). See relevant parts of methods for details. (EPS) S3 Fig. Phylogenetic topology of auxiliary dataset R (1805 sites) as revealed by Bayesian
inference under the GTR + C40 model as implemented in Phylobayes. Thickened lines
show branching supported by Bayesian posterior probabilities (dark, p.p. 1.0, grey p.p. above
0.94). See relevant parts of methods for details. (EPS) S3 Fig. Phylogenetic topology of auxiliary dataset R (1805 sites) as revealed by Bayesian
inference under the GTR + C40 model as implemented in Phylobayes. Thickened lines
show branching supported by Bayesian posterior probabilities (dark, p.p. 1.0, grey p.p. above
0.94). See relevant parts of methods for details. (EPS) S1 Table. Estimates of evolutionary divergence between sequences. The numbers of base
substitutions per site between sequences are shown. The analysis involved 50 nucleotide
sequences. All ambiguous positions were removed for each sequence pair. There were a total
of 1654 positions in the final dataset. The sequences for taxa in bold were derived from this
study, taxa highlighted in light grey belong to Selenidium s.s. and taxa highlighted in dark grey
are novel species for the first time described here. (XLSX) Acknowledgments SR and AH would like to thank the marine team from Station Biologique de Roscoff for col-
lecting some of the polychaete species. Both authors would like to thank the SEM facility at
the Institute of Parasitology, Biology Centre of CAS for their technical support and expertise. The authors would also like to thank Olga Flegontova, for her help in the field and in the
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Determination of the Performance of Some Apple Cultivars Matured in Different Periods in the Mediterranean Transitional Zone
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Yüzüncü Yıl Üniversitesi Tarım Bilimleri Dergisi/Yüzüncü Yıl Üniversitesi Tarım Bilimleri Dergisi
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Determination of the Performance of Some Apple Cultivars Matured in Different
Periods in the Mediterranean Transitional Zone Determination of the Performance of Some Apple Cultivars Matured in Different
Periods in the Mediterranean Transitional Zone Abstract: Apple is a temperate climate fruit grown in different ecology and a
wide range of area. However, to determine the suitable variety for every ecology,
it is of great importance to investigate the performance of these varieties. This
study was carried out in 2012-2013 in order to determine the performance of
Mondial Gala, Fuji and Scarlet Spur apple varieties grown in Mediterranean
transitional zone and matured in different periods. In this study, some
phenological properties of cultivars and yield and fruit quality characteristics
were investigated. According to the results, full bloom occurred in 21 to 25 April
and maturation on 11 of August and 5 October. In cultivars, fruit weights in
ranged between 137.50 g and 217.30 g, fruit diameter 66.57 mm and 80.67 mm. Yüzüncü Yıl Üniversitesi Tarım Bilimleri Dergisi Cilt 29, Sayı 2, 28.06. 2019 Yüzüncü Yıl Üniversitesi Tarım Bilimleri Dergisi Cilt 29, Sayı 2, 28.06. 2019 Yüzüncü Yıl Üniversitesi
Tarım Bilimleri Dergisi
(YYU Journal of Agricultural Science) Araştırma Makalesi (Research Article) Öz: Elma, farklı ekolojilerde ve oldukça geniş bir alanda yetiştiriciliği yapılan bir
ılıman iklim meyvesidir. Ancak, yetiştiricilik yapılacak ekolojiye uygun çeşit
önerisi için, o ekolojideki performansların incelenmesi büyük önem taşımaktadır. Çalışma, Akdeniz geçit kuşağında yetiştirilen ve değişik dönemlerde olgunlaşan
Mondial Gala, Fuji ve Scarlet Spur elma çeşitlerinin performanslarının
belirlenmesi amacıyla 2012-2013 yıllarında yürütülmüştür. Araştırmada,
çeşitlerin bazı fenolojik özellikleri ile verim ve meyve kalite özellikleri
incelenmiştir. Bulgulara göre çeşitlerde tam çiçeklenme 11 Nisan (Mondial
Gala)-25 Nisan (Fuji) ve meyve olgunlaşması ise 11 Ağustos (Mondial Gala) - 5
Ekim (Fuji) tarihleri arasında gerçekleşmiştir. Çeşitlerin meyve ağırlıkları 137.50
g (Scarlet Spur) - 217.30 g (Fuji), meyve boyu 61.65 mm (Mondial Gala) - 71.03
mm (Scarlet Spur) ve meyve çapı 66.57 mm (Scarlet Spur) - 80.67 mm (Fuji)
arasında değişim göstermiştir. Elma çeşitlerinde suda çözünebilir kuru madde
miktarı % 13.50 (Scarlet Spur) - % 15.17 (Mondial Gala), titre edilebilir asit
miktarı % 0.23 (Scarlet Spur) - % 0.40 (Fuji), indirgen şeker kapsamı % 8.93
(Scarlet Spur) - % 10.17 (Fuji), toplam şeker düzeyi % 12.08 (Scarlet Spur) – %
13.87 (Fuji) ve toplam fenol içerikleri ise 113.40 mg/100 g (Scarlet Spur) - 148.40
mg/100 g (Fuji) arasında yer almıştır. Öte yandan, elma çeşitlerinde ağaç başına
verim 4.92 (Scarlet Spur) – 9.32 kg/ağaç (Fuji) ve birim gövde kesit alanına düşen
verim 0.28 (Scarlet Spur) – 0.66 kg/cm2 (Mondial Gala) arasında değişim
göstermiştir. Yaz dönemi açısından Mondial Gala, erken güz dönemi açısından
Scarlet Spur ve güz dönemi için ise Fuji çeşidinin bölgede ürün çeşitliğinin
sağlanması açısından üreticilere katkı sağlayacağı düşünülmektedir. Geliş: 17.10.2018
Kabul: 11.04.2019
Online Yayınlanma 28.06. 2019
DOI: 10.29133/yyutbd.471552 Anahtar kelimeler
Fenoloji,
Malus domestica,
Meyve kalitesi,
Pomoloji,
Verim etkinliği 1. Giriş Dünya üzerinde Antarktika hariç, bütün kıtalarda, ılıman iklime sahip bölgelerde ve tropik
bölgelerin yüksek rakımlı yerlerinde doğal olarak yetişen ve 30 kadar türü bulunan elma (Malus
domestica Borkh.), ılıman iklim meyveleri içerisinde en fazla üretim ve tüketim miktarlarına sahip olan
türdür (Özongun ve ark., 2014). Dünyada elma yetiştiriciliği kuzey ve güney yarı kürenin 30°-50° enlemler arasındaki ılıman
iklime sahip bölgelerine yayılmıştır (Soylu, 2003). Türkiye’de ise Ege Bölgesi’nde 500 metreden,
Akdeniz ve Güneydoğu Anadolu Bölgesi’nde ise 800 metreden daha yüksek yerlerde yetişmektedir
(Kaygısız, 2004; Özçağıran ve ark., 2005). Dünya elma üretimi yaklaşık 83 milyon ton olup, Türkiye 3
032 164 ton ile dünya elma üretiminde Çin ve ABD’nin ardından 3. sırada gelmektedir (FAOSTAT
2017). Elma bitkisinin birçok kültür çeşidi ve yabani formları soğuk ve sert iklim koşullarına önemli
düzeyde dayanım göstermektedir. Elmanın odun dokuları kış döneminde - 35 ile - 40 oC’ye
dayanabilmektedir. Ancak, bahar döneminde açmış çiçekler - 2.2 ile - 2.3 oC, küçük meyveler ise - 1.1
ile - 2.2 oC düzeylerindeki sıcaklıklardan zarar görebilmektedir (Özcağıran ve ark., 2005; Gerçekcioğlu
ve ark., 2012). Elmada sağlıklı bir çiçeklenme meydana gelmesi ve buna dayalı olarak düzenli tozlanma ve
döllenme koşullarının oluşabilmesi için tür ve çeşitlere göre değişmekle birlikte + 7.2 oC’nin altında 2
000 ile 3 000 saat arasındaki kış soğuklanma ihtiyacının karşılanması gerekmektedir (Özbek 1978;
Kaygısız, 2004; Gerçekcioğlu ve ark., 2012). Ancak, son dönemlerde ıslah çalışmaları sonucunda düşük
soğuklanma isteğine sahip (400-600 saat gibi) oldukça fazla sayıda çeşit elde edilmiş ve bu sayede
elmadaki yetiştiricilik alanı subtropik bölgelere doğru önemli yayılma göstermiştir (Gerçekçioğlu ve
ark., 2012). Elma bitkisi yaz dönemindeki yüksek sıcaklıklara belirli düzeye kadar bir dayanım göstermekle
birlikte, genellikle 40 oC ve üzerindeki uzun süreli sıcaklıklardan ve düşük hava neminden olumsuz
olarak etkilenmektedir (Özbek, 1978; Özcağıran ve ark., 2005; Gerçekcioğlu ve ark., 2012). Son 25-30
yıllık dönemde ülkemizde elma tarımında önemli gelişmeler meydana gelmiştir. Elma yetiştiriciliğinde
uygulanan modern yetiştirme teknikleri sadece bu türün yetiştiriciliğindeki gelişmelerle kalmayıp, diğer
meyve türlerinde de modern teknoloji kullanımına öncülük etmiştir. Elma yetiştiriciliğinde en önemli
gelişme klasik çöğür anaçları yerine değişik kuvvette büyüme özelliğine sahip klonal anaçların
kullanımı alanında yaşanmıştır. Bu sayede ülkemizin hemen her bölgesinde bodur veya yarı bodur
büyüme özelliğine sahip klonal anaçlarla bahçeler kurulmuştur (Öztürk ve ark. 2005; Ikinci ve Bolat
2016; Atay ve Atay, 2018). Ülkemizde toplam elma üretiminin % 36.2’lik bölümü Akdeniz Bölgesi’nden sağlanmaktadır. Article Info Received: 17.10.2018
Accepted: 11.04.2019
Online Published 28.06.2019
DOI: 10.29133/yyutbd.471552 Keywords
Phenology,
Malus domestica, Keywords
Phenology,
Malus domestica, 258 YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267
Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267
Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi Fruit quality,
Pomology,
Yield efficiency In varieties, total soluble solids showed change as 13.50 % and 15.17 %, the
amount of titratable acidity 0.23 % and 0.40 %, reducing sugar content 8.93 %
and 10.17 %, total sugar level 12.08 % and 13.87 % and the total phenol content
113.40 mg/100 g and 148.40 mg/100 g. On the other hand, trunk cross-sectional
area of apple cultivars was found to vary between 13.20 cm2 and 22.67 cm2, yield
per tree: 4.92 kg/tree and 9.32 kg/tree and the yield efficiency: 0.28 kg/cm2 and
0.66 kg/cm2. Mondial Gala for the summer period, Scarlet spur for the early fall
period and Fuji apple cultivars for the fall period will contribute to producers in
the region in terms of product diversity is thought to contribute. **Mustafa YILMAZ’ın “Akdeniz Geçit Kuşağında Değişik Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin
Performanslarının Belirlenmesi” isimli tezinden üretilmiştir. 2.1. Yöntem Araştırmada ele alınan elma çeşitleri üzerinde yapılan gözlemler ve ölçümler ile uygulanan metotlar
aşağıda belirtilmiştir. Araştırmada ele alınan elma çeşitleri üzerinde yapılan gözlemler ve ölçümler ile uygulanan metotlar
aşağıda belirtilmiştir. 1. Giriş Bu bölgeyi % 18.0’lik üretim payları ile Batı ve Orta Anadolu Bölgeleri ve % 9.8 ile Ege Bölgesi takip
etmektedir (TÜİK, 2016). Akdeniz ve Güneydoğu Anadolu bölgeleri arasında bir geçiş noktasında
bulunan Osmaniye ilinde 2 079 da alanda 3 259 ton elma üretimi yapılmakta olup, ağaç başına ortalama
verim 37.4 kg’dır (TÜİK, 2016). Osmaniye ili Akdeniz Bölgesi’nde yer almakta ve etrafının büyük bir bölümü Toros Dağları ile
çevrili bulunmaktadır. Osmaniye ili Merkez ilçesi 123 m rakıma sahip olmakla birlikte, etrafını çeviren
dağlarda yükselti 1 575 m (Zorkun) - 2 246 m (Düldül Dağları) aralığında değişim göstermektedir. 259 YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267
Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi Osmaniye Merkez ilçesi ve yakın çevresi Akdeniz iklimi özelliğine sahiptir. İlin yüksek rakımlı
kısımlarında ise kışın daha soğuk ve yazın daha serin özelliğe sahip Akdeniz-Karasal bölgeler arasındaki
geçit iklimi hüküm sürmektedir (Anonim, 2018a). Osmaniye Merkez ilçesi ve yakın çevresi Akdeniz iklimi özelliğine sahiptir. İlin yüksek rakımlı
kısımlarında ise kışın daha soğuk ve yazın daha serin özelliğe sahip Akdeniz-Karasal bölgeler arasındaki
geçit iklimi hüküm sürmektedir (Anonim, 2018a). Osmaniye ili Merkez ilçesinde +7.2 oC’nin altındaki toplam soğuklama süresinin 679-970 saat
arasında değişim gösterdiği bildirilmektedir (Yelmen, 2007). İlde meyvecilik tarımının yoğun olarak
yapıldığı Toros dağları etekleri, Düziçi, Bahçe ve Hasanbeyli ilçeleri gibi daha yüksek rakımlı bölgelerin
soğuklama sürelerine ait veri bulunmamakla birlikte, bu kesimlerin Merkez ilçeden daha uzun bir
soğuklamaya sahip olduğu düşünülmektedir. Bu çalışmada Osmaniye ili Bahçe ilçesi sınırları içerisinde bulunan, geçit iklimi özelliğine sahip
alanda M9 anacı üzerine aşılı ve sık dikim uygulaması yapılan üç ayrı elma çeşidinin performanslarının
incelenmesi amaçlanmıştır. Toprak özellikleri Araştırmanın yürütüldüğü bahçe toprağının kimyasal özellikleri Çizelge 1.’de verilmiştir. Araştırmanın yürütüldüğü bahçe toprağının kimyasal özellikleri Çizelge 1.’de verilmiştir. Çizelge 1. Araştırmanın yürütüldüğü bahçenin toprak analizi sonuçları (Bozkurt ve ark. 2001) Çizelge 1. Araştırmanın yürütüldüğü bahçenin toprak analizi sonuçları (Bozkurt ve ark. 2001)
Toplam
Azot
(N)
(%)
Alınabilir
fosfor
(P2O5)
(kg/da)
Alınabilir
potasyum
(K2O)
(kg/da)
Alınabilir
kalsiyum
(CaO)
(kg/da)
Alınabilir
magnezyum
(MgO)
(kg/da)
Alınabilir
demir
(Fe)
(kg/da)
Alınabilir
çinko
(Zn)
(kg/da)
Toprak
(pH)
0.56
7.0
25.1
2720
213
12.5
0.71
8.00
Orta
Orta
Yeterli
Çok yüksek
Çok yüksek
Orta
Orta
Bazik 2. Materyal ve Yöntem Araştırma, 2012-2013 yılları arasında Osmaniye ili Bahçe ilçesi Burgaçlı köyü Payamlı
mevkiinde bulunan bir üretici bahçesinde (37o10’ Kuzey ve 36o25’ Doğu; rakımı 393 m) yürütülmüştür. Araştırma bahçesi, M9 klonal anacı üzerine aşılı Mondial Gala, Scarlet Spur ve Fuji çeşitleri ile 2008
yılı kış döneminde 3x1 m sıra arası ve sıra üzeri mesafede kurulmuştur. Tozlayıcı çeşit olarak Golden
Delicious kullanılmıştır. Ağaçlara merkezi lider terbiye sistemi uygulanmıştır. Bahçedeki ağaçların
üzeri güneş yanıklığından korunması için gölgeleme materyali ile kaplanmıştır. Araştırmanın
yürütüldüğü bahçe Nisan-Ekim ayları arasında damla sulama sistemi ile sulanmış, gübreleme ve
ilaçlama gibi teknik ve kültürel işlemler düzenli bir şekilde yerine getirilmiştir. İklim özellikleri Araştırmanın yürütüldüğü Osmaniye ilinde 1986-2014 yıllarında arasında belirlenen ortalama
sıcaklık 18.3 oC, maksimum sıcaklık 34.2 oC (Ağustos), minimum sıcaklık 3.4 oC (Ocak), toplam yağış
802 mm ve ortalama nispi nem değeri ise % 62.3 olarak belirtilmiştir (Anonim, 2018b). Verim Özellikleri Elma çeşitlerine ait ağaçlarda ağaç başına düşen verim (kg ağaç-1), birim gövde kesit alanına
düşen verim (kg cm-2) ve dekara verim (kg da-1) miktarı değerleri belirlenmiştir (Yaşasın ve ark., 2006,
Ikinci ve Bolat, 2016). Meyve Özelliklerin Belirlenmesi İncelenen elma çeşitlerinde, çeşidin özelliklerini temsil edecek şekilde her çeşide ait her bir
ağaçtan hasat döneminde alınan 10’ar meyvede; meyve ağırlığı (g) 0.01 g’a duyarlı hassas terazi (Precisa
BJ 6100D) ile; meyve eni (mm), meyve boyu (mm) 0.01 mm’ye duyarlı dijital kumpas (Mitutoyo CD-
20CPX) ile; meyve eti sertliği (kg cm-2) el penetrometresi ile; meyve hacmi, ağzına kadar saf su dolu
ölçü silindirinin içine teker teker elmalar bırakılarak, taşan suyun ölçü silindirinde ölçülmesi ile (cm3)
ve meyve tohum sayısı, meyvelerden çıkartılan tohumların sayılarak, ortalamalarının alınması ile (adet)
belirlenmiştir (Burak ve ark., 1998; Yaşasın ve ark. 2006, Ikinci ve Bolat, 2016). Fenolojik gözlemler Fenolojik özellikler olarak; tomurcuk kabarması, tomurcuk patlaması, çiçeklenme başlangıcı,
tam çiçeklenme, çiçeklenme sonu, meyvenin hasat olum tarihi ve yaprak döküm tarihleri tüm çeşitler
için ayrı ayrı belirlenmiştir. Çiçeklenme başlangıcı; çiçeklerin % 5’nin açtığı, tam çiçeklenme; çiçeklerin
% 60-70’inin açtığı ve çiçeklenme sonu; çiçeklerin taç yapraklarının % 90’dan fazlasının döküldüğü
tarihler olarak belirlenmiştir (Chapman and Catlin, 1976; Erdoğan ve Bolat, 2002; Yaşasın ve ark., 2006;
Ikinci ve Bolat, 2016). 260 YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267
Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi Kimyasal Özelliklerin Belirlenmesi Çeşitlere ait meyvelerde toplam suda eriyebilir kuru madde (SÇKM) miktarı el refraktometresi
ile % olarak ölçülerek (Burak ve ark., 1998; Karaçalı, 2004; Yaşasın ve ark., 2006; Özrenk ve ark., 2011;
Abacı ve Sevindik, 2014; Ikinci ve Bolat, 2016), titre edilebilir asitlik (TA) meyve suyu örneğinin 0.1N
NaOH ile titre edilmesi ve harcanan baz miktarına göre malik asit cinsinden hesaplanma ile % olarak
(Karaçalı, 2004; Ikinci ve Bolat, 2016), kabuklu meyve örneğinde toplam fenolik madde (mg/100 g taze
ağırlık) tayini Folin-Ciocalteu yöntemi kullanılarak (Hayoğlu ve Türkoğlu, 2007; Abacı ve Sevindik,
2014), indirgen ve toplam şeker tayinleri ise Lane-Eynon metoduna göre belirlenmiştir (Hayoğlu ve
Türkoğlu, 2007; Cemeroğlu, 2010). İstatistik Analiz Araştırma tesadüf blokları deneme desenine göre 3 tekerrürlü ve her tekerrürde 3 ağaç olacak
şekilde kurulmuştur. Denemeden elde edilen sonuçların değerlendirilmesinde JMP 8.0 paket programı
kullanılmıştır. Ortalamalar arasındaki farklılıkların karşılaştırılmasında ‘LSD Testi’ (p<0.05 ve p<0.01)
kullanılmıştır. YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267
Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi 3. Bulgular ve Tartışma Denemede yer alan çeşitlerde 2012 ve 2013 yıllarında yapılan fenolojik gözlemlerde; tomurcuk
kabarması 14 Mart (Mondial Gala)-23 Mart (Fuji) ve tomurcuk patlaması ise 20 Mart (Mondial Gala) -
31 Mart (Fuji) tarihleri arasında gerçekleşmiştir (Çizelge 2.). Çeşitler arasında en erken çiçeklenmeye
Mondial Gala (3 Nisan), en geç ise Fuji (17 Nisan) başlamıştır. İncelenen üç elma çeşidinde tam
çiçeklenme 11 Nisan (Mondial Gala) - 25 Nisan (Fuji) tarihleri arasında gerçekleşirken, çiçeklenme
sonu ise 17 Nisan (Mondial Gala) – 05 Mayıs (Fuji) tarihleri arasında meydana gelmiştir. y
(
j )
y
g
Bahçe (Osmaniye) İlçesi koşullarında yapılan gözlemlerde, elma çeşitlerinde ilk hasat
olgunluğuna 11 Ağustos’ta Mondial Gala çeşidi gelirken, en geç ise 5 Ekim’de Fuji çeşidi gelmiştir
(Çizelge 2.). Elma çeşitlerinde tam çiçeklenmeden hasada kadar geçen süre 122 gün (Mondial Gala) ile
167 gün (Fuji) arasında değişmiştir. Elma çeşitlerinin yaprak dökümü 8 Aralık - 27 Aralık tarihleri
arasında meydana gelmiştir. 261 YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267
Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi Çizelge 2. Mondial Gala, Fuji ve Scarlet Spur elma çeşitlerinde 2012 ve 2013 yılına ait bazı fenolojik
gözlem sonuçları
Çeşitler
Yıl
T.K. T.P. Ç.B. T.Ç. Ç.S. H.T. T.Ç.H.G. Mondial
Gala
2012
20.03
27.03
12.04
21.04
30.04
22.08
123
2013
14.03
20.03
03.04
11.04
17.04
11.08
122
Scarlet
Spur
2012
21.03
29.03
14.04
21.04
29.04
10.09
142
2013
15.03
21.03
05.04
12.04
20.04
30.08
141
Fuji
2012
23.03
31.03
17.04
25.04
05.05
05.10
163
2013
17.03
24.03
08.04
15.04
24.04
29.09
167
T.K: Tomurcuk kabarması. T.P: Tomurcuk patlaması, Ç.B: Çiçeklenme başlangıcı, T.Ç: Tam çiçeklenme, Ç.S: Çiçekleneme
sonu, H.T: Hasat tarihi, T.Ç.G.S: Tam çiçeklenmeden hasada kadar geçen gün sayısı Mondial Gala, Fuji ve Scarlet Spur elma çeşitlerinde 2012 ve 2013 yılına ait bazı fenolojik
gözlem sonuçları 2013
17.03
24.03
08.04
15.04
24.04
29.09
167
T.K: Tomurcuk kabarması. T.P: Tomurcuk patlaması, Ç.B: Çiçeklenme başlangıcı, T.Ç: Tam çiçeklenme, Ç.S: Çiçekleneme
sonu, H.T: Hasat tarihi, T.Ç.G.S: Tam çiçeklenmeden hasada kadar geçen gün sayısı Tomurcuk kabarması. T.P: Tomurcuk patlaması, Ç.B: Çiçeklenme başlangıcı, T.Ç: Tam çiçeklenme, Ç.S: Çiçeklenem
H.T: Hasat tarihi, T.Ç.G.S: Tam çiçeklenmeden hasada kadar geçen gün sayısı Burak ve ark. (1994), Yalova koşullarında çöğür anacına aşılı Jerseymac çeşidinde tomurcuk
kabarmasının 15 Mart, tomurcuk patlamasının 29 Mart, ilk çiçeklenmenin 16 Nisan, tam çiçeklenmenin
19 Nisan ve çiçeklenme sonunun ise 30 Nisan tarihlerinde gerçekleştiğini bildirmişlerdir. 3. Bulgular ve Tartışma Benzer
ekolojide yapılan bir başka araştırmada ise elma çeşitlerinin çiçeklenme başlangıcının 16 Mart - 21
Nisan tarihleri arasında meydana geldiği tespit edilmiştir (Yaşasın ve ark., 2006). Tokat ekolojik
koşullarında yetiştirilen dört farklı elma çeşidinde ilk çiçeklenme 29 Mart -26 Nisan tarihleri arasında
(Polat, 1997) olurken, Eğirdir (Isparta) ekolojik koşullarında ise ilk çiçeklenme 16 Nisan - 18 Nisan
tarihleri arasında meydana gelmiştir (Atay ve ark., 2010). Atay ve ark. (2010), Eğirdir koşullarında MM
106 anacı üzerine aşılı Jerseymac, Galaxy Gala ve Braeburn çeşitlerinin 2006 yılında sırasıyla; 20, 22
ve 20 Nisan tarihlerinde tam çiçeklenme aşamasında olduğunu, 2007 yılında ise sırasıyla; 28 Nisan, 1
Mayıs ve 28 Nisan tarihlerinde tam çiçeklenme aşamasına ulaştıklarını bildirmişlerdir. Van ekolojik
koşulları altında 2005-2007 yılları arasında yöresel elma genotiplerinde yapılan gözlemlerde tam
çiçeklenmenin 30 Nisan - 11 Mayıs (Kaya ve Balta, 2009), Çoruh Vadisi’nde yetişen elma çeşitlerinde
8 - 22 Mayıs (Erdoğan ve Bolat, 2002) ve Tokat ekolojik koşullarında yetiştirilen elma çeşitlerinde ise
5-29 Nisan (Polat, 1997) tarihleri arasında meydana geldiği belirlenmiştir. Özongun ve ark. (2014), Eğirdir/Isparta ekolojisinde MM 106 anacına aşılı 10 elma çeşidinde
2003-2009 yılları arasındaki fenolojik gözlem sonuçlarına göre çiçeklenme sonunun 01-05 Mayıs
tarihlerinde olduğunu bildirmişlerdir. Eğirdir koşullarında yıllara göre yapılan fenolojik gözlem
sonuçlarına göre M9, M26, MM 106 ve MM 111 anacı üzerine aşılı Mondial Gala elma çeşidinde tomurcuk
kabarma tarihi 20-25 Mart, tomurcuk patlaması 29 Mart-5 Nisan, ilk çiçeklenme 17-21 Nisan, tam çiçeklenme
23-29 Nisan ve çiçeklenme sonu 1-7 Mayıs olarak belirlenmiştir (Özongun ve ark., 2016). Benzer şekilde
ülkemizin farklı bölgelerinde değişik elma çeşitleri üzerinde yapılan çalışmalarda da çiçeklenme
sonunun bölgeye ve çeşit özelliğine bağlı olarak değişim gösterdiği saptanmıştır (Polat. 1997; Atay ve
ark.. 2010). Tokat koşullarında farklı elma çeşitlerinden meyve olgunlaşmasının 5-19 Eylül (Polat 1997),
Van koşullarında 22 Ağustos - 10 Ekim (Kaya ve Balta, 2009), Ordu koşullarında 25 Eylül - 17 Ekim
(Yarılgaç ve ark., 2009) ve Eğirdir koşullarında 25 Ağustos- 1 Eylül (Özongun ve ark., 2016) tarihleri
arasında gerçekleştiği saptanmıştır. Araştırmamızda incelemiş olduğumuz elma çeşitlerinde 2012 yılında tam çiçeklenmeden -
hasada kadar geçen süre 123 gün (Mondial Gala) ile 163 gün (Fuji), 2013 yılında ise 122 gün (Mondial
Gala) ile 167 gün (Fuji) olarak tespit edilmiştir (Çizelge 2.). 3. Bulgular ve Tartışma Ülkemizin farklı bölgelerinde değişik elma
çeşitleri üzerinde yapılan çalışmalarda tam çiçeklenmeden hasada kadar geçen süre Niğde ekolojik
koşullarında ise 122 - 164 gün (Ceylan, 2008), Van koşullarında 102 - 150 gün (Kaya ve Balta, 2009),
Tokat koşullarında 98 - 180 gün (Atay ve ark. 2010) ve Eğirdir koşullarında 142-169 gün (Seymen ve
Polat, 2015) arasında değişim gösterdiği saptanmıştır. Ülkemizin değişik yörelerinde yapılan çalışmalarda, fenolojik gözlemler bakımından
birbirinden farklı sonuçlar elde edilmiştir. Meyve yetiştiriciliğinde çiçeklenme zamanı ve süresi türlere,
çeşitlere, anaçlara, ekolojiye, yetiştiriciliğin yapıldığı yerin enlem-boylamına, rakımına, sıcak ve soğuk
rüzgarlara, yıllara ve kültürel uygulamalara göre değişkenlik göstermektedir (Karaçalı, 2004). Osmaniye koşullarında değişik dönemlerde olgunlaşan 3 elma çeşidi ait bazı meyve özellikleri
Çizelge 3.'te verilmiştir. İncelenen meyve özellikleri bakımından çeşitler arasında istatistiksel olarak
önemli (p<0.01 ve p<0.05) düzeyde farklılıkların olduğu tespit edilmiştir. Çeşitlerin ortalama meyve
ağırlığı 157.5 g (Scarlet Spur) – 212.8 g (Fuji), ortalama meyve eni 68.2 mm (Mondial Gala) – 79.3 mm 262 YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267
Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi (Fuji), ortalama meyve boyu 62.7 g (Mondial Gala) -67.5 (Scarlet Spur), ortalama meyve hacmi 166.7
ml (Mondial Gala) – 260 ml (Fuji) ve ortalama çekirdek sayısı 6.6 adet (Scarlet Spur) – 8.7 adet (Fuji)
arasında saptanmıştır. Meyve eti sertliği bakımından Fuji çeşidi (8.2 kg/cm2) en yüksek değeri, Scarlet Spur çeşidi ise
en düşük değeri (5.1 kg/cm2) vermiştir (Çizelge 3.). Suda çözünebilir kuru madde yönünden en yüksek
değer Mondial Gala çeşidinden (% 14.7) elde edilirken, bu çeşidi Fuji (% 14.1) ve Scarlet Spur (% 13.9)
çeşitleri takip etmiştir. En yüksek titre edilebilir asit miktarı Fuji çeşidinde (% 0.39), en düşük ise Scarlet
Spur (% 0.28) çeşidinde belirlenmiştir. Eğirdir (Isparta) koşullarında MM 106 anacı üzerine aşılı 10 elma
çeşidinin yer aldığı bir çeşit adaptasyon denemesinde, çeşitlerin meyve ağırlıkları 162 (Rubinstein) - 287
g (Crown Gold), meyve eni 69 (Early Red One) - 84 mm (Novaja), meyve boyu 57 (Gala Selecta)
– 77 mm (Topred), meyve eti sertliği 6.94 (Crown Gold) - 9.55 kg/cm2 (Cripps Pink (Pink LadyTM)),
SÇKM içeriği % 12.20 (Scarlet spur) – 16.40 (Cripps Pink (Pink LadyTM)) ve asitlik değeri % 0.31
(Topred) – % 1.12 (Cripps Pink (Pink LadyTM)) arasında belirlenmiştir (Özongun ve ark., 2014). 3. Bulgular ve Tartışma Konya ekolojisine uygun yeni elma çeşitlerinin belirlenmesi amacıyla yürütülen bir çalışmada, M9
anacına aşılı Fuji elma çeşidinde ortalama meyve ağırlığı 142.33 g, meyve eni 68.43 mm, meyve boyu
58.59 mm, çekirdek sayısı 8.83 adet, meyve eti sertliği 4.96 kg/cm², SÇKM miktarı % 12.40 ve titre
edilebilir asit miktarı ise % 0.48 olarak belirlenmiştir (Arıkan ve ark., 2015). Samsun ekolojik
şartlarında yetiştirilen bazı elma çeşitlerinde ortalama meyve ağırlığı 112.3 (Jersey Mac) - 173.9 g
(Starkrimson Delicious), meyve eni 64.83 (Golden Delicious) - 74.27 mm (Granny Smith), meyve boyu
54.55 (Jersey Mac) - 63.74 mm (Red Chief), meyve eti sertliği 4.94 (Jersey Mac)-7.98 kg/cm² (Granny
Smith), SÇKM % 10.46 (Starkrimson Delicious) - % 13.45 (Cooper 7 SB2 ve Golden Delicious) ve TA
% 0.39 (Süper Chief) - % 0.90 (Granny Smith) arasında değişim göstermiştir (Öztürk ve Öztürk, 2016). Eğirdir koşullarında M9 anacı üzerine aşılı Mondial Gala çeşidinin meyve ağırlığı, meyve eni, meyve
boyu, meyve eti sertliği, SÇKM ve TA sırasıyla; 147 g, 69.04 mm, 61.73 mm, 9.25 kg/cm2, % 13.03,
3.70 ve % 0.30 arasında tespit edilmiştir (Özongun ve ark., 2016). p
ş
(
g
,
)
En önemli sekonder metabolitlerinden olan toplam fenolik madde içeriği incelenen çeşitlerde
119.10 mg/100 g (Scarlet Spur) – 145.25 mg/100 g (Fuji) arasında değişim göstermiştir (Çizelge 3.). Benzer şekilde Ardahan ekolojik koşullarında yetiştirilen elma çeşitlerinin meyve kabuğunda fenolik
madde miktarları 209.7-578.9 mg/100 g, meyve etinde - ise 50.2 ile 112.2 mg/100 g arasında değişim
gösterdiği saptanmıştır (Abacı ve Sevindik, 2014). Diğer taraftan Özden ve Özden (2014), olgunluk
dönemindeki elma meyvelerindeki toplam fenolik madde içeriğini 698.67 mg/GAE/kg (Granny Smith)
– 810.48 mg/GAE/kg (Gala) arasında yer aldığını belirlemişlerdir. Üç farklı elma çeşidinde indirgen şeker kapsamının % 9.13 (Scarlet Spur) - % 10.09 (Fuji)
arasında değiştiği belirlenmiştir (Çizelge 3.). İndirgen şeker kapsamı üzerine yapılan istatistiki
analizlerde, Mondial Gala ve Scarlet Spur çeşitleri aynı grupta, Fuji çeşidi ise farklı grupta yer almıştır. Ülkemizin farklı bölgelerinde değişik elma çeşitlerinde yapılan çalışmalarda da meyvedeki indirgen
şeker kapsamı bakımından çeşitlere göre farklılıklar olduğu tespit edilmiştir. Nitekim, indirgen şeker
kapsamı Çoruh Vadisi’nde yetişen elma çeşitlerinde % 5.30 (Fındık) - % 8.69 (Golden Delicious)
(Erdoğan ve Bolat, 2002) arasında; Erzurum, Kars, Erzincan ve Gümüşhane koşullarında yetiştirilen
elmalarda % 8.11 (Amasya) - % 10.06 (Göbek) (Keleş, 1979) arasında ve Erzincan koşullarında
yetiştirilen elmalarda ise % 6.96 (Granny Smith) - % 8.97 (Golden Delicious) (Güleryüz ve ark., 2001)
arasında değiştiği belirlenmiştir. YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267
Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi Bahçe (Osmaniye) İlçesi koşullarında M9 anacı üzerine aşılı 4 yaşlı Mondial Gala, Scarlet Spur
ve Fuji elma çeşitleri arasında, ağaçlardan elde edilen veya hesaplama ile belirlenen tüm verim değerleri
arasında istatistiksel anlamda önemli fark bulunmuştur (Çizelge 4.). Elma çeşitlerinden 2012-2013
yıllarında elde edilen ağaç başına ortalama verim değerlerinin 6.40 kg/ağaç (Scarlet Spur) – 7.89 kg/ağaç
(Fuji) arasında değişim gösterdiği saptanmıştır. Ağaç başına verim değerini Niğde koşullarında değişik
elma çeşitleri üzerinde yapılan benzer bir çalışmada -10.48 kg/ağaç (Galaxy Gala/M9) - 17.34 kg/ağaç
(Granny Smith/M9) (Ceylan, 2008), Tokat koşullarında 3.24 kg/ağaç - 6.82 kg/ağaç (Gala/M9)
(Küçüker ve ark., 2011) ve Çorum koşullarında 10.10 kg/ağaç (Red Chief/M9) - 16.60 kg/ağaç (Granny
Smith/M9) (Çulha, 2010) arasında yer aldığı tespit edilmiştir. Denemede yer alan elma çeşitlerine ait ağaçlarda 14.51 cm2 (Mondial Gala) – 20.74 cm2 (Fuji)
arasında gövde enine kesit alanı değerleri ölçülmüştür. Farklı elma çeşitlerinde Tokat koşullarında 2.09
cm2 (Delcorf) - 3.69 cm2 (Red Star) (Özkan ve ark., 2009), Yalova koşullarında 28.10 cm2 (Red Chief)
- 494.63 cm2 (Mutsu) (Akçay ve ark., 2009) ve Tokat koşullarında 4.36 cm2 (Gala) - 20.51 cm2 (Gala)
(Küçüker ve ark., 2011) arasında gövde enine kesit alanı tespit edilmiştir. Birim gövde kesit alanına düşen verim miktarları (verim etkisi =etkili verim) göz önüne
alındığında, en yüksek değer Mondial Gala (0.50 kg/cm2) çeşidinden elde edilirken, en düşük ise Fuji
çeşidinde (0.39 kg/cm2) tespit edilmiştir. Görükle (Bursa) ekolojik koşulları altında farklı elma
çeşitlerinin etkili verim değerinin 0.15 kg/cm2 (Elstar, Topred) - 0.39 kg/cm2 (Granny Smith) (Soylu ve
ark., 2003), Tokat koşullarında 0.16 kg/cm2 (Jonagold) - 0.40 kg/cm2 (Gala) (Baytekin ve Akça 2011),
Yalova koşullarında 0.49 kg/cm2 (Stark Earliest) - 2.79 kg/cm2 (Starkrimson Delicious) (Akçay ve ark.,
2009) arasında yer aldığı bildirilmiştir. Elma çeşitlerinde iki yılın ortalamasına göre belirlenen dekara verim değerlerine göre; en
yüksek dekara verim 2628 kg ile Fuji çeşidinde belirlenirken, bu çeşidi Mondial Gala (2367 kg) ve
Scarlet Spur (2135 kg) çeşitleri izlemiştir. Tokat koşullarında elma ağaçlardan elde edilen dekara verim
değerleri 1 238 - 1 925 kg/da (Küçüker ve ark., 2011) arasında, Niğde koşullarında 2 324 kg/da (Scarlet
Spur) – 3 326 kg/da (Super Chief) (Özdemir ve ark. 2009) ve Samsun koşullarında ise 791 kg/da
(Skyline Supreme/MM 111) – 6 655 kg/da (Granny Smith/MM 106) (Kaplan ve Macit, 2009) arasında
değişim gösterdiği belirtilmiştir. 3. Bulgular ve Tartışma Çeşitlerin toplam şeker içerikleri % 12.38 (Scarlet Spur) –% 13.74
(Fuji) aralığında değişim göstermiştir (Çizelge 3.). Ülkemizin farklı bölgelerinde değişik elma çeşitleri
üzerinde yapılan çalışmalarda; Çoruh Vadisi koşullarında yetiştirilen elma çeşitlerinde % 8.38 (Fındık)
- % 12.72 (Golden Delicious) (Erdoğan ve Bolat, 2002) ve Erzincan koşullarında yetiştirilen elma
çeşitlerinde ise % 9.04 (Granny Smith) - % 11.84 (Sakı) (Güleryüz ve ark., 2001) arasında toplam şeker
içeriği tespit edilmiştir. Diğer taraftan Teletar (1985), olgunluk dönemindeki elma meyvelerindeki
toplam şeker içeriğinin % 9.56 (Amasya) - % 13.09 (Hüryemez) arasında değiştiğini saptamıştır. Ü Ülkemizin farklı ekolojilere sahip olan yörelerinde yürütülen araştırmalarda, aynı çeşit ve anaç
bileşenleri kullanılmış olsa dahi, elma çeşitlerinin meyve kalite özellikleri ve kimyasal içeriklerinde
kısmen birbirinden farklı sonuçlar elde edilmiştir. Değişik çalışmalarla, yürütülmüş olan bu çalışma
arasındaki farklılığın çeşit, anaç, meyve tutum oranlarının farklı olmasından, kültürel uygulamalardan,
iklim ve toprak özelliklerinden, yetiştiriciliğin yapıldığı yerin enlem-boylam ve rakımından
kaynaklandığı düşünülmektedir. 263 YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267
Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267
Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi Çalışmamızda; verim değerleri ve gövde enine kesit alanı ile ilgili elde edilen bulgular,
ülkemizin farklı yörelerinde yürütülen çalışmalarda elde edilen bulgularla bazı durumlarda benzerlik,
bazı durumlarda ise farklılık gösterdiği belirlenmiştir. Bulgular arasındaki farklılığın; çalışmalardaki
anaç, çeşit, kültürel uygulamalar, iklim, toprak ve ağacın yaşı gibi özelliklerin farklılık göstermesinden
kaynaklandığı düşünülmektedir. 264 YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267
K
ğ d F kl Dö
l d Ol
l
B
El
Ç
i l i i P YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267
Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin P Çizelge 3. Mondial Gala, Scarlet Spur ve Fuji elma çeşitlerinin bazı meyve özellikleri (2012-2013 yılı ortalaması)
Çeşitler
Meyve
ağırlığı
(g)
Meyve eni
(mm)
Meyve
boyu
(mm)
Meyve
hacmi
(cm3)
Çekirdek
sayısı
(adet)
Meyve eti
sertliği
(kg/cm2)
SÇKM
(Briks)
Titre
edilebilir
asitlik
(%)
Toplam
fenol
(mg/100g
taze ağırlık)
İndirgen
şeker
kapsamı
(%)
Toplam
şeker
(%)
Mondial Gala
169.8 bz
68.2 b
62.7 b
166.7 c
7.3 b
7.4 b
14.7 a
0.36 ab
122.80 b
9.40 b
12.97 ab
Scarlet Spur
157.5 b
68.7 b
67.5 a
225.5 b
6.6 b
5.1 c
13.9 b
0.28 b
119.10 b
9.13 b
12.38 b
Fuji
212.8 a
79.3 a
65.4 ab
260.0 a
8.7 a
8.2 a
14.1 b
0.39 a
145.25 a
10.09 a
13.74 a
LSD 0.05
15.17*
6.60*
3.63**
34.36*
1.06**
0.75**
0.34**
0.07**
17.30**
0.43**
0.86**
z Aynı sütunda aynı harfle işaretlenmiş ortalamalar arasında istatistiksel bakımdan fark yoktur (P<0.05). *:% 5 ve **: % 1 düzeylerinde önemli. Ö.D.:Önemli Değil ve Fuji elma çeşitlerinin bazı meyve özellikleri (2012-2013 yılı ortalaması) z Aynı sütunda aynı harfle işaretlenmiş ortalamalar arasında istatistiksel bakımdan fark yoktur (P<0.05). *:% 5 ve **: % 1 düzeylerinde önemli. Ö.D.:Önemli Değil Çizelge 4. Mondial Gala, Scarlet Spur ve Fuji elma çeşitlerine ait ağaçların gövde enine kesit
alanı, ağaç başına verimi, dekara verimi ve etkili verim değerleri (2012-2013 yılı ort.) Çizelge 4. YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267
Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi Mondial Gala, Scarlet Spur ve Fuji elma çeşitlerine ait ağaçların gövde enine kesit
alanı, ağaç başına verimi, dekara verimi ve etkili verim değerleri (2012-2013 yılı ort.)
Çeşitler
Verim
(kg/ağaç)
Gövde
enine
kesit alanı
(cm2)
Birim
gövde
kesit
alanına
düşen verim
(kg cm-2)
Birim
alana
düşen verim
(kg da-1)
Mondial Gala
7.10 b z
14.51 c
0.50 a
2 367.0 b
Scarlet Spur
6.40 c
15.99 b
0.41 b
2 135.5 c
Fuji
7.89 a
20.74 a
0.39 b
2 628.0 a
LSD 0.05
0.36**
0.46**
0.065**
120.19**
z Aynı sütunda aynı harfle işaretlenmiş ortalamalar arasında istatistiksel bakımdan fark yoktur (P<0.05). **: % 1 düzeylerinde önemli. 265 YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267
Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267
Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi 4. Sonuç Doğu Akdeniz Bölgesi’nde yer alan Osmaniye ilinde genellikle kışlar ılıman geçmektedir. Osmaniye’de öteden beri ağırlıklı olarak tarla tarımı yapılmakta, meyvecilik sektörünün tarımsal
üretimdeki payı ise oldukça düşük düzeydedir. İlde eskiden elma yetiştiriciliği hemen hemen hiç
yapılmamaktaydı. Ancak, son yıllarda ülkemize giren yeni çeşitler, Osmaniye ili üreticilerinin de
dikkatini çekmiş ve uygun alanlarda özellikle bodur anaçlarının kullanıldığı, soğuklama isteği fazla
olmayan elma çeşitlerinin kullanıldığı ve sık dikim sistemlerinin uygulandığı elma bahçeleri tesis
edilmeye başlanmıştır. Mondial Gala, Scarlet Spur ve Fuji elma çeşitleri ülkemizde son dönemlerde önemli derecede
yaygınlık gösteren ve pazar değerleri yüksek olan çeşitlerdir. Bu çeşitler ülkemizde hemen her bölgede
yetiştirilmekle birlikte, Akdeniz Bölgesi ve İç Anadolu Bölgesi arasındaki geçit konumu özelliği
gösteren Doğu Akdeniz havzasının yüksek kesimleri için (özellikle soğuklama ve renklenme ile ilgili
sorunların ortadan kalkması nedeniyle) çok daha büyük bir potansiyel taşımaktadır. Araştırmanın yürütüldüğü geçit iklimi özelliği gösteren lokasyonda, üzerinde çalışılan üç elma
çeşidinde de bitki gelişimi, verim ve meyve kalitesi açısından önemli bir sorunla karşılaşılmamıştır. Gerek kendi verilerimizden ve gerekse diğer bölgelerde yapılan çalışmalardan elde edilen bulguların
ışığında, yaz dönemi açısından Mondial Gala, erken güz dönemi açısından Scarlet Spur ve güz dönemi
açısından ise Fuji elma çeşidinin, bölgedeki elma tarımı yapılabilecek benzer alanlarda ürün çeşitliğinin
sağlanması açısından üreticilere katkı sağlayacağı düşünülmektedir. YYÜ TAR BİL DERG (YYU J AGR SCI) Cilt (sayı):258-267
Bolat ve ark., / Akdeniz Geçit Kuşağında Farklı Dönemlerde Olgunlaşan Bazı Elma Çeşitlerinin Performanslarının Belirlenmesi Kaynakça Abacı, Z. T., & Sevindik, E. (2014). Ardahan bölgesinde yetiştirilen elma çeşitlerinin biyoaktif
bileşiklerinin ve toplam antioksidan kapasitesinin belirlenmesi. Yüzüncü Yıl Üniversitesi Tarım
Bilimleri Dergisi, 24(2), 175-184. Akçay, M. E., Doğan, A., Burak, M., Yaşasın, A. S., & Öz, F. (2009). Bazı elma çeşitlerinin Marmara
Bölgesinde yapılan adaptasyon çalışmaları. Tarım Bilimleri Araştırma Dergisi, 2(2), 65-71. Anonim. (2018a). http, //www.osmaniye.gov.tr Erişim tarihi: 06 Ekim 2018. İ
İ Anonim. (2018b). Osmaniye Meteoroloji İl Müdürlüğü İklim verileri. İ Arıkan, Ş., İpek, M., & Pırlak, L. (2015). Konya ekolojik şartlarında bazı elma çeşitlerinin fenolojik ve
pomolojik özelliklerinin belirlenmesi. Türk Tarım – Gıda Bilim ve Teknoloji Dergisi, 3(10), 811-
815. Atay, A. N., & Atay, E. (2018). Elma ıslahında ve çeşit yönetiminde yenilikçi eğilimler. Yüzüncü Yıl
Üniversitesi Tarım Bilimleri Dergisi, 28(2), 234-240. Atay, E., Pırlak, L., & Atay, A. N. (2010). Determination of fruit growth in some apple varieties. J. of
Agric. Sci., 16, 1-8. Baytekin, S., & Akça, Y. (2011). M9 elma anacı üzerine aşılı farklı elma çeşitlerinin performanslarının
belirlenmesi. Gaziosmanpaşa Üniversitesi Ziraat Fakültesi Dergisi, 28(1), 45-51. Burak, M., Büyükyılmaz, M., & Öz, F, (1998). Marmara Bölgesi için ümitvar elma çeşitlerinin seçimi. Bahçe, 27, 107-119. Bozkurt, M. A., Yarılgaç, T., & Çimrin, K. M. (2001). Çeşitli meyve ağaçlarında beslenme durumlarının
belirlenmesi. Yüzüncü Yıl Üniversitesi Tarım Bilimleri Dergisi, 11(1), 39-45. Ceylan, F. B., (2008). Bodur ve yarı bodur anaçlar üzerine aşılı bazı elma çeşitlerinin Niğde ekolojik
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(Malus domestica Borkh.) elma çeşidinde erken dönem performansının belirlenmesi. Batı
Akdeniz Tarımsal Araştırma Enstitüsü Derim Dergisi, 28(1), 25-36. Akdeniz Tarımsal Araştırma Enstitüsü Derim Dergisi, 28(1), 25 36. Özbek, S. (1978). Elma yetiştiriciliği. Özel meyvecilik (Kışın yaprağını döken meyve türleri) (s. 15-94). Çukurova Üniversitesi Ziraat Fakültesi Yayınları Yayın No: 128 Ders Kitabı: 11 Adana g
Özbek, S. (1978). Elma yetiştiriciliği. Özel meyvecilik (Kışın yaprağını döken meyve türleri) (s. 15-94). Çukurova Üniversitesi Ziraat Fakültesi Yayınları, Yayın No: 128, Ders Kitabı: 11, Adana. Ö
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elma çeşitlerinin pomolojik özellikleri. Tarım Bilimleri Dergisi, 21(1), 57-63. Kaynakça Özongun, Ş., Dolunay, E. M., Pektaş, M., Öztürk, G., & Çalhan, Ö. (2016). Farklı klon anaçları üzerinde
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belirlenmesi ve morfolojik karakterizasyonu. Harran Tarım ve Gıda Bil. Derg., 19(3), 1 Seymen, T., & Polat, M. (2015). Bazı Amasya elma tiplerinin fenolojik, pomolojik özelliklerinin
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Görükle koşullarındaki verim ve kalite özelliklerinin incelenmesi II. Uludağ Üniversitesi Ziraat
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ümitvar elma çeşitleri - V. Bahçe, 35(1-2), 75-82. Yelmen, H. (2007). Doğu Akdeniz Bölgesinde farklı soğuklama yöntemleri kullanılarak olasılıklı
soğuklama süre haritalarının çıkarılması (Yayımlanmamış yüksek lisans tezi). Çukurova
Üniversitesi, Fen Bilimleri Enstitüsü, Adana, 119s. 267
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In vitro–in silico correlation of three-dimensional turbulent flows in an idealized mouth-throat model
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PLOS COMPUTATIONAL BIOLOGY PLOS COMPUTATIONAL BIOLOGY RESEARCH ARTICLE
In vitro–in silico correlation of three-
dimensional turbulent flows in an idealized
mouth-throat model Eliram NofID1¤a, Saurabh Bhardwaj1¤b, Pantelis Koullapis2, Ron Bessler1,
Stavros Kassinos2, Josue´ SznitmanID1* Eliram NofID1¤a, Saurabh Bhardwaj1¤b, Pantelis Koullapis2, Ron Bessler1,
Stavros Kassinos2, Josue´ SznitmanID1*
1 Department of Biomedical Engineering, Technion—Israel Institute of Technology, Haifa, Israel,
2 Computational Sciences Laboratory (UCY-CompSci), Department of Mechanical and Manufacturing
Engineering, University of Cyprus, Nicosia, Cyprus 1 Department of Biomedical Engineering, Technion—Israel Institute of Technology, Haifa, Israel,
2 Computational Sciences Laboratory (UCY-CompSci), Department of Mechanical and Manufacturing
Engineering, University of Cyprus, Nicosia, Cyprus a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 ¤a Current address: Department of Medical Physics, Memorial Sloan Kettering Cancer Center, New York,
New York, United States of America
¤b Current address: Department of Biomedical Engineering, Pennsylvania State University, State College,
Pennsylvania, United States of America
* sznitman@bm.technion.ac.il a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 ¤a Current address: Department of Medical Physics, Memorial Sloan Kettering Cancer Center, New York,
New York, United States of America
¤b Current address: Department of Biomedical Engineering, Pennsylvania State University, State College,
Pennsylvania, United States of America
* sznitman@bm.technion.ac.il Editor: Alison L. Marsden, Stanford University,
UNITED STATES Received: September 5, 2022
Accepted: February 8, 2023
Published: March 23, 2023 Received: September 5, 2022
Accepted: February 8, 2023
Published: March 23, 2023 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.pcbi.1010537 Copyright: © 2023 Nof et al. This is an open access
article distributed under the terms of the Creative
Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in
any medium, provided the original author and
source are credited. OPEN ACCESS There exists an ongoing need to improve the validity and accuracy of computational fluid
dynamics (CFD) simulations of turbulent airflows in the extra-thoracic and upper airways. Yet, a knowledge gap remains in providing experimentally-resolved 3D flow benchmarks
with sufficient data density and completeness for useful comparison with widely-employed
numerical schemes. Motivated by such shortcomings, the present work details to the best of
our knowledge the first attempt to deliver in vitro–in silico correlations of 3D respiratory air-
flows in a generalized mouth-throat model and thereby assess the performance of Large
Eddy Simulations (LES) and Reynolds-Averaged Numerical Simulations (RANS). Numeri-
cal predictions are compared against 3D volumetric flow measurements using Tomographic
Particle Image Velocimetry (TPIV) at three steady inhalation flowrates varying from shallow
to deep inhalation conditions. We find that a RANS k-ω SST model adequately predicts
velocity flow patterns for Reynolds numbers spanning 1’500 to 7’000, supporting results in
close proximity to a more computationally-expensive LES model. Yet, RANS significantly
underestimates turbulent kinetic energy (TKE), thus underlining the advantages of LES as
a higher-order turbulence modeling scheme. In an effort to bridge future endevours across
respiratory research disciplines, we provide end users with the present in vitro–in silico cor-
relation data for improved predictive CFD models towards inhalation therapy and therapeu-
tic or toxic dosimetry endpoints. Citation: Nof E, Bhardwaj S, Koullapis P, Bessler R,
Kassinos S, Sznitman J (2023) In vitro–in silico
correlation of three-dimensional turbulent flows in
an idealized mouth-throat model. PLoS Comput
Biol 19(3): e1010537. https://doi.org/10.1371/
journal.pcbi.1010537 Editor: Alison L. Marsden, Stanford University,
UNITED STATES a link to a repository: https://doi.org/10.6084/m9.
figshare.20239125. a link to a repository: https://doi.org/10.6084/m9. figshare.20239125. a link to a repository: https://doi.org/10.6084/m9. figshare.20239125. resources, fluid dynamics (CFD) simulations are widely sought to predict deposition out-
comes but often lack detailed experimental data to first validate the three-dimensional
(3D) flow structures anticipated to arise in the upper respiratory tract. In an effort to rec-
oncile such data scarcity, we deliver experimental-numerical correlations of 3D respira-
tory airflows in an idealized 3D printed mouth-throat model against two widely-
established numerical schemes with varying computational costs, namely coarse RANS
and finer LES technique. Our time-resolved 3D flow data underline the complexity of
these physiological inhalation flows, and discuss advantages and drawbacks of the differ-
ent numerical techniques. With an outlook on future respiratory applications geared
towards broad preclinical inhaled aerosol deposition studies, our open source data are
made available for future benchmark comparisons for a broad range of end users in the
respiratory research community. Funding: This work was supported by the Israel
Science Foundation (ISF) (grant no. 1840/21 to
JS), the European Research Council (ERC) under
the European Union’s Horizon 2020 research and
innovation program (grant agreement no. 677772
to JS) as well as the PBC Fellowship Program by
the Israeli council for supporting SB with partial
funding. The funders had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript. Competing interests: N/A. PLOS COMPUTATIONAL BIOLOGY PLOS COMPUTATIONAL BIOLOGY Correlation of three-dimensional turbulent flows in an idealized mouth-throat model Author summary The dispersion and ensuing deposition of inhaled airborne particulate matter in the lungs
are strongly influenced by the dynamics of turbulent respiratory airflows in the mouth-
throat region during inhalation. To cirumvent costly in vitro experimental measurement Data Availability Statement: Relevant data are
within the manuscript and its Supporting
information files, and in addition we have provided 1 / 16 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537
March 23, 2023 Introduction Respiratory airflow characteristics are known to strongly influence the transport and deposi-
tion of inhaled aerosols in the human airways. The intricate geometry of the extra-thoracic
that includes bends, expansions and constrictions leads to transition to turbulent airflows in
the pharynx, larynx and trachea with Reynolds numbers on the order of several thousands
(i.e., 2’000–10’000), depending on inhalation regimes [1–4]. Detailed modeling of these com-
plex flows is required to determine the fate of particle-laden airflows and ensuing deposition
patterns towards predicting for example pulmonary dosimetry [5] or the dispersion of air-
borne pathogen in the lungs [6, 7]. p
g
g
In recent years, computational fluid dynamics (CFD) have spearheaded advances to over-
come some of the prohibitive costs of in vitro experimental campaigns. Numerical methods
solving the Reynolds-Averaged Navier-Stokes (RANS) equations are the most widely adopted
for modeling non-laminar flows due to their lower computational cost. RANS typically
involves applying a turbulence model to a stationary solution at steady breathing conditions
[8]. Coupled with the low-Reynolds number (LRN) k–ω turbulence model (e.g. k–ω SST),
RANS is often used to predict laminar–transitional–turbulent flows in the respiratory tract [9,
10]. While RANS simulations are popular, they often come short of determining model con-
stants, making clinical relevance and comparison with other studies challenging [11–15]. Ria-
zuddin et al. [16] used a k-ω SST turbulence model to investigate breathing in a nasal cavity,
demonstrating the model’s accuracy for modeling flows with unfavourable pressure gradients
via good agreement with experimental and numerical data. Ma et al. simulated airflows and
aerosol transport in patient-derived human airways with a k- model, showing good agree-
ment with coarse (i.e., regionally averaged) in vivo deposition data [17]. Stapleton et al. simi-
larly studied aerosol deposition using a k- model in an idealized mouth–throat validated with
in vitro regional deposition measurements from gamma scintigraphy [18]. The authors found
good agreement for laminar but not turbulent conditions, suggesting that particle deposition
may be sensitive to pressure drop and flow recirculation. Longest et al. investigated local air-
way aerosol deposition using different variants of the k-ω turbulence model [19], finding best
agreement with in vitro deposition patterns using the low Reynolds number (LRN) approxi-
mation. Despite such popularity, most RANS studies have used (sub-)regionally averaged
deposition metrics for experimental validation, whereas more accurate and spatially-resolved
deposition models would first require experimental validation of the underlying turbulent
flows. Introduction In recent years, computational fluid dynamics (CFD) have spearheaded advances to over-
come some of the prohibitive costs of in vitro experimental campaigns. Numerical methods
solving the Reynolds-Averaged Navier-Stokes (RANS) equations are the most widely adopted
for modeling non-laminar flows due to their lower computational cost. RANS typically
involves applying a turbulence model to a stationary solution at steady breathing conditions
[8]. Coupled with the low-Reynolds number (LRN) k–ω turbulence model (e.g. k–ω SST), 2 / 16 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537
March 23, 2023 PLOS COMPUTATIONAL BIOLOGY Correlation of three-dimensional turbulent flows in an idealized mouth-throat model As RANS modelling is not sufficiently accurate to capture small-scale fluctuations in turbu-
lent flows in extra-thoracic airways, a more adequate choice lies in Large Eddy Simulations
(LES). There, only the smallest flow scales containing a small fraction of the kinetic energy are
discarded, thereby retaining significantly more features of the underlying turbulence physics
compared with RANS [20, 21]. Lin et al. [20] were among the first to use LES in assessing the
effect of laryngeal jet-induced turbulence on airflow characteristics and tracheal wall shear
stress. Their study revealed that turbulence generated by the laryngeal jet can significantly
affect the downstream flow patterns, highlighting the importance of including the extrathor-
acic airways in a model. Choi et al. [2] performed LES in two CT-derived upper airway models
to examine the effect of inter-subject variabilities on the overall flow characteristics, finding
that the glottis constriction ratio and the curvature and shape of the airways have significant
effect on the generated flows. More recently, Koullapis et al. used LES to investigate inlet flow
conditions in a CT-reconstructed geometry of the human airways [21] where flow field differ-
ences largely dissipated just a short distance downstream of the mouth inlet. Furthermore,
increasing the inhalation flowrate from sedentary to active breathing conditions left the mean
flow field structures largely unaffected. Recent LES-based deposition studies have established
the trustworthiness of the method using generalized geometries [22, 23] meanwhile constant
gains in computing power have made LES more affordable. However, the underlying compu-
tational expense of LES is still considerably higher than RANS, precluding its accessibility for
broader use. Furthermore, imaging modalities are generating larger and more intricate patient
geometries, requiring first the validation of simpler models for practical and patient-specific
applications. Experimental method We briefly describe the experimental setup (Figs 1 and S1. A closed-loop perfusion system
comprising a centrifugal pump, 15 l reservoir tank and digital flow rate sensor, supplies
water/glycerol (58:42 mass ratio with a density of ρf = 1’150 kg/m3 and a dynamic viscosity
of μf = 9.66 × 10−3 kg/m-s) through the phantom model (fabricated using previously
described methods [34, 35]). We measure the three-dimensional, three-component (3D-3C)
velocity fields in the phantom model using a high-speed, tomographic particle image veloci-
metry setup (TPIV) (LaVision GmbH, Germany) consisting of four CMOS cameras (Fas-
tcam Mini UX100, 1’280 x 1’024 pixel, 12 bit, Photron USA, Inc.) equipped with 100 mm
focal length lenses (Zeiss Milvus, Germany). Volume illumination is provided by a 70 mJ
dual-head Nd:YLF laser (DM30–527DH, Photonics Industries, USA) and continuous image
acquisition was conducted using a frame straddling technique at a fixed 1’250 frames per
second (fps), where the time separation between laser pulses was set to 25, 40 and 65 μs,
respectively for the high, mid and low Re cases investigated. The field of view (FOV) spans
18.5 × 45 × 9 mm (x-y-z). These experimental settings were carefully optimized to ensure
that individual seeded particles correspond to 3 to 5 pixels imaged in the instantaneous
images so to avoid any peak-locking effects for PIV while maximum particle displacements
betwee consecutive images range between 3 and 8 pixels depending on the flow case. The
laser beam is introduced through the side of the model and shaped into a thick slab by an
optical arrangement consisting of a beam expander and cylindrical lens, followed by a knife
edge aperture (Fig 1a); the latter is commonly used in PIV to reduce light reflections from
regions void of tracer particles, contributing amongst other to the rectangularity of the
processed vector maps relative to the actual elliptical shape of the experimental model (see
results). Details on the TPIV methodology, including refractive index matching and scaling following
dynamic similarity can be found in our previous work [35], where raw images and TPIV pro-
cessing are performed with Davis 10 (LaVision GmbH, Germany) and further analyzed in
Matlab (Mathworks Inc., USA). Briefly, red fluorescent polystyrene particles (PS-FluoRed,
microParticles GmbH, Germany) are seeded and act as flow tracers where optical filters are
fitted to each camera lens to reduce non-fluorescent light reflection thereby increasing signal-
to-noise (SNR) ratio. Geometry and flow conditions The oral airway is based on a simplified elliptic model extending from the mouth through the
larynx and previously used in aerosol inhalation studies [31]. This elliptic mouth-throat (MT)
model generalizes a patient-specific geometry derived from a healthy adult’s computed tomog-
raphy (CT) scan [32]. Comparisons between in vitro measurements and in silico simulations are carried out at
three distinct Reynolds numbers, namely Re = 1’500, 4’500 and 7’000 based on the inlet
mouth diameter of the model. Values of Re correspond to steady inhalation air flowrates of
approximately 11.5, 32.8 and 52 l/min; such inhalation conditions are comparable to seden-
tary, light and heavy exercise conditions, respectively. Moreover, the two latter inhalation
flowrates are specifically relevant for pulmonary drug delivery via a dry powder inhaler
(DPI) [33]. Introduction To this end, a European Cooperation in Science and Technology (COST) action pub-
lished a much needed benchmark case, known as the Siminhale benchmark [23], comparing
several numerical schemes in an idealized airway geometry spanning mouth to the fourth
bronchial generation at an inhalation flowrate of 60 l/min. In silico predictions were vali-
dated against 2D particle image velocimetry (PIV) measurements [24] with good agreement
between PIV and LES, with a slight over-prediction of turbulent kinetic energies (TKE) in
the simulations. The experimental and numerical data in [24], along with two additional LES
and a RANS datasets, are currently part of a common publicly accessible ERCOFTAC data-
base providing best practice advice for setting up a computational fluid-particle dynamics
(CFPD) model of the human upper airways with available validation data. However, the
PIV data are limited to six orthogonal 2D planes and the laryngeal constriction, known to
strongly modulate the inlet flow [25], was not captured due to obstruction from optical
access. Several studies have measured flow in the laryngeal site using planar PIV alone [26,
27], while others employed numerical methods without comparable experimental measure-
ments. Jayaraju et al. validated their LES simulations in a mouth-throat model with planar
PIV, but measured only at the mid-sagittal plane [22]. In turn, a knowledge gap still remains
in providing an experimental benchmark with sufficient data density and completeness for
useful comparison to numerical schemes. Recently, volumetric flow measurement tools have been leveraged to study respiratory
flows in 3D with tomographic particle image velocimetry (TPIV) and magnetic resonance
velocimetry (MRV) [28, 29]. Notably, Kenjeres and Tijn validated their RANS and LES simula-
tions with available 3D MRV data [28] in an identical upper airway geometry [30] but the use
of a patient-specific model limits validations for future comparative studies. Motivated by
these ongoing shortcomings, the present work details the first attempt to deliver in vitro–in
silico flow correlations in a generalized mouth-throat model capturing 3D airflow patterns. RANS and LES predictions are compared against 3D TPIV flow measurements at three steady
inhalation flowrates varying from shallow to deep inhalation conditions. We share our model 3 / 16 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537
March 23, 2023 PLOS COMPUTATIONAL BIOLOGY Correlation of three-dimensional turbulent flows in an idealized mouth-throat model geometry and TPIV measurement data https://doi.org/10.6084/m9.figshare.20239125 as open-
source material towards future benchmark references. Experimental method The mean particle diameter dp = 10 μm and particle density ρp = 1’050 kg/ 4 / 16 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537
March 23, 2023 PLOS COMPUTATIONAL BIOLOGY Correlation of three-dimensional turbulent flows in an idealized mouth-throat model Workstation
Flowrate
sensor
Centrifugal
pump
Inlet
Thermometer
Outlet
Ball
valves
Cylindrical
lens
Beam
expander
High speed
cameras
15 liter
reservoir
°C
Nd:YLF laser
Knife
edges
Phantom model
Experimental setup
pharynx
oral
cavity
larnyx
trachea
Phantom model
10 m/s
PIV
1 mm
(c)
(b)
(a)
Fig 1. Model geometry and tomographic particle image velocimetry (TPIV) setup. (a) Experimental setup
consisting of a laser, optical equipment, flow system, four high-speed cameras and a phantom idealized mouth-throat
model. (b) The phantom model is illustrated schematically in three-dimensional view with inlet and outlet ports
marked. Note that the laryngeal constriction is illuminated by laser light, maximizing spatial resolution in the specific
region of interest (ROI). (c) A representative vector field is plotted along the mid-sagittal plane, following TPIV
processing algorithms. Workstation
Flowrate
sensor
Centrifugal
pump
Inlet
Thermometer
Outlet
Ball
valves
Cylindrical
lens
Beam
expander
High speed
cameras
15 liter
reservoir
°C
Nd:YLF laser
Knife
edges
Phantom model
Experimental setup
(a) Flowrate
sensor
Centrifugal
pump
Inlet
Thermometer
Outlet
Ball
valves
High speed
cameras
15 liter
reservoir
°C
fe
es
Phantom model
Experimental setup Experimental setup High speed
cameras Ball
valves Flowrate
sensor 10 m/s
PIV
1 mm
(c) pharynx
oral
cavity
larnyx
trachea
Phantom model
(
(b) (c) (b) Phantom model Phantom model Fig 1. Model geometry and tomographic particle image velocimetry (TPIV) setup. (a) Experimental setup
consisting of a laser, optical equipment, flow system, four high-speed cameras and a phantom idealized mouth-throat
model. (b) The phantom model is illustrated schematically in three-dimensional view with inlet and outlet ports
marked. Note that the laryngeal constriction is illuminated by laser light, maximizing spatial resolution in the specific
region of interest (ROI). (c) A representative vector field is plotted along the mid-sagittal plane, following TPIV
processing algorithms. https://doi.org/10.1371/journal.pcbi.1010537.g001 https://doi.org/10.1371/journal.pcbi.1010537.g001 m3 yield a corresponding particle relaxation time ts ¼ d2
prp=18mf of 0.6 μs and equivalent to
a particle Stokes number much smaller than unity. Concurrently, particle drift due to buoyancy
effects resulting from density differences between the working fluid and the particle, are
largely negligible as the (buoyant) terminal velocity is estimated to be ut = 5.7 × 10−7 m/s (i.e. Numerical methods For the CFD simulations, we introdude the governing equations and computational methodol-
ogy used to describe the motion of air based on the Eulerian approach. The governing equa-
tions for incompressible fluid flow are composed of the Navier-Stokes’ (momentum) and
continuity equations. Two distinct numerical methodologies (RANS and LES) are adopted to
model turbulent flows in the mouth-throat geometry. RANS details. A commercial software (ANSYS Fluent, ANSYS Inc.) was used to perform
the transient flow simulations using a RANS approach in which mass and momentum (i.e. Navier-Stokes) conservation equations are solved numerically by using the finite volume
method (FVM) in the 3D domain (see S1 Text. for RANS equations). Turbulent flow phenom-
ena were modeled using the Shear Stress Transport (SST) k-ω model with Low Reynolds Num-
ber (LRN) correction; considered the most suitable RANS model for predicting low
turbulence flow in the respiratory system, in particular with limited available computational
resources. The popular SST k-ω turbulence model [36, 37] is a two-equation eddy-viscosity
model. The use of a k-ω formulation in the inner parts of the boundary layer renders the
model directly usable all the way down to the wall through the viscous sub-layer. Hence the
SST k-ω model can be used as a Low-Re turbulence model without any extra damping func-
tions. The SST formulation also switches to a k- behaviour in the free-stream and thus avoids
the common k-ω problem whereby the model is too sensitive to the inlet free-stream turbu-
lence properties. Furthermore, the SST k-ω model is often credited for its good behaviour in
adverse pressure gradients and separated flows [3, 38]. Note that the SST k-ω model is
acknowledged to produce slightly high turbulence levels in regions with large normal strains,
e.g. stagnation regions and regions with strong acceleration. However, this tendency is much
less pronounced than with a normal k- model [39, 40]. The computational geometry was discretized in ICEM (ANSYS Inc., Canonsburg, PA)
using tetrahedral elements with prism layers at the walls. The final mesh was transformed into
a polyhedral mesh in Fluent (ANSYS, Inc.). A rigorous mesh convergence study was first car-
ried out (i.e. ranging from 2M to 6M tetrahedral cells) to eventually select the final mesh of
*900’000 polyhedral cells (converted from *2.4M tetrahedral cells), with up to 10 prism lay-
ers for near-wall refinements (see S2 Fig). Experimental method ut ¼ gd2
pðrf rpÞ=18mf), compared with characteristic velocities of the flow on the order of
Oð1Þ m/s at the mouth inlet. m3 yield a corresponding particle relaxation time ts ¼ d2
prp=18mf of 0.6 μs and equivalent to
a particle Stokes number much smaller than unity. Concurrently, particle drift due to buoyancy
effects resulting from density differences between the working fluid and the particle, are
largely negligible as the (buoyant) terminal velocity is estimated to be ut = 5.7 × 10−7 m/s (i.e. ut ¼ gd2
pðrf rpÞ=18mf), compared with characteristic velocities of the flow on the order of
Oð1Þ m/s at the mouth inlet. 5 / 16 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537
March 23, 2023 PLOS COMPUTATIONAL BIOLOGY Correlation of three-dimensional turbulent flows in an idealized mouth-throat model PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537
March 23, 2023 Numerical methods For each Re condition, a fully-developed parabolic
velocity profile is provided at the inlet surface whereas outflow condition is prescribed at outlet
surface and guaranteeing no-slip velocity conditions at the walls. A second-order implicit
scheme is used for the transient formulation, with a time step of 10−3 s to ensure good accu-
racy. To decrease numerical diffusion in the unstructured three-dimensional mesh, a second-
order upwind approach is utilized to discretize the advection terms. A segregated solver is then
used to solve the resultant system of equations. The SIMPLE algorithm is used to solve the gov-
erning equations by coupling velocity and pressure. LES details. Large Eddy Simulations (LES) are performed using the dynamic version of
the Smagorinsky-Lilly subgrid scale model [41] in order to examine the unsteady flow in the
upper airways geometry. Previous studies have shown that this model performs well in transi-
tional flows in the human airways [24]. The airflow is described by the filtered set of incom-
pressible Navier-Stokes equations. In order to generate appropriate inlet velocity conditions for the CFD model, a mapped
inlet (or recycling) boundary condition is used [42]. To apply this boundary condition, the
pipe at the inlet is extended by a length equal to ten times its diameter. The pipe section is ini-
tially fed with an instantaneous turbulent velocity field generated in a separate pipe flow LES. During the simulation, the velocity field from the mid-plane of the pipe domain is mapped to
the inlet boundary. Scaling of the velocities is applied to enforce the specified bulk flow rate. In 6 / 16 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537
March 23, 2023 PLOS COMPUTATIONAL BIOLOGY Correlation of three-dimensional turbulent flows in an idealized mouth-throat model Fig 2. Large Eddy Simulation (LES) grid sensitivity analysis (Re = 7’000). Comparison of 1D profiles of normalized time-averaged velocity
magnitudes (first column) and turbulent kinetic energy (second column), normalized by the mean inlet velocity uin, are presented at three stations (see
inset; top right panel) along the larynx (A-A’), glottis (B-B’) and upper trachea (C-C’), respectively, for two grid resolutions: (i) coarse (9.2M cells) and
(ii) fine (25M cells). Fig 2. Large Eddy Simulation (LES) grid sensitivity analysis (Re = 7’000). https://doi.org/10.1371/journal.pcbi.1010537.g002 Results and discussion
Glottal jet characteristics Flow through the human larynx, i.e. the passageway connecting the mouth to the respiratory
airways, is modulated by a valve-like constriction known as the glottis. During inhalation,
this constriction forms a jet of air that extends into the trachea known as the glottal or laryn-
geal jet. The glottal jet embodies the most dominant flow feature in the upper airways and
has been studied extensively in the production of voice and speech [44] as well as in its role
in mixing and dissipating boluses of inhaled aerosols [3, 20, 45]. By plotting the velocity mag-
nitude contours for the highest flowrate case (Re = 7’000), we observe the characteristic
structure clearly, namely a high velocity flow region extending within and downstream of
the constriction and dissipating further downstream (Fig 3a–c). In our idealized geometry,
we observe a mostly symmetric glottal jet, while asymmetries would be expected in more
realistic patient-specific anatomies; a phenomenon also known as glottal jet skewing [44]. We briefly note that typically, limited velocity data are experimentally resolved in the vicinity
of the model’s walls (Fig 3a and 3d); this is a well-known limitation of PIV techniques and
generally due to the decreasing concentration or loss of tracer particles at the walls, in con-
junction with significant flow gradients resulting from the no-slip condition (and possibly
wall reflections) [46]. The glottal constriction has been known to represent a source of turbulent flow, despite the
relatively low Reynolds numbers (O(103)) [10]. Shear flows such as the glottal free jet can
reduce the critical Reynolds threshold and nevertheless generate turbulent kinetic energy
(TKE); a key characteristic associated with the formation of eddies and other coherent flow
structures used in classical turbulence analyses. In the examined cases, airflow enters the
mouth in the laminar regime at the low flow rate case (Re = 1’500). However, low levels of tur-
bulence develop downstream due to geometrical effects such as bends and constrictions. In
contrast, both for the intermediate and high flow rate cases (Re = 4’500 and Re = 7’000), air-
flow now enters the mouth under turbulent flow conditions. In Fig 3d–f, we plot mean flow
TKE contours along the mid-sagittal plane for the three modalities (TPIV, RANS and LES) at
the higher Reynolds number case (Re = 7’000). While the velocity magnitude contours agree
rather closely between all three modalities, we observe discrepences between TKE results. Numerical methods Comparison of 1D profiles of normalized time-averaged velocity
magnitudes (first column) and turbulent kinetic energy (second column), normalized by the mean inlet velocity uin, are presented at three stations (see
inset; top right panel) along the larynx (A-A’), glottis (B-B’) and upper trachea (C-C’), respectively, for two grid resolutions: (i) coarse (9.2M cells) and
(ii) fine (25M cells) https://doi.org/10.1371/journal.pcbi.1010537.g002 this manner, turbulent flow is sustained in the extended pipe section, and a turbulent velocity
profile enters the mouth inlet. At the outlet of the model (lower trachea), uniform pressure is
prescribed. A no-slip velocity condition is imposed on the airway walls. The governing equations are discretized using a finite volume method and solved using
OpenFOAM, an open-source CFD code [43]. The scheme is second-order accurate in both
space and time. To ensure numerical stability the final time step used is 5 × 10−7s. A total of
9.2M cells was used to have sufficient grid resolution for LES, based on a grid sensitivity analy-
sis (see S3 Fig). Specifically, normalised time-averaged velocity and turbulent kinetic energy
(TKE) predictions for two grid resolutions, namely coarse (9.2M cells) and fine (25M cells),
were compared at an inlet Re = 7’000. The comparisons of 1D velocity magnitude profiles at
three stations along the larynx (A-A’), glottis (B-B’) and upper trachea (C-C’) are shown in
Fig 2. We find very good agreement between the two grid resolutions, ensuring the adequacy
of the selected resolution for LES. 7 / 16 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537
March 23, 2023 PLOS COMPUTATIONAL BIOLOGY Correlation of three-dimensional turbulent flows in an idealized mouth-throat model Results and discussion
Glottal jet characteristics Firstly, maximum values of TKE differ in the downstream wake of the jet, at the shear interface
with the resting fluid; this observation was previously reported for example by Lin et al. [20]
in a patient-specific geometry and concurrently by Das et al. using the same identicl idealized
mouth-throat geometry [3]. For all three modalities, we observe a common feature with the presence of a thin streak of
maximum TKE values originating at the glottic that gradually widens and dissipiates down-
stream into the trachea. In both experiments and RANS, the shape appears to be in strong
agreement whereas for the LES the peak TKE streak is more underlined. However, the RANS
simulation reports more than *60% lower peak TKE values relative to both TPIV and LES,
while a background base level of TKE upstream of the glottis is resolved in the TPIV measure-
ment (i.e. 1–2 m/s) yet absent in RANS (i.e. baseline of near zero TKE) and much lower in LES
(i.e. <1 m/s). It is known that differences in TKE hold potential ramifications towards predic-
tions of inhaled aerosols dispersed in the lungs [8]. Notably, the significantly lower TKE levels
obtained with RANS are acknowledged to lead to overpredictions of particle deposition when
only the (time-averaged) RANS velocity field is used [23, 47]. Indeed, in regions where there
is significant large-scale anisotropy in turbulence, turbulent dispersion plays an important
role in particle transport and tends to decrease deposition. Hence, RANS are typically used
together with a turbulent dispersion model to provide improved deposition predictions. In PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537
March 23, 2023 8 / 16 PLOS COMPUTATIONAL BIOLOGY Correlation of three-dimensional turbulent flows in an idealized mouth-throat model Fig 3. Comparison of flow characteristics between tomographic PIV (TPIV), Reynolds Averaged Navier Stokes (RANS) and Large
Eddy Simulation (LES) for the high flowrate case (Re = 7’000). Top row: Velocity magnitude contours along the mid-sagittal plane shown
along with several orthogonal transverse planes to illustrate 3D characteristics of the flow. Bottom row: Turbulent kinetic energy (TKE)
contours are plotted on the mid-sagittal plane. Results are generally in good agreement with strong similarity but derivative TKE values
reveal more subtle differences. Specifically, RANS underestimates the peak TKE values near the jet’s wake while the TPIV data introduce
background noise. https://doi.org/10.1371/journal.pcbi.1010537.g003 Fig 3. Secondary flows In a next step, we compare ensuing secondary flows downstream of the glottal constriction. Fig 5 plots velocity magnitude contours and velocity vectors for each of the three modalities
and flow rates, resulting in a 3 by 3 matrix. Similarly, as seen along the mid-sagittal plane (Fig
4), the basic flow features are largely invariant with respect to Re variation, whereas flow mag-
nitudes vary as anticipated with higher Re number. Here, we observe a pair of counter-rotating vortices classically refered to as Dean vortices,
originating from the curvature of the laryngeal geometry. The Dean number is defined as
Dn = Re
ffiffiffiffiffiffiffiffiffiffiffi
D=2r
p
, where r denotes the curvature radius and D the cross-sectional diameter of
the airway. In our analysis, the Dean number changes only with Re because of the statically
defined geometry (i.e., r and D are constants). Therefore our measurements of increasingly
stronger Dean vortex flow correlate with increased Re, as plotted in Fig 5. We note here that
the cross-section in 2D appears rectangular, in contrast to the circular cross-sections in the
numerical simulations (RANS and LES); a consequence of the volume illumination technique
which involves knife-edges to form the laser light into a prism (see Fig 1, and subsequent post-
processing masking steps that are performed in 2D. Results and discussion
Glottal jet characteristics With such differences accounted for, we nevertheless consitently 10 / 16 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537
March 23, 2023 PLOS COMPUTATIONAL BIOLOGY Correlation of three-dimensional turbulent flows in an idealized mouth-throat model observe the common laryngeal jet structure described above and present across all flowrates,
underlining how the jet phenomenon scales consistently. In the low flowrate case (Re = 1’500)
plotted in Fig 4a–c, we observe the weakest jet, with peak velocity values of *2 m/s and a *1
m/s upstream and downstream flow, with near zero counter flow at the shear interface. For the
intermediate flow rate (Re = 4’500) plotted in Fig 4d–f, we observe the same jet structure, with
higher peak velocity values (*5 m/s) and the same near zero counter flow to the right of the
shear interface. Lastly, we plot the high flow rate case (Re = 7’000) in Fig 4g–i, with the highest
peak velocity values at >9 m/s. We note that the near zero counter flow to the right of the
shear interface is nearly identical between the mid and high Re cases, and slightly lower in the
low Re case. While the three modalities agree well for the low Re case, we observe in the mid
and high Re flows that the RANS simulations does not capture the zero-flow interface as well,
which also explains the lower TKE values discussed earlier in Fig 3e, as a common source of
energy for turbulent velocity fluctuations lies in the presence of shear in the mean flow. Results and discussion
Glottal jet characteristics Comparison of flow characteristics between tomographic PIV (TPIV), Reynolds Averaged Navier Stokes (RANS) and Large
Eddy Simulation (LES) for the high flowrate case (Re = 7’000). Top row: Velocity magnitude contours along the mid-sagittal plane shown
along with several orthogonal transverse planes to illustrate 3D characteristics of the flow. Bottom row: Turbulent kinetic energy (TKE)
contours are plotted on the mid-sagittal plane. Results are generally in good agreement with strong similarity but derivative TKE values Fig 3. Comparison of flow characteristics between tomographic PIV (TPIV), Reynolds Averaged Navier Stokes (RANS) and Large
Eddy Simulation (LES) for the high flowrate case (Re = 7’000). Top row: Velocity magnitude contours along the mid-sagittal plane shown
along with several orthogonal transverse planes to illustrate 3D characteristics of the flow. Bottom row: Turbulent kinetic energy (TKE)
contours are plotted on the mid-sagittal plane. Results are generally in good agreement with strong similarity but derivative TKE values
reveal more subtle differences. Specifically, RANS underestimates the peak TKE values near the jet’s wake while the TPIV data introduce
background noise https://doi.org/10.1371/journal.pcbi.1010537.g003 contrast, since LES resolve the large scale eddies, these do not need an additional dispersion
model when addressing particle-laden flows [48]. Next, we compare measurements for the low (Re = 1’500) and intermediate (Re = 4’500)
flow rate cases, plotted as velocity magnitude contours overlaid with velocity vectors in Fig 4. 9 / 16 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537
March 23, 2023 PLOS COMPUTATIONAL BIOLOGY Correlation of three-dimensional turbulent flows in an idealized mouth-throat model Fig 4. Flow characterization of the laryngeal jet variation on the mid-sagittal plane in the region of interest (ROI). Results are presented as a function of the inlet Reynolds number for TPIV, RANS and LES, respectively. https://doi.org/10.1371/journal.pcbi.1010537.g004 Fig 4. Flow characterization of the laryngeal jet variation on the mid-sagittal plane in the region of interest (ROI). Results are presented as a function of the inlet Reynolds number for TPIV, RANS and LES, respectively. https://doi.org/10.1371/journal.pcbi.1010537.g004 https://doi.org/10.1371/journal.pcbi.1010537.g004 We briefly note that a small size deviation in the width of the experimental model relative to
the numerical ones shown (i.e. a deviation on the order of <5%), likely introduced by the
volume calibration step and the physical compliance of the silicone phantom model itself
(and also seen in Fig 3d–f). One-dimensional velocity curves In a final step, we compare detailed flow characteristics between the experimental and numeri-
cal approaches via the simultaneous plotting of 1D velocity magnitude curves. To this end, we
first identify the mid-sagittal plane that bisects the geometry (see dashed-dot line in Fig 6a)
and the region imaged via TPIV experiments (see Figs 1 and S1). Positioned in this orienta-
tion, the mouth inlet is viewed in the normal direction, with a smaller isometric view given as
a reference. Four transverse lines (labeled A through D) spanning the mid-sagittal plane are
chosen for plotting 1D velocity curves, as shown in Fig 6b. Good agreement is observed between all three modalities (i.e, TPIV, RANS and LES) for
each of the four lines, with variations slightly more pronounced in Line A due to the shorter
y-axis range relative to Lines B-D. We observe that TPIV and LES velocity magnitude curves
along line A are very similar (i.e. LES exceeds the TPIV values by a maximum of 1–2%), char-
acterized by asymmetric twin peaks, with the higher peak on the left side of the dimensionless
x-axis. The RANS curve, by contrast, features a more symmetric pair of peaks and deviates
from the TPIV measurements by <4%. For lines B-D, excellent agreement (<1% deviation) is
observed between all modalities. 11 / 16 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537
March 23, 2023 PLOS COMPUTATIONAL BIOLOGY Correlation of three-dimensional turbulent flows in an idealized mouth-throat model C
l
i
Fig 5. Flow characterization of the laryngeal jet with Reynolds variation on a transverse plane bisecting the jet’s wake. Results are presented
as a function of the inlet Reynolds number for TPIV, RANS and LES, respectively and exemplifies reconstructed flow streamlines in the 2D cut
plane (see Line C in Fig 6a for the location of the cut plane). https://doi.org/10.1371/journal.pcbi.1010537.g005 Fig 5. Flow characterization of the laryngeal jet with Reynolds variation on a transverse plane bisecting the jet’s wake. Results are presented
as a function of the inlet Reynolds number for TPIV, RANS and LES, respectively and exemplifies reconstructed flow streamlines in the 2D cut
plane (see Line C in Fig 6a for the location of the cut plane). https://doi.org/10.1371/journal.pcbi.1010537.g005 https://doi.org/10.1371/journal.pcbi.1010537.g005 Conclusion The present work has been motivated by the ongoing need for experimentally-resolved 3D
flow data to improve the valididty and accuracy of computational fluid dynamics (CFD) simu-
lations resolving turbulent airflows in the upper and extra-thoracic airways towards various Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537
March 23, 2023 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537
March 23, 2023 12 / 16 PLOS COMPUTATIONAL BIOLOGY Correlation of three-dimensional turbulent flows in an idealized mouth-throat model |u| (m/s)
|u| (m/s)
|u| (m/s)
|u| (m/s)
2.5
2.3
2.9
2.7
3.1
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non-dimensional axial position
0
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RANS
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Line A
Line B
Line C
Line D
(a)
(b)
Line A
Line B
Line C
Line D
Midsagittal plane
Isometric view
10 mm
5 mm
Fig 6. 1-dimensional velocity magnitude curves for Re = 7’000 case. (a): Schematic of the full mouth-throat model
geometry (see isometric view) used in the fabrication of the experimental phantom and computational mesh. The area
illuminated via laser (Region of Interest) and imaged using tomographic particle image velocimetry (TPIV) is noted,
along with four lines (labeled A-D) spanning the mid-sagittal plane. (b) Comparison of 1D velocity profiles along the
anotated Lines A-D. https //doi org/10 1371/jo rnal pcbi 1010537 g006 |u| (m/s)
|u| (m/s)
|u| (m/s)
|u| (m/s)
2.5
2.3
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3.1
3.3
0
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0
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RANS
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Line A
Line B
Line C
Line D
(b) (a)
(
Line A
Line B
Line C
Line D
Midsagittal plane
Isometric view
10 mm
5 mm Fig 6. 1-dimensional velocity magnitude curves for Re = 7’000 case. (a): Schematic of the full mouth-throat model
geometry (see isometric view) used in the fabrication of the experimental phantom and computational mesh. The area
illuminated via laser (Region of Interest) and imaged using tomographic particle image velocimetry (TPIV) is noted,
along with four lines (labeled A-D) spanning the mid-sagittal plane. (b) Comparison of 1D velocity profiles along the
anotated Lines A-D. https://doi.org/10.1371/journal.pcbi.1010537.g006 https://doi.org/10.1371/journal.pcbi.1010537.g006 https://doi.org/10.1371/journal.pcbi.1010537.g006 inhalation therapy and therapeutic or toxic dosimetry applications. Conclusion To the best of our knowl-
edge, the findings presented herein are the first detailed 3D in vitro–in silico correlations of
respiratory airflows in a benchmark anatomical mouth-throat model. We find that a RANS k-
ω SST model adequately predicts velocity flow patterns for Re numbers spanning 1’500 to
7’000, supporting results in close proximity to a more computationally-costly LES model. Yet,
RANS significantly underestimates turbulent kinetic energy (TKE), thus demonstrating the
superiority of LES as a higher-order turbulence modeling scheme. With a keen eye on end-
user applications across various respiratory disciplines, researchers can leverage such valida-
tion data in conjunction with open-access files (see SM) for improved predictive CFD models. Supporting information S1 Fig. Tomographic particle image velocimetry (TPIV) experimental setup. A: Photo-
graphed in the lab B: a schematic illustration. (EPS) Author Contributions Data curation: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis. Formal analysis: Saurabh Bhardwaj, Pantelis Koullapis. Investigation: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis, Josue´ Sznitman. Methodology: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis, Ron Bessler. Project administration: Eliram Nof, Josue´ Sznitman. Resources: Stavros Kassinos. Supervision: Stavros Kassinos, Josue´ Sznitman. Validation: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis. Validation: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis. Visualization: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis. Writing – original draft: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis, Josue´ Sznitman. Writing – review & editing: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis, Stavros Kassi-
nos, Josue´ Sznitman. Writing – review & editing: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis, Stavros Kassi-
nos, Josue´ Sznitman. S3 Fig. Large eddy simulation (LES) mesh refinement and model scheme comparison.
(EPS) 13 / 16 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1010537
March 23, 2023 PLOS COMPUTATIONAL BIOLOGY Correlation of three-dimensional turbulent flows in an idealized mouth-throat model S1 Data. Mouth-throat geometry STL file. (STL)
S2 Data. Modified mouth-throat geometry file used in 3D printing the mold for fabricat-
ing the silicon phantom. (STL) S1 Data. Mouth-throat geometry STL file. (STL)
S2 Data. Modified mouth-throat geometry file used in 3D printing the mold for fabricat-
ing the silicon phantom. (STL) Author Contributions
Conceptualization: Eliram Nof, Josue´ Sznitman. Data curation: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis. Formal analysis: Saurabh Bhardwaj, Pantelis Koullapis. Funding acquisition: Josue´ Sznitman. Investigation: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis, Josue´ Sznitman. Methodology: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis, Ron Bessler. Project administration: Eliram Nof, Josue´ Sznitman. Resources: Stavros Kassinos. Supervision: Stavros Kassinos, Josue´ Sznitman. Validation: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis. Visualization: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis. Writing – original draft: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis, Josue´ Sznitman
Writing – review & editing: Eliram Nof, Saurabh Bhardwaj, Pantelis Koullapis, Stavros Kass
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TIP30 regulates lipid metabolism in hepatocellular carcinoma by regulating SREBP1 through the Akt/mTOR signaling pathway
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Oncogenesis
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cc-by
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1PLA General Hospital Cancer Center Key Lab, PLA Postgraduate School of Medicine, Beijing, China; 2Molecular Pathology Laboratory, College of Basic Medicine, Inner Mongolia
Medical University, Hohhot, China; 3Department of Orthopedics, PLA General Hospital, Beijing, China; 4International Joint Cancer Institute, The Second Military Medical University,
Shanghai, China; 5Changhai Hospital, The Second Military Medical University, Shanghai, China and 6Shanghai University Of Medicine & Health Sciences, Shanghai, China.
Correspondence: Dr W Jing, Changhai Hospital, The Second Military Medical University, Changhai Road, Shanghai 200433, China.
E-mail: jingwei7777@163.com
or Professor J Zhao, International Joint Cancer Institute, The Second Military Medical University, 800 Xiangyin Road, Library Building 9-11th Floor, Shanghai 200433, China.
E-mail: zhaojian@smmu.edu.cn
7These authors contributed equally to this work.
Received 24 October 2016; revised 18 March 2017; accepted 2 May 2017 TIP30 regulates lipid metabolism in hepatocellular carcinoma
by regulating SREBP1 through the Akt/mTOR signaling F Yin1,7, G Sharen1,2,7, F Yuan3,7, Y Peng1, R Chen4, X Zhou5, H Wei1, B Li4, W Jing5 and J Zhao1,4,6 Lipid reprogramming has been considered as a crucial characteristic in hepatocellular carcinoma (HCC) initiation and progression. However, detailed molecular mechanisms have yet to be clearly defined. Here, we examined the effects of tumor suppressor TIP30 on
the regulation of HCC lipid metabolism. We found that decreased TIP30 expression leads to elevated fatty acid synthesis and enhanced
levels of lipogenic enzymes SCD and FASN in HCC cells. Moreover, SREBP1 is one of the key transcription factors regulating liver lipid
metabolism, and TIP30 deficiency significantly increased SREBP1 expression and nuclear accumulation. Small interfering RNAs
targeting SREBP1 could reverse fatty acid synthesis induced by TIP30 deficiency. Furthermore, downregulating TIP30 activated the
Akt/mTOR signaling pathway to upregulate SREBP1 expression, which promoted lipid metabolism by activating gene transcription of
lipogenesis, including fasn and scd. We also showed that TIP30 deficiency-regulated lipid metabolism promoted proliferation of HCC
cells. Clinically, our data revealed that TIP30 expression significantly correlated with SREBP1 in patients with HCC and that a
combination of TIP30 and SREBP1 is a powerful predictor of HCC prognosis. Together, our data suggested a novel function of TIP30 in
HCC progression and indicate that TIP30 regulation of SREBP1 may represent a novel target for HCC treatment. Oncogenesis (2017) 6, e347; doi:10.1038/oncsis.2017.49; published online 12 June 2017 OPEN OPEN OPEN Citation: Oncogenesis (2017) 6, e347; doi:10.1038/oncsis.2017.49 www.nature.com/oncsis 1PLA General Hospital Cancer Center Key Lab, PLA Postgraduate School of Medicine, Beijing, China; 2Molecular Pathology Laboratory, College of Basic Medicine, Inner Mongolia
Medical University, Hohhot, China; 3Department of Orthopedics, PLA General Hospital, Beijing, China; 4International Joint Cancer Institute, The Second Military Medical University,
Shanghai, China; 5Changhai Hospital, The Second Military Medical University, Shanghai, China and 6Shanghai University Of Medicine & Health Sciences, Shanghai, China.
Correspondence: Dr W Jing, Changhai Hospital, The Second Military Medical University, Changhai Road, Shanghai 200433, China.
E mail: jingwei7777@163 com INTRODUCTION metastatic human variant cells and less metastatic classic cells.6
Subsequently, TIP30 was found to be downregulated in various
tumors and is considered to be a tumor suppressor due to its
pro-apoptotic activity and its anti-metastatic and anti-angiogenic
capacities.7–11 Our previous research reported that TIP30-regulated
tumor metastasis and chemoresistance in various cancers.12–18
Moreover, we also found downregulated TIP30 induces epithelial–
mesenchymal transition in HCC and pancreatic cancer.19,20 The morbidity and mortality of hepatocellular carcinoma (HCC)
ranks top5 and top3 respectively among common malignant
tumors worldwide. The poor outcomes of HCC patients are mainly
due to high recurrence of HCC after surgery and resistance to
chemotherapy.1 Meanwhile, sorafenib was used as first line
targeted drugs in treating advanced HCC, which can only prolongs
the survival period of less than 3 months.2 Consequently, to
elaborate the pathogenesis and progression of HCC and to
develop new therapeutic strategies seem extremely crucial. y
Considering the crucial role of lipid metabolic reprogramming
in cancer development, identifying new molecules and pathways
that are involved in this process is vital. Recently, TIP30 has been
preliminarily revealed to affect fatty acid storage and oxidation in
hepatocytes21 and we will extensively investigate the role of TIP30
in lipid metabolism deregulation of HCC. We indicate decreased
TIP30 promotes lipid metabolism via Akt/mTOR/SREBP1 signaling
and that the combination of TIP30 and SREBP1 is an effective
predictor for HCC prognosis. Major risk factors of HCC are viral hepatitis, exposure to
hepatotoxins and alcohol abuse, whereas recent clinic and
epidemiology
researches
indicate
nonalcoholic
fatty
liver
disease (NAFLD) increases HCC incidence.3 Recently, metabolic
reprogramming,
especially
lipid
metabolism
alteration,
is
considered to be the initiating factor of tumor occurrence
and progression. Continuous de novo cholesterogenesis and
lipogenesis are frequently activated in tumors for providing extra
lipids and lipid precursors during rapid cell proliferation.4 Several
key enzymes have been identified to promote de novo lipid
synthesis, including fatty acid synthase (FASN), stearoyl-CoA
desaturase (SCD) and acetyl-CoA carboxylase (ACC).5 However,
detailed mechanisms of abnormal lipid metabolism have not yet
been comprehensively identified during HCC progression. nt Cancer Institute, The Second Military Medical University, 800 Xiangyin Road, Library Building 9-11th Floor, Shanghai 200433, China. International Joint Cancer Institute, The Second Military Medical University, 800 Xiangyin Road, Library Building 9-11th Floor, Shangh
mmu.edu.cn E mail: jingwei7777@163.com
or Professor J Zhao, International Joint Cancer Institute, The Second Military Medical University, 800 Xiangyin Road, Library Building 9-11th Floor, Shanghai 200433, China.
E-mail: zhaojian@smmu edu cn RESULTS TIP30 is a negative regulator of lipid metabolism in HCC cells
To determine whether TIP30 regulates lipid metabolism of HCC
cells, microarray analysis was firstly applied for comparing gene
expression profiles of HCC-LM3 infected with shNon or shTip30. 837 genes were differentially expressed upon TIP30 knockdown
(fold change ⩾2, Po0.05). Through GO and KEGG enrichment y
TIP30, namely HTATIP2 or CC3, was firstly discovered in small cell
lung
carcinoma,
using
differential
analyses
between
highly TIP30 regulates lipid metabolism in HCC
F Yin et al TIP30 regulates lipid metabolism in HCC 2 analysis,
these
genes
were
enriched
in
several
biological
pathways. The results indicated that genes that significantly
correlated to TIP30 expression were involved in fatty acid
metabolism (Supplementary Figure 1A), which suggested the
important role of TIP30 involved in lipid metabolism regulation. Furthermore, effects of TIP30 on cellular lipid levels were explored
in HCC-LM3, SMMC-7721 and HepG2 cell lines using the lipophilic
dye BODIPY 493/503. We employed lentiviruses to knockdown or
overexpress TIP30 expression in HCC cell lines, and the lentiviral
infection efficiency is showed in Figure 1d. We observed that
silencing TIP30 led to increased levels of neutral lipid staining in
HCC-LM3 and SMMC-7721 cells, whereas the staining was
significantly decreased in TIP30-overexpressed cells (Figure 1a). Similar results were obtained when the lipid accumulation inducer
oleate was added to the culture medium (Supplementary
Figure 1B). Consistently, intracellular triglyceride levels were
further determined to support the negative effects of TIP30
on lipid metabolism regulation (Figure 1b). Downregulating TIP30 enhances SREBP1 expression by activating
the Akt/mTOR signaling pathway Loss of TIP30 can activate EGFR/AKT signaling of human lung
adenocarcinoma and mammary cancer.25,26 We have previously
confirmed that TIP30 deficiency can activate AKT signaling in HCC
and laryngeal carcinoma.16,20 Previous studies have indicated
activation of AKT/mTOR signaling had critical effects on lipid
metabolism regulation.27,28 Here, we find downregulation of TIP30
expression activates AKT and causes elevated mTOR phosphoryla-
tion in HCC-LM3 cells, whereas up-regulating TIP30 expression
reduced AKT and mTOR phosphorylation levels (Figure 3a). Additionally, we found that blockade of AKT by its inhibitor,
MK-2206, dramatically attenuated the TIP30 deficiency-induced
up-regulation of p-mTOR and SREBP1 levels (Figure 3b). Moreover,
MK-2206 could reverse the upregulated FASN and SCD expression
in TIP30-deficient HCC-LM3 cells (Figure 3c). Intracellular triglycer-
ide levels and the intensity of BODIPY staining were also reduced
after MK-2206 treatment (Figures 3d and e). SREBP1 is essential for TIP30 deficiency-mediated lipogenesis-
promoting effects The previous analysis showed that SREBPs (sterol regulatory
element-binding proteins) were critical transcription factors that
control lipogenesis and lipid uptake.22 SREBPs firstly located in
endoplasmic reticulum membrane, which was considered as its
inactive precursors. Once the sterol levels drop, SREBPs transferred
from endoplasmic reticulum to Golgi apparatus, where mature
forms were released by proteases (site-1 and site-2). Thereafter,
mature SREBPs entered nucleus to bind SRE-containing gene
promoters to induce transcription.23 Emerging evidence indicates
that SREBP1 is a crucial linkage of oncogenic signaling transduc-
tion and cancer metabolism.24 To elucidate the molecular
mechanisms
of
the
TIP30
deficiency-mediated
upregulation
of lipogenic enzymes, cellular SREBP1 levels were examined. qRT–PCR and western blot analyses revealed that SREBP1
mRNA levels were significantly increased after TIP30 knockdown
(Figures 1c and 2a). Additionally, immunostaining showed nuclear
accumulation
of SREBP1 with TIP30 deficiency (Figure 2b;
Supplementary Figure 2B). Meanwhile, overexpression of TIP30
leaded to adverse effects on SREBP1 levels in HCC cells (Figures 1c
and 2a,b). SREBP1 expression was reduced in TIP30 knockdown
cells which TIP30 are re-expressed in (Supplementary Figure 2A). We then used small interfering RNAs (siRNAs) targeting SREBP1 to
explore whether TIP30 deficiency upregulated lipogenic enzyme
expression through SREBP1. SREBP1 depletion of TIP30-deficient
HCC-LM3 and SMMC-7721 cells leaded to decreased expression of
FASN and SCD (Figure 2c). Moreover, intracellular triglyceride
levels and the intensity of BODIPY staining were remarkably
reduced by siSREBP1 in TIP30-deficient HCC cells (Figure 2d;
Supplementary Figure 2C). These results demonstrate that SREBP1
is involved in TIP30 deficiency related lipogenesis-promoting
effects. Decreased TIP30 is associated with elevated SREBP1 levels in HCC
samples, and combinational biomarkers provide powerful
prognostic value for HCC patients To better understand the correlation between TIP30 and SREBP1
expression, immunohistochemical staining of 80 clinical HCC
samples was performed. SREBP1 protein levels in HCC tissues
negatively correlated with TIP30 expression (r = −0.473, Po0.001)
(Figures 5a and b), suggesting that SREBP1 may be upregulated by
TIP30 deficiency in HCC. We then measured TIP30 and SREBP1
mRNA levels in 30 HCC tissue samples investigated to provide
further support for our research. Results showed the negative
association(r = −0.37, P = 0.039) between TIP30 and SREBP1 mRNA
levels (Figure 5c). Using NCBI GEO databases to analyze the HCC
sample array (GEO dataset accession GSE36376),29 we also found
the negative correlation of TIP30 and SREBP1 in 240 HCC samples
(r = −0.24, Po0.001), showing the same tendency as our results
(Supplementary Figure 3). RESULTS These data show
that AKT/mTOR/SREBP1 signaling is required for TIP30 to regulate
lipid metabolism in HCC cells. p
g
g
To well explain the mechanisms of TIP30 regulating de novo
lipid
synthesis,
we
analyzed
lipogenesis-related
enzymes
(FASN, SCD, and ACC) levels of HCC cells with different TIP30
expressions. Results demonstrated both the mRNA and protein
levels of SCD and FASN notablely elevated in TIP30-depleted cells,
whereas overexpression of TIP30 reduced SCD and FASN levels
comparing to control (Figures 1c and d). However, ACC expression
remained unchanged with TIP30 knockdown or overexpression
(Figure 1c). As fatty acid oxidation (FAO) played an important role
in lipid metabolism reprogramming in several types of cancer, we
also analyzed the levels of critical FAO-related factors, including
CPT1A and ACOX1. However, TIP30 exerted no effects on these
two oxidative enzymes in HCC cells (Figure 1c). Taken together,
our research indicates TIP30 can regulate de novo fatty acid
synthesis of HCC cells. Decreased TIP30 promotes the proliferation of HCC cells via
SREBP1-related lipid metabolism in vitro and in vivo Given important effects of lipid metabolism on tumor progression,
we examined whether TIP30-regulated HCC cell growth via SREBP1-
mediated lipid metabolism. As shown in Figure 4a, depletion of
SREBP1 signaling with siRNA inhibited TIP30 deficiency-induced
enhanced HCC-LM3 and SMMC-7721 cell growth. Using colony-
forming assays, we found that decreasing the TIP30 deficiency-
induced HCC cell colony formation was also dependent on SREBP1
(Figure 4b). To better understand the role of lipid metabolism
reprogramming in TIP30-regulated HCC growth in vivo, xenograft
studies were performed. We established stable HCC-LM3 cell lines
with both TIP30 and SREBP1 deficiency and the corresponding cells
were subcutaneously injected into 4 weeks old BALB/c nude mice. Results showed decreased SREBP1 significantly abolished the
accelerated
tumor
growth
of
TIP30-deficient
HCC-LM3
cells
(Figures 4c and d). Interestingly, the levels of triglycerides were
increased in the tumor tissues with reduced TIP30 expression,
whereas silencing SREBP1 reversed this effect (Figure 4e), further
supporting the conclusion that lipid metabolism deregulation
contributed to HCC cell growth. Meanwhile, immunohistochemical
staining showed tumors originated from TIP30-silenced HCC-LM3
cells exhibited increased SREBP1, SCD and FASN expression
(Figure 4f), which was coincident with results obtained in HCC cell
lines in vitro. Taken together, our results suggest that TIP30 can
modulate SREBP1-related lipid metabolism, which contributes to
tumor growth in HCC. SREBP1 is essential for TIP30 deficiency-mediated lipogenesis-
promoting effects SREBP1 is essential for TIP30 deficiency-mediated lipogenesis-
promoting effects Meanwhile, patients with low TIP30 Oncogenesis (2017), 1 – 9 Figure 1. TIP30 negatively regulates lipid metabolism of hepatocellular cancer cell lines. (a) HCC cells (HCC-LM3, SMMC-7721 and HepG2) we
infected with lentivirus to knockdown or overexpress TIP30. BODIPY 493/503 (green staining) was used to stain neutral lipids of each cell. DA
(blue staining) was used to stain nuclear. (magnification, × 200). For quantification of the mean integrated optical density (IOD) of BODIP
staining, Image J software was applied and values were analyzed with unpaired t-test. (b) Intracellular triglyceride levels were detected in eac
cell, with or without lipid accumulation inducer, oleate (0.05 mM), added in the culture medium. (c) qRT–PCR methods were applied to te
mRNA levels of lipogenic enzymes (ACC, FASN, SCD and SREBP1) and fatty acid oxidation enzymes (ACOX1 and CPT1A) in the indicated cel
(d) Western blot was applied to detect the SCD and FASN expression in each cell. Protein levels of TIP30 were also detected to determine th
efficiency of lentivirus infection. *Po0.05. TIP30 regulates lipid metabolism in HCC
F Yin et al TIP30 regulates lipid metabolism in HCC
F Yin et al Figure 1. TIP30 negatively regulates lipid metabolism of hepatocellular cancer cell lines. (a) HCC cells (HCC-LM3, SMMC-7721 and HepG2) were
infected with lentivirus to knockdown or overexpress TIP30. BODIPY 493/503 (green staining) was used to stain neutral lipids of each cell. DAPI
(blue staining) was used to stain nuclear. (magnification, × 200). For quantification of the mean integrated optical density (IOD) of BODIPY
staining, Image J software was applied and values were analyzed with unpaired t-test. (b) Intracellular triglyceride levels were detected in each
cell, with or without lipid accumulation inducer, oleate (0.05 mM), added in the culture medium. (c) qRT–PCR methods were applied to test
mRNA levels of lipogenic enzymes (ACC, FASN, SCD and SREBP1) and fatty acid oxidation enzymes (ACOX1 and CPT1A) in the indicated cells. (d) Western blot was applied to detect the SCD and FASN expression in each cell. Protein levels of TIP30 were also detected to determine the
efficiency of lentivirus infection. *Po0.05. Oncogenesis (2017), 1 – 9 TIP30 regulates lipid metabolism in HCC
F Yin et al l
d h h
l
l
h b
d h
fi
d
d
d
igure 2. SREBP1 is essential for TIP30 deficiency-mediated lipogenesis-promoting effects. SREBP1 is essential for TIP30 deficiency-mediated lipogenesis-
promoting effects (a) Western blot was performed for detecting
cytosolic precursor (P) and nucleic mature (M) forms of SREBP1 in indicated HCC cells lysates. (b) Results of immunofluorescence analysis were
howed in indicated HCC cells. Red staining represented SREBP1 protein. DAPI (blue staining) was used to stain nuclear. (magnification, × 200
For quantification of the mean integrated optical density (IOD) of BODIPY staining, Image J software was applied and values were analyzed
with unpaired t-test. (c) Indicated protein levels were detected in HCC-LM3-shTip30 and SMMC-7721-shTip30 cells transfected with si-SREBP
si-SREBPl-1 or si-SREBPl-2) or si-NC using western blot. (d) Intracellular triglyceride levels were detected in cells as decreased in c. *Po0.05
F Yin et al 4 Figure 2. SREBP1 is essential for TIP30 deficiency-mediated lipogenesis-promoting effects. (a) Western blot was performed for detecting
cytosolic precursor (P) and nucleic mature (M) forms of SREBP1 in indicated HCC cells lysates. (b) Results of immunofluorescence analysis were
showed in indicated HCC cells. Red staining represented SREBP1 protein. DAPI (blue staining) was used to stain nuclear. (magnification, × 200)
For quantification of the mean integrated optical density (IOD) of BODIPY staining, Image J software was applied and values were analyzed
with unpaired t-test. (c) Indicated protein levels were detected in HCC-LM3-shTip30 and SMMC-7721-shTip30 cells transfected with si-SREBPl
(si-SREBPl-1 or si-SREBPl-2) or si-NC using western blot. (d) Intracellular triglyceride levels were detected in cells as decreased in c. *Po0.05. TIP30 was first discovered as a metastasis suppressor in 1997 and
as a tumor suppressor in 2003.6,7 The tumor suppressor function of
TIP30 has been extensively demonstrated in various types of human
tumors, including HCC. TIP30 exerts its tumor-suppressive role by
influencing cell apoptosis, growth, metastasis and angiogenesis.32
Recently, TIP30 has also been confirmed to regulate the metabolic
adaptation to glucose limitation of HeLa cells, which contributes to
tumor metastasis and aggressiveness.33 For the first time, our study
demonstrated that TIP30 is a negative regulator of lipid metabolism
in HCC. We also demonstrated that decreased TIP30 may facilitate
lipid metabolism through the AKT/mTOR/SREBP1 signaling pathway
to promote tumor growth in HCC. levels and high SREBP1 levels exhibited the poorest recurrence-
free survival (RFS) as well as overall survival (OS), indicating that
combinational detection of the two molecules may have a
powerful prognostic value (Figures 5d and e). Oncogenesis (2017), 1 – 9 DISCUSSION chemoresistance
and
self-renewal
of
laryngeal
carcinoma.16
Additionally, downregulation of TIP30 could activate AKT to regulate
the levels of epithelial–mesenchymal transition related transcription
factors in HCC-LM3 cells.20 mTOR activation by Akt contributes to
regulation of de novo lipogenesis. Through up-regulating SREBP1
transcription, processing and nucleic accumulation, mTOR signaling
senses nutrients for growth and accelerates de novo lipogenesis.42
In particular, Calvisi has reported that AKT-mTORC1 signaling-
induced lipogenesis accelerated HCC development from transcrip-
tional and post-transcriptional aspects, including downregulation of
FASN ubiquitination and interruption of SREBPs degradation.40
Consistently, in our study, SREBP1 was revealed to be upregulated
by TIP30 deficiency-mediated Akt/mTOR activation. Meanwhile,
CD147 has been reported to form a complex with integrinβ to
activate PI3K/Akt pathway and then reprogram lipid metabolism
through Akt/mTOR/SREBP1 signaling in HCC.43,44 As a cancer-
associated biomarker for detection and an effective target for
treatment, CD147 also forms complexes with CD44 and EGFR to
induces EGFR downstream signaling (ERK, pSTAT3) in breast cancer
and pancreatic cancer.45,46 Also, loss of TIP30 can improve EGFR
activity
in
various
tumors
and
Tip30
knockout
in
primary
hepatocytes of mouse leads to trapping of EGF-EGFR complex,
which contributes to prolonged EGFR signaling.25,26,41,47 Consider-
ing both TIP30 and CD147 could regulate EGFR related signaling, it
would be interesting to figure out whether downregulated TIP30
expression may synergistically act with increased CD147 expression
in HCC in future research. cells during their rapid growth and proliferation.34 Thus, several
key lipogenic enzymes are activated to increase de novo
lipogenesis
of
cancer
cells. SREBP1-regulated
downstream
lipogenic enzymes (FASN and SCD), have been confirmed to be
elevated in various tumors.35,36 Consistently, our results demon-
strated that TIP30 deficiency could promote the lipid synthesis
of HCC cells via the up-regulation of FASN and SCD. We also
found that the mRNA levels of two oxidative enzymes (CPT1A and
ACOX1) were not affected by TIP30 in HCC cells. However, a recent
report
preliminarily
suggested
that
TIP30-regulated
fatty
acid oxidations in normal hepatocytes by evaluating the CO2
production of cells labeled with [14C] palmitate.21 Considering
the different cell lines and research methods applied in these
data, the role of TIP30 in fatty acid oxidations of HCC needs
further evaluation from transcriptional and post-transcriptional
regulation. As the main regulator of hepatic lipogenesis, SREBP1 is highly
activated in cancers and activates the fatty acid pathway in human
HCC cell lines. DISCUSSION Increasing evidences showed lipid metabolism was a key player
in tumor growth, metastasis and resistance to therapies. As the
main metabolic organ, liver is crucial for carrying out lipid
metabolism, and aberrant activation of lipogenesis has been
considered as an oncogenic event in human HCC.30,31 In the
present research, we evaluated whether TIP30 participates in
abnormal lipid metabolism of HCC. Increasing evidences showed lipid metabolism was a key player
in tumor growth, metastasis and resistance to therapies. As the
main metabolic organ, liver is crucial for carrying out lipid
metabolism, and aberrant activation of lipogenesis has been
considered as an oncogenic event in human HCC.30,31 In the
present research, we evaluated whether TIP30 participates in
abnormal lipid metabolism of HCC. Normal tissues often utilize circulating lipids, while more than
90% of fatty acids are produced from de novo synthesis in tumors TIP30 regulates lipid metabolism in HCC
F Yin et al gure 3. Downregulating TIP30 enhanced SREBP1 expression through activating Akt/mTOR signaling. (a) Western blotting showed p-AKT and
mTOR levels of HCC-LM3 cells infected with shNon, shTip30, LV-Non and LV-Tip30, respectively. (b) HCC-LM3-shNon and HCC-LM3-shTip30
ere treated with AKT inhibitor MK-2206 (0.5 μM) for 24 h, indicated protein expressions were analyzed using western blot. (c) MK-2206
.5 μM) was added in HCC-LM3-shTip30 cells. After 24 h of the treatment, mRNA levels of TIP30 and lipogenic enzymes were tested using
RT–PCR. BODIPY 493/503 staining (magnification, × 200) (d) and intracellular triglyceride levels (e) were performed in cells as decreased in c. or quantification of the mean integrated optical density (IOD) of BODIPY staining, Image J software was applied and values were analyzed
th unpaired t-test. *Po0.05. 5 Figure 3. Downregulating TIP30 enhanced SREBP1 expression through activating Akt/mTOR signaling. (a) Western blotting showed p-AKT and
p-mTOR levels of HCC-LM3 cells infected with shNon, shTip30, LV-Non and LV-Tip30, respectively. (b) HCC-LM3-shNon and HCC-LM3-shTip30
were treated with AKT inhibitor MK-2206 (0.5 μM) for 24 h, indicated protein expressions were analyzed using western blot. (c) MK-2206
(0.5 μM) was added in HCC-LM3-shTip30 cells. After 24 h of the treatment, mRNA levels of TIP30 and lipogenic enzymes were tested using
qRT–PCR. BODIPY 493/503 staining (magnification, × 200) (d) and intracellular triglyceride levels (e) were performed in cells as decreased in c. For quantification of the mean integrated optical density (IOD) of BODIPY staining, Image J software was applied and values were analyzed
with unpaired t-test. *Po0.05. DISCUSSION Genetic or pharmacological inhibition of SREBP1
resulted in cell growth arrest and decreased cell proliferation.24
Recent study reported that inhibition of de novo lipid biosynthesis
by suppressing the SREBP pathway prevented HCC progression.37
Our present data also confirmed that decreased TIP30 could
promote HCC cell growth via SREBP1-related lipid metabolism
in vitro and in vivo, which was also responsible for elevated FASN
and SCD expression induced by TIP30 deficiency. Several experimental models have revealed critical effects of Akt
on lipogenesis regulation. It has been recently found that liver
tumors induced by AKT/c-Met displayed increased lipogenesis and
genetic deletion of the main lipogenic enzyme, FASN, suppressed
the in vivo hepatocarcinogenesis driven by AKT and c-Met
oncogenes.38,39 Another research reported that excessive activation
of AKT in mice liver accelerated fatty acid synthesis as well as tumor
development.40 In human lung adenocarcinoma, breast tumors
and glioma, p-AKT and p-ERK1/2 were upregulated by TIP30
deficiency.25,26,41 We previously revealed that loss of TIP30 activated
AKT/GSK-3β/β-catenin
signaling,
which
was
vital
to
growth, In addition, we confirmed that TIP30 expression was negatively
associated with SREBP1 expression in clinical HCC samples. TIP30
is an important prognostic predictor for various cancers.19,48,49
Upregulated SREBP1 associated with a poor prognosis of HCC
patients.50 Remarkably, when the combined effects of TIP30
and SREBP1 were evaluated, the sensitivity for survival analysis of
HCC patients was improved. In summary, we linked TIP30 to lipid metabolism through
SREBP1 in HCC, which revealed alternative mechanisms under-
lying
TIP30-induced
growth
regulation
in
hepatocellular Oncogenesis (2017), 1 – 9 TIP30 regulates lipid metabolism in HCC
F Yin et al Decreased TIP30 promotes HCC cells growth via modulating SREBP1-related lipid metabolism. (a) Cell proliferation was
-shNon and HCC-LM3-shTip30 transfected with si-SREBP1 (si-SREBPl-1 or si-SREBPl-2) or si-NC using MTS. Similar metho
in SMMC-7721 cells. (b) Clone formation was generated in cells as decreased in a. (c) Pictures showed the tumors dissec
e, which were transplanted with HCC-LM3 cells infected with shNon, shTip30, or shTip30 and shSrebp1. (d) Average weight o
uated in each group. (e) Levels of triglycerides were individually measured in tumor tissues of each group using a tissue tri
(f) Tumors derived from nude mice were immunostained for TIP30, SREBP1, SCD and FASN (magnification, × 200). *Po0
F Yin et al 6 Figure 4. Decreased TIP30 promotes HCC cells growth via modulating SREBP1-related lipid metabolism. DISCUSSION After the concentration of RNA was tested, reverse transcription
PCR was done as the PrimeScript RT reagent Kit (Takara Bio, Tokyo, Japan)
guided. SYBR Green-based real-time PCR was operated on a 7500 Fast
Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) and Gapdh
was detected for endogenous control. Primer sequences are listed in
Supplementary Table 1. BODIPY staining HCC cells were fixed for 15 min using 4% paraformaldehyde. Thereafter,
cells were washed then dyed by BODIPY 493/503 (10 μg/ml) (Thermo
Fisher Scientific, Waltham, MA, USA) at 37 ℃for 15 min.Then, the nucleus
was counterstained with DAPI (Invitrogen, Carlsbad, CA, USA) for 10 min. We
captured
images
using
a
fluorescence
microscope
(Olympus,
Lake Success, NY, USA). DISCUSSION (a) Cell proliferation was tested in
HCC-LM3-shNon and HCC-LM3-shTip30 transfected with si-SREBP1 (si-SREBPl-1 or si-SREBPl-2) or si-NC using MTS. Similar methods were
repeated in SMMC-7721 cells. (b) Clone formation was generated in cells as decreased in a. (c) Pictures showed the tumors dissected from
nude mice, which were transplanted with HCC-LM3 cells infected with shNon, shTip30, or shTip30 and shSrebp1. (d) Average weight of tumors
was evaluated in each group. (e) Levels of triglycerides were individually measured in tumor tissues of each group using a tissue triglyceride
assay kit. (f) Tumors derived from nude mice were immunostained for TIP30, SREBP1, SCD and FASN (magnification, × 200). *Po0.05. Oncogenesis (2017), 1 – 9 TIP30 regulates lipid metabolism in HCC
F Yin et al Figure 5. TIP30 expression negatively associates with SREBP1 level in tumor tissues of HCC patients and the combinational biomarkers provide
powerful prognostic value for HCC. (a) TIP30 and SREBP1 were detected by immunostaining in 80 HCC samples. Representative pictures
of immunostaining were shown for two patients. (magnification, × 200). (b) The correlation between TIP30 and SREBP1 expression was
analyzed in HCC tissues according to the scores of immunohistochemistry staining. (c) Scatter plot showed correlations between TIP30
and SREBP1 mRNA level in tumors of 30 HCC patients investigated. Recurrence-free survival rates and overall survival rates were
analyzed between TIP30-high expression group and TIP30-low expression group (d), as well as between four subgroups (TIP30low/SREBP1low;
TIP30low/SREBP1high; TIP30high/SREBP1low; TIP30high/SREBP1high) (e). F Yin et al 7 Figure 5. TIP30 expression negatively associates with SREBP1 level in tumor tissues of HCC patients and the combinational biomarkers provide
powerful prognostic value for HCC. (a) TIP30 and SREBP1 were detected by immunostaining in 80 HCC samples. Representative pictures
of immunostaining were shown for two patients. (magnification, × 200). (b) The correlation between TIP30 and SREBP1 expression was
analyzed in HCC tissues according to the scores of immunohistochemistry staining. (c) Scatter plot showed correlations between TIP30
and SREBP1 mRNA level in tumors of 30 HCC patients investigated. Recurrence-free survival rates and overall survival rates were
analyzed between TIP30-high expression group and TIP30-low expression group (d), as well as between four subgroups (TIP30low/SREBP1low;
TIP30low/SREBP1high; TIP30high/SREBP1low; TIP30high/SREBP1high) (e). carcinoma. Meanwhile, we also expanded the novel function of
TIP30 in HCC metabolism regulation, which should be extensively
studied in other types of tumors. RNA extraction and real-time quantitative PCR
NucleoSpin RNA kit (Macherey-Nagel, Germany) was used for total RNA
extraction. MATERIALS AND METHODS Cell lines, antibodies and reagents Cell lines, antibodies and reagents HCC-LM3 was supported by the Liver Cancer Institute of Zhong Shan
Hospital (Shanghai, China). HepG2 was available from American Type
Culture Collection. SMMC-7721 was acquired from Cell Bank of Shanghai
Institutes for Biological Sciences (Shanghai, China). Cell lines have been
tested for mycoplasma contamination and were cultured at 37 °C in a
humidified condition with 5% CO2. The Dulbecco’s modified Eagle’s
medium (DMEM) was used for cell culture, supplemented with 10% fetal
bovine serum. Details of primary antibodies and their sources are as
follows: TIP30 (produced by our lab as previously reported);17 p-mTOR
(Ser2448), p-AKT (Ser473), mTOR, AKT, FASN and GAPDH (Cell Signaling
Technology, Danvers, USA); SCD and SREBP1 (Abcam, Cambridge, USA). Additionally, we purchased horseradish peroxidase-conjugated secondary
antibodies (anti-rabbit or anti-mouse) from Santa Cruz Biotechnology
(SCBT, CA, USA). MK-2206 was from Selleck Chemicals (Houston, TX, USA). Oleate was acquired from Sigma (St Louis, MO, USA). TG measurements The cellular TG content was tested using a Triglyceride Quantification Kit
(Abcam, Cambridge, UK) with detailed experimental procedure provided
in the kit. Oncogenesis (2017), 1 – 9 Immunofluorescence staining Lentivirus and small interfering RNA
HCC cells were infected with lentivirus expressing Tip30 cDNA or shRNA
targeting Tip30 as literature described.17 A siRNA targeting SREBP1 were
designed by
GenePharma
(Shanghai, China). Briefly,
the
following
sequences
were
used:
for
SREBP1-homo-523
(si-SREBP1-1),
sense
5′-GCUCCUCUCUUGAAGCCUUTT-3′
and
antisense
5′-AAGGCUUCAAG-
AGAGGAGCTT-3′; for SREBP1-homo-1403 (si-SREBP1-2), sense 5′-GCA-
ACACAGCAACCAGAAATT-3′ and antisense 5′-UUUCUG GUUGCUGUG-
UUGCTT-3′. HCC cells were incubated on 24-well plates for 24 h and then were
fixed using paraformaldehyde with the final concentration 4%. Triton
X-100 was prepared in PBS for the final concentration 0.2% and then was
used for permeabilization.10% BSA/PBS was used as blocking buffer. For immunofluorescence staining, primary antibodies were added for
incubation for 24 h on a shaker setting at 4 °C. After washing three times, a
goat anti-rabbit Alexa Fluor 555 antibody (Thermo Fisher Scientific) was
mixed and for 1 h incubation. Thereafter, cells were counterstained using
DAPI (Invitrogen). All matched samples were photographed (control and
test) using confocal laser scanning microscopy (FLUOVIEW FV-1000). Protein extraction and western blotting Proliferation detection and colony-formation assays Statistical analysis in hepatocellular carcinoma. Oncogene 2015; 34: 1420–1431. 21 Liao BM, Raddatz K, Zhong L, Parker BL, Raftery MJ, Schmitz-Peiffer C. Proteomic
analysis of livers from fat-fed mice deficient in either PKCdelta or PKCepsilon
identifies Htatip2 as a regulator of lipid metabolism. Proteomics 2014; 14:
2578–2587. We repeated in vitro experiments in triplicate. SPSS software (version 16.0,
Chicago, IL, USA) was used for statistical analysis. Pearson chi-square test and
Student’s t-test were applied for analysis of dichotomous variables and
continuous variables, respectively. Correlations of two variables were
determined using Spearman rank test. Survival analyses of investigated
patients were achieved using Kaplan–Meier analysis with log-rank test. When
more than two data sets were analyzed, variance analysis was performed. The results are showed as the mean± s.e.m. The above statistical analyses
were all two-sided with Po0.05 deemed statistically significant. 22 Horton JD. Sterol regulatory element-binding proteins: transcriptional activators
of lipid synthesis. Biochem Soc Trans 2002; 30: 1091–1095. 23 Horton JD, Goldstein JL, Brown MS. SREBPs: activators of the complete program
of cholesterol and fatty acid synthesis in the liver. J Clin Invest 2002; 109:
1125–1131. 24 Guo D, Bell EH, Mischel P, Chakravarti A. Targeting SREBP-1-driven lipid
metabolism to treat cancer. Curr Pharm Design 2014; 20: 2619–2626. 25 Li A, Zhang C, Gao S, Chen F, Yang C, Luo R et al. TIP30 loss enhances cytoplasmic
and nuclear EGFR signaling and promotes lung adenocarcinogenesis in mice. Oncogene 2013; 32: 2273–2281. Tumor xenograft mouse model 11 NicAmhlaoibh R, Shtivelman E. Metastasis suppressor CC3 inhibits angiogenic
properties of tumor cells in vitro. Oncogene 2001; 20: 270–275. Animal studies are authorized by medical ethics committee of PLA General
Hospital. Male Balb/c nude mice (4 weeks old) are randomly allocated into
three groups (6 mice/group) and the number of mice is determined
according to prior experience of in vivo studies in our laboratory. We
subcutaneously inject 5 × 106 indicated cells into each mouse. Investigators
were not blinded for the animal studies. During the experiment, mice were
monitored and euthanized for histopathology examination after cell
inoculation for 28 days. Then, the tumor weight and their triglycerides
levels were measured. 12 Zhao J, Lu B, Xu H, Tong X, Wu G, Zhang X et al. Thirty-kilodalton Tat-interacting
protein suppresses tumor metastasis by inhibition of osteopontin transcription in
human hepatocellular carcinoma. Hepatology 2008; 48: 265–275. 13 Zhao J, Ni H, Ma Y, Dong L, Dai J, Zhao F et al. TIP30/CC3 expression in breast
carcinoma:
relation
to
metastasis,
clinicopathologic
parameters,
and
P53
expression. Hum Pathol 2007; 38: 293–298. 14 Zhao J, Zhang X, Shi M, Xu H, Jin J, Ni H et al. TIP30 inhibits growth of HCC cell
lines and inhibits HCC xenografts in mice in combination with 5-FU. Hepatology
2006; 44: 205–215. 15 Zhao J, Chen J, Lu B, Dong L, Wang H, Bi C et al. TIP30 induces apoptosis under
oxidative stress through stabilization of p53 messenger RNA in human hepato-
cellular carcinoma. Cancer Res 2008; 68: 4133–4141. Patients, immunohistochemistry and scoring Samples of 80 patients who had radical resection of HCC were collected from
2003 to 2007 at Guangxi Cancer Hospital (Nanning, China). Radical surgery
was defined as previously reported.20 Informed consent authorized by Ethics
Committee of Guangxi Cancer Hospital was acquired from patients when
specimen collection was performed. Supplementary Table 2 showed
clinicopathological features of the above patients. All the patients were
monitored for recurrence every 1–6 months after the curative resection
depending on the post-operative time. Immunohistochemistry of clinical
samples were performed as previously reported.17,20 Two experienced
pathologists independently evaluated the staining scores. According to the
staining intensity and distribution, immunostaining scores were semiquanti-
tatively estimated.17 Immunohistochemical scores of ⩽4 and scores of ⩾5
were classified as low and high expression, respectively. 16 Zhu M, Yin F, Yang L, Chen S, Chen R, Zhou X et al. The authors declare no conflict of interest. The authors declare no conflict of interest. 26 Zhang C, Mori M, Gao S, Li A, Hoshino I, Aupperlee MD et al. Tip30 deletion in
MMTV-Neu mice leads to enhanced EGFR signaling and development of estrogen
receptor-positive and progesterone receptor-negative mammary tumors. Cancer
Res 2010; 70: 10224–10233. Microarray analysis HCC-LM3 cell line was infected with shTip30 or control lentivirus. After
7 days, TRIzol (Thermo Fisher Scientific) was used to extract cellular RNA,
which was then purified by an RNeasy kit (Qiagen, Hilden, Germany). NimbleGen Gene Expression Microarray was applied in microarray analysis. Axon GenePix 4000B microarray scanner was used for scanning and raw
data were extracted by NimbleScan software 2.5. Gene Ontology (GO) and
Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of
the differentially expressed genes (fold change ⩾2, Po0.05) were
performed for predicting biologic effects of TIP30. Such analysis was
achieved by the ClueGo plugin of Cytoscape (Software version 3.2.3,
INSERM UMRS1138, Paris, France), which is a functional annotation way to
evaluate over-representation of functional categories in interested genetic
sets.51 Enrichment analysis was performed via functional annotation chart
and annotation clustering options, which was limited to GO terms and
KEGG pathways in ‘Biologic Process’ categories. Functional annotation was
deemed significant with P-valueo0.05, using Fisher’s exact test. multicenter prospective study. Hepatology 2016; 63: 827–838. 4 Martinez-Outschoorn UE, Peiris-Pages M, Pestell RG, Sotgia F, Lisanti MP. Cancer 4 Martinez-Outschoorn UE, Peiris-Pages M, Pestell RG, Sotgia F, 4 Martinez-Outschoorn UE, Peiris-Pages M, Pestell RG, Sotgia F, Lisanti MP. Ca
metabolism: a therapeutic perspective. Nat Rev Clin Oncol 2016; 14: 113. metabolism: a therapeutic perspective. Nat Rev Clin Oncol 2016; 14: 113. 5 Rohrig F, Schulze A. The multifaceted roles of fatty acid synthesis in cancer. Nat Rev Cancer 2016; 16: 732–749. 6 Shtivelman E. A link between metastasis and resistance to apoptosis of variant
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9373–9381. ACKNOWLEDGEMENTS 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
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domain of HAb18G/CD147 binds to metal ion-dependent adhesion site (MIDAS) © The Author(s) 2017 © The Author(s) 2017 Supplementary Information accompanies this paper on the Oncogenesis website (http://www.nature.c tary Information accompanies this paper on the Oncogenesis website (http://www.nature.com/oncsis) Oncogenesis (2017), 1 – 9 Oncogenesis (2017), 1 – 9
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Use of English Phrasal Verbs of Chinese Students Across Proficiency Levels: A Corpus-Based Analysis
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English phrasal verbs, Chinese learner corpus, proficiency level, single-word synonyms English phrasal verbs, Chinese learner corpus, proficiency level, single-word synonyms Use of English Phrasal Verbs of Chinese Students Across
Proficiency Levels: A Corpus-Based Analysis Yuanyuan Wei
Renmin University of China, China Abstract A number of studies have investigated phrasal verbs from various aspects (Liu, 2006; Yu, 2011; Liao
& Fukuya, 2004). However, those studies lack generality, because only a couple of phrasal verbs
were chosen as examples, or too much content prompt was offered in task response of L2 learners. The current study applies a corpus-based approach to investigate English phrasal verbs in free
conversations from a Chinese learner corpus, SWECCL (Wen & Wang, 2008), which demonstrates
Chinese students’ actual use of phrasal verbs. This study focuses on analysis of two phenomena:
development of phrasal verb uses and avoidance of phrasal verbs across different proficiency levels
in Chinese students’ oral English conversations. Results showed that students at higher proficiency
level used more phrasal verbs in oral communication, and the progress of phrasal verb use was
evident from middle proficiency level to advanced proficiency level. The study also found that phrasal
verbs occurred less frequently overall in L2 learners’ oral conversations compared with frequency of
their single-word synonyms. International Journal of TESOL Studies (2021)
Vol. 3 (4) 25-41 https://doi.org/10.46451/ijts.2021.12.03 International Journal of TESOL Studies (2021)
Vol. 3 (4) 25-41 https://doi.org/10.46451/ijts.2021.12.03 Address: School of Foreign Languages, University of Renmin University, Beijing, China
Email: wei_yuanyuan23@163.com 2.1 Semantic and syntactical studies on phrasal verbs Relevant micro-linguistic studies demonstrated several special features of phrasal verbs (Zhao, 1984;
Wang, 1993; Wang, 1997). Complicated multiple meanings and complex syntactical structures were two
main concerns of those semantic and syntactic studies. On the one hand, phrasal verbs usually contain
multiple meanings, of which literal meaning is at the core of semantic field, and metaphorical meanings
are connected with it (Bolinger, 1971; Liu, 2006). On the other hand, phrasal verbs are syntactically
complex because they can be transitive or intransitive. In a transitive construction, an object can be
placed between a verb and a particle to construct a VOP or after a particle to be a VPO (Fan, 1995). Also,
sometimes a prepositional phrase (PP) follows a particle to provide more information, such as fall out of
the window (Han, 2018). In terms of their collocational association with other words, it is not fixed by
regular rules, for example, map out a plan is proper but map out people is an inappropriate use. Most researchers are aware of syntactical and semantic difficulties in acquisition of verb-particle
constructions, and phrasal verbs are pervasively used in English especially in many informal conversations
(McArthur, Wang & Zhu, 1992). In order to tackle issues of acquiring phrasal verbs, researchers investigate
phrasal verbs from a pedagogical perspective and emphasize conceptual meanings of them, and some
progress was made. White (2012) encouraged denotation and metaphorical meaning of particles in phrasal
verbs by asking them to draw pictures of them and proved this method was useful for acquisition. Ding and
Yang (2016) introduced a new pedagogy of phrasal verbs’ acquisition—summing up figurative meanings of
particles and generating verb patterns. For example, “at” means “attempt to”, so when it is used with a verb
like “shoot”, the construction refers to “attempt to shoot somebody”. 1 Introduction Chinese students are prone to use high-frequency verbs repetitively, which leads to the lack of variety in
language use. Native speakers also frequently use those verbs, but they often combine verbs with adverbs
or prepositions to express various meanings. The combination which native speakers frequently use in
their conversations is called phrasal verb (e.g., put on, take off). A phrasal verb is made up of a verb and a prepositional or adverbial particle (Waibel, 2007). Their
meanings are not constituted through simply combining words’ meanings, and they can be replaced by
single verbs. Appropriate use of phrasal verbs becomes a distinguishing feature of a proficient L2 learner. However, Chinese L2 learners are faced with challenges of acquirement of phrasal verbs, because
verb-particle combinations are flexible rather than regulated by specific rules. Another difficulty is the
polysemy of those phrasal verbs, a prevailing phenomenon in verb-particle combinations (Han, 2018). Due to those two major obstacles, Chinese students usually learn phrasal verbs through rote learning, International Journal of TESOL Studies 3 (4) 26 (4) which is extremely ineffective (Ding & Yang, 2016). While a considerable amount of literature has been
published on semantic and syntactical analysis of phrasal verbs, and there has been little quantitative
analysis which investigates Chinese college students’ phrasal verb use. Since empirical research on
phrasal verbs is still not fully developed, corpora can be useful tools which contribute to demonstrating
L2 learners’ performance on phrasal verb use. 2.2 Avoidance of phrasal verbs Semantic studies and error analysis on propositions usually focused on case study through the interpretation
of cognitive theory. Thorough syntactical analysis of phrasal complex structure also heavily relied on
giving examples (Zhao, 1984; Wang, 1993; Wang, 1997; Han, 2018). Another branch delves L2 learners’
preference for single-word verbs over phrasal verbs. Avoidance of verb-particle construction was confirmed
by many studies. Dagut and Laufer (1985) conducted translation and multiple-choice tests. They concluded
that Hebrew-speaking students used more single-word verbs over phrasal verbs and used more frequently
their literal meanings over figurative meanings. Laufer and Eliasson (1993) studied advanced Swedish-
speaking learners through similar assessments and found that cross-linguistic difference, specifically,
the lack of phrasal verb structures in L1, mainly accounted for the underuse of this structure. Liu (2006)
focused on assessing correct particle use of Chinese students through multiple choice questions and
filling blanks and generalized three reasons why errors occurred when Chinese learners chose appropriate
particles for phrasal verbs: (a) only considering literal meanings of phrasal verbs; (b) negative transfer of
L1; (c) ignoring the context when students infer meanings of phrasal verbs. Controversially, Guo (2013)
used multiple choice tests to count the frequency of phrasal verbs and single words and found that Chinese
college students have preference for phrasal verbs over single-word verbs and underused phrasal verbs only
under the condition of unfamiliarity with multiple meanings of them rather than interference of L1. With
proficiency as a variable, studies with similar tests concluded that Chinese learners at middle level avoided 27 Yuanyuan Wei using phrasal verbs but used single-word verbs instead and students at middle level and advanced level
avoid using phrasal verbs in figurative meanings (Liao & Fukuya, 2004; Zhang, 2007). However, those
studies offered options for participants to choose. Therefore, the items used in tasks can be content prompts
for participants, and those multiple-choice tasks do not mimic students’ language output in real life. Another
potential problem of those studies was that researchers determined literal meanings and figurative meanings
of the use of phrasal verbs. However, categories of phrasal verbs in figurative meanings and literal meanings
are not completely distinguished like those researchers established in their studies, but a continuum with
varying degrees of semantic transparency (Bolinger, 1971; Liu, 2006). In order to address those issues,
corpora can be a helpful tool. 2.2 Avoidance of phrasal verbs Sung (2020) searched the frequencies of 150 most frequently used phrasal
verbs and their single-word synonyms and concluded that frequency of underused phrasal verbs by L2
learners was statistically significantly lower than their single-word counterparts. Since only written data was
taken into consideration, oral data may get different results and thus it is worth studying. The 150 phrasal
verb list, S&AW PHaVE List (Liu & Myers, 2020), can be a useful reference which provides most common
phrasal verbs and their meanings in both spoken and written registers. 2.3 L2 Leaners’ use of phrasal verbs across proficiency levels With the help of corpus method, various aspects of phrasal verbs can be analyzed effectively, such as
collocational constraints of verb-particle combinations, multiplicity of phrasal verbs’ meanings, and
frequent use of phrasal verbs among native speakers (Gardner & Davies, 2007). However, L2 learners’
use of phrasal verbs is not fully analyzed in corpus linguistics across proficiency levels. Regarding
English proficiency, Chen explained why subjects in her research did not show avoidance of phrasal
verbs, which was in conflict with Waibel (2007) who generated more frequent use of phrasal verbs
by German students than L2 learners of other L1s. Furthermore, Yu (2011) focused on oral English
performance of English majors and illuminated that the ability of using phrasal verbs developed with the
improvement of English proficiency but it stopped developing from middle proficiency level to advanced
proficiency level. However, since only the number of oral records was taken into consideration, and the
text length of every record was also important to work as a basis for relative frequency, this research
may lack accuracy in comparing performance of groups at different proficiency levels. Proficiency level,
which works as an important variable in related corpus studies, can bring diversified findings to studies
on phrasal verbs. Inspired by previous studies, the present study is concerned with actual performance of Chinese L2
learners on phrasal verb use. Corpora provide more scientific data than isolated linguistic examples,
researchers’ intuitions or random group of phrasal verbs, because they deal with verb-particle
combinations by utilizing frequency of occurrences in language calculated by computers and phrasal verb
usage in real context (Gardner & Davies, 2007). Furthermore, unlike case studies which were frequently
used to analyze Chinese students’ use (Ma & Shang, 2011; Yu, 2011), such as prepositions with frequent
use, “in” “on” “over”, “come”, “do” “get” “give” “go” “make” “put” and “take” working as verbs in the
frequency analysis, this paper adopted S&AW PHaVE List. The list was proposed by Liu and Myers (2020)
based on COCA. It offered 150 phrasal verbs of most frequent use and their frequently-used meanings. Phrasal verb frequency difference among groups at different proficiency and frequency difference
between phrasal verbs and single-word verbs were investigated in SWECCL to reveal Chinese learners’
performance. 3.2 Learner population Though there are a few combinations of verb + adverb or preposition
in Chinese, such as “hui lai (come back)”, they differentiate from English phrasal verbs in that the
amount is much smaller and most of them do not have any figurative meanings (Zhang, 2007). Because of cross-linguistic differences, Chinese learners find it challenging to appropriately use
multiple meanings of English phrasal verbs. 3.1 Research questions The current study is intended to address the following two research questions: International Journal of TESOL Studies 3 (4) 28 (4) 1. How does frequency of phrasal verb use among Chinese L2 learners vary across different pro-
ficiency levels? 1. How does frequency of phrasal verb use among Chinese L2 learners vary across different pro-
ficiency levels? 1. How does frequency of phrasal verb use among Chinese L2 learners vary across different pro-
ficiency levels? 2. How do Chinese students show avoidance of phrasal verbs in L2 oral communication? 2. How do Chinese students show avoidance of phrasal verbs in L2 oral communicatio 3.2 Learner population Learner population in this study was 1148 Chinese sophomores whose majors were English. Their
speeches and free conversations were ranked by two professional scorers. And they were classified into
four groups based on their ranks with rank 4 being the most proficient (Wang & Wen, 2007). In terms of similarities of phrasal verbs in Chinese and English, firstly, they are constructed by
two components and one is complementary to the other. And they all work as predicates. Also, two
components can be separable. (1) English: V + particle e.g., come over
Chinese: V + particle e.g., guo lai
come-PRS-DECL over-ADV
(2) English: take a book out
Chinese: na chu yi ben shu lai
take-PRS-DECL one-CLF book-OBJ out-ADV Whereas, the difference is that a phrasal verb in English usually contains multiple meanings and some
idiomatical meanings cannot be inferred easily through literal meaning of components. However,
it is not the case in Chinese. For example, “come out” in English refers to “come out a book” or
“come out from somewhere”. Phrasal verb use also undergoes negative influence of native language. Whereas, the difference is that a phrasal verb in English usually contains multiple meanings and some
idiomatical meanings cannot be inferred easily through literal meaning of components. However,
it is not the case in Chinese. For example, “come out” in English refers to “come out a book” or
“come out from somewhere”. Phrasal verb use also undergoes negative influence of native language. For example, “eat completely”, “drink thoroughly”, “use completely” are translated directly from
Chinese to English. However, “eat up”, “drink up”, “use up” are more frequently used among the
native speakers (Wang, 1987). Though there are a few combinations of verb + adverb or preposition
in Chinese, such as “hui lai (come back)”, they differentiate from English phrasal verbs in that the
amount is much smaller and most of them do not have any figurative meanings (Zhang, 2007). Because of cross-linguistic differences, Chinese learners find it challenging to appropriately use
multiple meanings of English phrasal verbs. Whereas, the difference is that a phrasal verb in English usually contains multiple meanings and some
idiomatical meanings cannot be inferred easily through literal meaning of components However For example, “eat completely”, “drink thoroughly”, “use completely” are translated directly from
Chinese to English. However, “eat up”, “drink up”, “use up” are more frequently used among the
native speakers (Wang, 1987). 3.3 The corpora The Spoken and Written English Corpus of Chinese Learners (SWECCL) was employed for the
investigation of the Chinese L2 learners’ use of phrasal verbs. SWECCL is a two-million-word corpus
compiled by Nanjing University, Foreign Language Teaching and Research Press and Beijing Foreign
Study University. SECCL is a part of the corpus which only compiles spoken data. The spoken data was
collected from Test for English Majors-Band 4 (TEM-4) during 1996-2002. It contains 1148 transcripts
of audio samples which are 1,460,042 words. All subjects were sophomore English majors from colleges around the country. They were asked
to do three tasks: listening to and retelling a story, impromptu speech and conversation between two
students. And their recordings were scored by two raters. Every task performance was given a rank,
rank 1 referred to the worst performance and rank 4 meant the best performance. In this study, only 713
free conversations were chosen because they could reflect actual use of phrasal verbs in communication
of daily life. For the convenience of research, this paper calculated words of each rank: 122306 words,
218430words, 27556 words, and 3922 words. 29 Yuanyuan Wei 3.4 The extraction of phrasal verbs and their single-word synonyms In order to address the issue which only focused on certain verbs and particles, this paper adopted S&AW
PHaVE List (Liu & Myers, 2020) as a criterion to extract data in the learner corpus. The list generated
the most common meanings of the most frequently used phrasal verbs in spoken and academic written
English derived from COCA. Meanwhile, Wordnet (Princeton University, 2010) provided words related
to phrasal verbs. Then words which matched the top two most common meanings of each phrasal verb in
spoken register were determined as single-word synonyms. AntConc (Anthony, 2020) was used to do the
searches, with verbs as search terms and particles as context words as shown in Figure 1. The searches
for single-word synonyms were also conducted in AntConc, as shown in Figure 2. For parents to keep
up to bring up their children is already very hard could be a concordance example of the phrasal verb,
bring up. And but I think you know firstly the pay is good, you can to some extent raise your family was
a concordance line of raise, one of the synonyms of bring up. Similar concordance examples like bring
up their children and raise your family can also be found in COCA (Davies, 2008) as shown in Figure 3
and Figure 4. Those examples demonstrate that Chinese college students can use phrasal verbs and their
single-word synonyms correctly in oral communications in English. Figure 1 Figure 1 Figure 1
An Example of Searching Phrasal Verbs
Figure 2
An Example of Searching Single-Word Synonym An Example of Searching Phrasal Verbs An Example of Searching Phrasal Verbs An Example of Searching Phrasal Verbs Figure 2 An Example of Searching Single-Word Synonym An Example of Searching Single-Word Synonym An Example of Searching Single-Word Synonym International Journal of TESOL Studies 3 (4) 30 Figure 3
Concordance of the Phrasal Verb Bring up Searched in COCA Figure 3
Concordance of the Phrasal Verb Bring up Searched in COCA Concordance of the Phrasal Verb Bring up Searched in COCA Figure 4 gu e
Concordance of Raise, the Synonym of Bring Up Searched in COCA
Since in a phrasal verb, a verb and a particle can be either adjacent to each other or separable from each
other, collection of data should take both usages into consideration. A maximum of five words between a
verb and a particle was used in this paper since Gardner and Davies (2007) illuminated that even seven-
word phrasal verbs exist in BNC such as send your certificate of motor insurance back. Next, because
verbs have various inflectional forms, these forms were counted under the frequency of the original
form, for example, goes, went, going were all grouped under go. Another challenge associated with data
extraction was the similarity of form between verb-particle combinations and verb-preposition phrases Concordance of Raise, the Synonym of Bring Up Searched in COCA
Since in a phrasal verb, a verb and a particle can be either adjacent to each other or separable from each
other, collection of data should take both usages into consideration. A maximum of five words between a
verb and a particle was used in this paper since Gardner and Davies (2007) illuminated that even seven-
word phrasal verbs exist in BNC such as send your certificate of motor insurance back. Next, because
verbs have various inflectional forms, these forms were counted under the frequency of the original
form, for example, goes, went, going were all grouped under go. Another challenge associated with data
extraction was the similarity of form between verb-particle combinations and verb-preposition phrases Concordance of Raise, the Synonym of Bring Up Searched in COCA Concordance of Raise, the Synonym of Bring Up Searched in COCA Since in a phrasal verb, a verb and a particle can be either adjacent to each other or separable from each
other, collection of data should take both usages into consideration. A maximum of five words between a
verb and a particle was used in this paper since Gardner and Davies (2007) illuminated that even seven-
word phrasal verbs exist in BNC such as send your certificate of motor insurance back. Next, because
verbs have various inflectional forms, these forms were counted under the frequency of the original
form, for example, goes, went, going were all grouped under go. 4.1 Frequency difference among Chinese L2 learners at different proficiency levels Following extracting procedure, this paper firstly calculated absolute token frequency and relative
frequency of 150 phrasal verbs in the task three, free conversation, of SECCL, the spoken corpus, as
found in Appendix A. The frequency analysis of Chinese students’ phrasal verb use revealed a difference among rank 1,
rank 2, rank3 and rank 4. Table 1 below demonstrated that relative token of rank 1 students was lower
than rank 2 students, and rank 2 students had a lower relative token than rank 3 students. And rank 4
students had the highest relative token among students of four ranks. Table 1
Token Frequencies of Phrasal Verb in the Spoken Corpus of SWECCL
Proficiency level
Size
Absolute token
Relative token
rank 1
122,306
181
147.99
rank 2
218,430
334
152.91
rank 3
27,556
47
170.56
rank 4
3,922
8
203.98
Note. Relative token frequency was calculated through the absolute token frequency divided by token
size of each rank in the spoken corpus and then rounding it to the nearest tenth. Token Frequencies of Phrasal Verb in the Spoken Corpus of SWECCL
P
fi i
l
l
Si
Ab
l t t k
R l ti
t k Token Frequencies of Phrasal Verb in the Spoken Corpus of SWECCL Note. Relative token frequency was calculated through the absolute token frequency divided by token
size of each rank in the spoken corpus and then rounding it to the nearest tenth. Because word sizes of each rank are different, relative token frequencies of every phrasal verb used by
students of four ranks rather than absolute token frequencies were used to compare difference. Non-para
metric Kruskal-Wallis H test was conducted to compare difference of phrasal verb use frequency among
four rank groups. And the Kruskal-Wallis H test found that there was a statistically significant difference
on relative token frequencies among the different rank groups, χ2(3) = 51.751, p = .000, η²=0.082, with
a mean rank frequency of 317.74 for rank 1 group, 349.77 for rank 2 group, 282.2 for rank 3 group and
252.29 for rank 4 group. The findings were in conflict with previous findings that phrasal verb use of
students at rank 2, 3 and 4 was similar. Instead, statistically significant differences were found between
rank 4 group and rank 3 group, rank 3 group and rank 2 group. Figure 4 All those exceptions were manually checked by the researcher and only phrasal verb
structures were coded in this paper. Extracting single-word synonyms of phrasal verbs from AntConc also encountered great challenges
that there were no complete synonyms. Those single word alternatives chosen from Wordnet contain
multiple meanings. Therefore, in some cases, single-word verbs cannot replace their phrasal verb
counterparts. For example, raise, as the single word alternative of bring up, also means increase, such as
the concordance I think helping others can also raise our sense of, er, raise our sense of social life, then
we can, er, then we can make ourselves to help others in SWECCL. In order to maintain correctness of
results, every concordance was counted into frequency only when it was manually checked. Figure 4 Another challenge associated with data
extraction was the similarity of form between verb-particle combinations and verb-preposition phrases Since in a phrasal verb, a verb and a particle can be either adjacent to each other or separable from each
other, collection of data should take both usages into consideration. A maximum of five words between a
verb and a particle was used in this paper since Gardner and Davies (2007) illuminated that even seven-
word phrasal verbs exist in BNC such as send your certificate of motor insurance back. Next, because
verbs have various inflectional forms, these forms were counted under the frequency of the original
form, for example, goes, went, going were all grouped under go. Another challenge associated with data
extraction was the similarity of form between verb-particle combinations and verb-preposition phrases Since in a phrasal verb, a verb and a particle can be either adjacent to each other or separable from each
other, collection of data should take both usages into consideration. A maximum of five words between a
verb and a particle was used in this paper since Gardner and Davies (2007) illuminated that even seven-
word phrasal verbs exist in BNC such as send your certificate of motor insurance back. Next, because
verbs have various inflectional forms, these forms were counted under the frequency of the original
form, for example, goes, went, going were all grouped under go. Another challenge associated with data
extraction was the similarity of form between verb-particle combinations and verb-preposition phrases 31 Yuanyuan Wei structures in the learner corpus and the research on phrasal verb analysis had to differentiate phrasal
verbs from verb-preposition phrases structures (Kim & Baldwin, 2006). For example, take over works
as a phrasal verb in he takes over the accounts, but it becomes a verb - preposition phrase in He takes the
lamp over the table. All those exceptions were manually checked by the researcher and only phrasal verb
structures were coded in this paper. structures in the learner corpus and the research on phrasal verb analysis had to differentiate phrasal
verbs from verb-preposition phrases structures (Kim & Baldwin, 2006). For example, take over works
as a phrasal verb in he takes over the accounts, but it becomes a verb - preposition phrase in He takes the
lamp over the table. 4.1 Frequency difference among Chinese L2 learners at different proficiency levels It proved that capability of using phrasal
verb in oral English conversation of Chinese college students was also improving from middle-level to
advanced level. International Journal of TESOL Studies 3 (4) 32 Table 2
The Kruskal-Wallis H test: Ranks for Phrasal Verb Use’ Frequency of Different Proficiency Groups
Ranks
group
N
Mean Rank
alt
1
150
317.74
2
150
349.77
3
150
282.2
4
150
252.29
Total
600 The Kruskal-Wallis H test: Ranks for Phrasal Verb Use’ Frequency of Different Proficiency Table 3
Pairwise Comparisons of Group for Phrasal Verb Use’ Frequency at Rank 1, 2, 3, 4
Sample 1-Sample 2
Test Statistic
Std. Error
Std. Test Statistic
Sig. Adj. Sig.a
4.00-3.00
29.913
14.424
2.074
0.038
0.229
4.00-1.00
65.45
14.424
4.538
0
0
4.00-2.00
97.477
14.424
6.758
0
0
3.00-1.00
35.537
14.424
2.464
0.014
0.083
3.00-2.00
67.563
14.424
4.684
0
0
1.00-2.00
-32.027
14.424
-2.22
0.026
0.158
Each row tests the null hypothesis that the Sample 1 and Sample 2 distributions are the same. Asymptotic significances (2-sided tests) are displayed. The significance level is .05. a Significance values have been adjusted by the Bonferroni correction for multiple tests. Pairwise Comparisons of Group for Phrasal Verb Use’ Frequency at Rank 1, 2, 3, 4 4.2 Avoidance of phrasal verbs in L2 oral communication across proficiency levels In order to demonstrate phrasal verb avoidance in oral English, this study chose 10 phrasal verbs
which were frequently used in free conversations compared with other 140 phrasal verbs. Meanwhile,
if a complete synonym counterpart of a phrasal verb can be found in Wordnet was also taken into
consideration when this paper chose phrasal verbs as typical examples. In the end, as Table 4 shows, 10
phrasal verbs and 20 single-word synonyms were picked out for analysis. And as Table 5 shows, phrasal
verb use is less frequent than their single-word synonyms overall. Frequency of phrasal verb use at four
separate rank groups are demonstrated in Appendix B. Ten Relatively Frequent-Used Phrasal Verbs in SWECCL and Their Single-Word Synonyms
Phrasal verbs
single-word synonyms
bring up
mention
raise
get in
enter
arrive
go on
happen
continue
build up
develop
enlarge
get off
escape
leave
Set up
establish
arrange
take up
start
discuss
get down
lower
begin
get on
board
progress
pick up
collect
learn Ten Relatively Frequent-Used Phrasal Verbs in SWECCL and Their Single-Word Synonym 33 Yuanyuan Wei Table 5
Frequencies of Phrasal Verbs and Single-Word Synonyms
Phrasal verbs
Absolute Frequency of phrasal verbs
Absolute Frequency of single-word synonyms
bring up
26
59
get in
19
92
go on
27
200
build up
7
119
get off
5
42
set up
7
19
take up
7
114
get down
6
33
get on
16
2
pick up
6
834 Then the analysis of phrasal verb use of Chinese college students at different proficiency levels was
conducted. Because data was not normally distributed (skewness and kurtosis statistics were greater than
±2 when they divided by their respective errors), non-parametric independent samples test on relative
frequency was adopted to assess frequency difference between phrasal verbs and single-word synonyms
at each rank in the spoken corpus of SWECCL. The Wilcoxon Signed-Ranks test for rank 1 indicated
that single-word synonyms (mean rank = 40.881) was rated more favorably than phrasal verbs (mean
rank = 3.025), Z = 2.601, p = .009< .05, r = 0.58. The Wilcoxon Signed-Ranks test for rank 2 indicated
that single-word synonyms (mean rank = 41.341) was rated more highly than phrasal verbs (mean rank
= 3.388), Z = 2.701, p = .007< 0.05, r = 0.60. 4.2 Avoidance of phrasal verbs in L2 oral communication across proficiency levels The Wilcoxon Signed-Ranks test for rank 3 showed that
single-word synonyms (mean rank = 36.653) was rated more highly than phrasal verbs (mean rank =
1.451), Z = 2.803, p = .005< .05, r = 0.63. Statistically significant differences were found for rank 1
group, rank 2 group and rank 3 group. The findings above showed phrasal verb avoidance in Chinese students’ oral English conversations
at primary level and middle-level. However, at advanced level, no statistically significant difference was
shown between the uses of phrasal verbs and single-word synonyms, the Wilcoxon Signed-Ranks test
for rank 4 showed that single-word synonyms (mean rank = 28.047) was rated more highly than phrasal
verbs (mean rank = 2.55), Z = 1.604, p = .109> .05, r = 0.36. Therefore, students at advanced level do not
avoid using phrasal verbs. Table 6 Table 6
Descriptive Statistics of phrasal verb use and single-word synonym use for rank 1, 2, 3, 4 groups
Descriptive Statistics for rank 1
N
Minimum
Maximum Mean
Std. Error
Std. Deviation
Skewness Std. Error
Kurtosis
Std. Error
Phrasal
verbs
10
0.82
7.36
3.03
0.67904
2.15
1.05
0.69
0.41
1.33
Single-
word
10
0
247.74
40.88
23.26
73.56
3.02
0.69
9.36
1.33
Valid N
(listwise)
10 ve Statistics of phrasal verb use and single-word synonym use for rank 1, 2, 3, 4 groups Descriptive Statistics of phrasal verb use and single-word synonym use for rank 1, 2, 3, International Journal of TESOL Studies 3 (4) 34 Descriptive Statistics for rank 2
N
Minimum
Maximum
Mean
Std. Error
Std. Deviation
Skewness Std. Error
Kurtosis
Std. Error
Phrasal
verbs
10
0.92
8.24
3.39
0.97
3.07
0.98
0.69
-1.17
1.33
Single-
word
10
0.46
219.29
41.34
20.63
65.23
2.72
0.69
7.84
1.33
Valid N
(listwise)
10
Descriptive Statistics for rank 3
N
Minimum
Maximum
Mean
Std. Error
Std. Deviation
Skewness Std. Error
Kurtosis
Std. Error
Phrasal
verbs
10
0
7.25
1.45
0.80
2.54
1.66
0.69
2.03
1.33
Single-
word
10
3.63
177.82
36.65
16.48
52.13
2.66
0.69
7.50
1.33
Valid N
(listwise)
10
Descriptive Statistics for rank 4
N
Minimum
Maximum
Mean
Std. Error
Std. Deviation
Skewness Std. Error
Kurtosis
Std. Error
Phrasal
verbs
10
0
25.50
2.55
2.55
8.06
3.16
0.69
10.00
1.33
Single-
word
10
0
178.48
28.05
18.41
58.20
2.37
0.69
5.59
1.33
Valid N
(listwise)
10 5 Discussion The first question concerns phrasal verb use of Chinese L2 learners at different proficiency level in oral
English. The corpus analysis identified frequency difference among four groups of students at different
proficiency levels. The most proficient L2 learners used phrasal verbs most frequently, as the relative
token frequency of phrasal verbs in rank 4 was found to be the highest among four ranks. And another
finding, statistically significant differences between rank 2 and rank 3, rank 3 and rank 4, is inconsistent
with previous research that Chinese students at middle level and advanced level did not show a striking
difference in phrasal verb use (Yu, 2011). Consequently, this paper believes that phrasal verb use still
develops when L2 learners at middle level are approaching advanced level. The second research question explores avoidance of phrasal verbs in L2 oral communication through
a corpus approach. For phrasal verbs, their single-word synonyms convey similar meanings and can
replace them in sentences. However, the frequency difference between them was statistically significant
at lower proficiency level. And single words appeared more frequently in the learner corpus. The
avoidance phenomenon did not show among advanced-level students. Why did Chinese students use less
phrasal verbs than single-word synonyms in L2 daily communication? One reasonable explanation
may be that they are not equipped with too much knowledge about phrasal verbs in oral English study 35 Yuanyuan Wei (Dagut & Laufer, 1985), although phrasal verbs are more colloquial and informal in style and they
should be used more in oral English (Chen, 2013). Phrasal verbs are notoriously difficult for L2 learners,
and because of its flexible and complex structure, teachers tend to refer it as chunks and require students
to memorize it mechanically. Additionally, multiple meanings, especially figurative meanings are
acquired by rote learning, actual contexts of using phrasal verbs are not provided in pedagogy. However,
using phrasal verbs appropriately in spoken and written registers can make L2 learners more native-
like, knowing the characteristics of them, such as structural variance and collocation with particles, is
prerequisite to perform better in English oral communication (Gilquin, 2015). 6 Conclusion In conclusion, with the development of English proficiency, Chinese students gradually use phrasal verbs
more frequently. And the phrasal verb use is still developing from middle level to advanced level. As
for the reasons of avoiding using phrasal verbs in oral English communication, replacement of single-
word synonyms should be taken into consideration because students at lower proficiency level use fewer
phrasal verbs than their single-word synonyms. However, the statistically significant frequency difference
does not show among advanced L2 learners. These findings bring light to future research on phrasal verbs, such as demonstrating use of phrasal
verbs with corpus and quantitative method of phrasal verb analysis. The findings about relationship
between development of English proficiency level and phrasal verbs use encourages L2 learners to
acquire phrasal verbs in order to improve their English proficiency. Teachers are also required to adopt
effective teaching pedagogy because of difficulties students are encountering during phrasal verb
learning. There are some limitations in this paper. Firstly, Wordnet provided lists of words related to phrasal
verbs, only two of them were chosen by the author. Also, the corpus used in this paper, Spoken English
Corpus of Chinese Learners (SECCL), collected spoken data only from Chinese English majors, it
may not represent Chinese L2 learners in general. Data collection is not flawless because phrasal verbs
and their single-word counterparts cannot perfectly replace each other in terms of meaning, but every
concordance of single-word synonyms had been manually checked before it was taken into account. When the meaning of a single-word synonym was different from the meaning of its phrasal verb, the
concordance would be eliminated. Token frequency of 150 phrasal verbs in the spoken corpus of SWECCL Phrasal verbs
Rank 1
Rank 2
Rank 3
Rank 4
Back up
0(0)
0(0)
0(0)
0(0)
Blow up
0(0)
0(0)
0(0)
0(0)
Break down
0(0)
0(0)
0(0)
0(0)
Break off
0(0)
0(0)
0(0)
0(0)
Break out
0(0)
0(0)
0(0)
0(0)
Break up
0(0)
0(0)
0(0)
0(0)
Bring about
0(0)
0(0)
0(0)
0(0)
Bring back
0(0)
0(0)
1(3.62)
0(0)
Bring down
0(0)
0(0)
0(0)
0(0) International Journal of TESOL Studies 3 (4) 36 Bring in
0(0)
0(0)
0(0)
0(0)
Bring out
0(0)
0(0)
0(0)
0(0)
Bring up
7(5.72)
17(7.78)
2(7.25)
0(0)
Build up
5(4.09)
2(0.92)
0(0)
0(0)
Call out
0(0)
0(0)
0(0)
0(0)
Carry on
0(0)
2(0.92)
0(0)
0(0)
Carry out
0(0)
4(1.83)
0(0)
0(0)
Catch up
1(0.82)
2(0.92)
0(0)
0(0)
Check out
0(0)
0(0)
0(0)
0(0)
Clean up
0(0)
0(0)
0(0)
0(0)
Close down
0(0)
0(0)
0(0)
0(0)
Come about
0(0)
0(0)
0(0)
0(0)
Come along
0(0)
0(0)
0(0)
0(0)
Come around
0(0)
0(0)
0(0)
0(0)
Come back
2(1.63)
16(7.33)
4(14.52)
0(0)
Come down
0(0)
0(0)
0(0)
0(0)
Come in
1(0.82)
0(0)
0(0)
0(0)
Come off
0(0)
1(0.46)
0(0)
0(0)
Come on
3(2.45)
15(6.87)
0(0)
1(25.5)
Come out
0(0)
1(0.46)
0(0)
1(25.5)
Come over
0(0)
0(0)
0(0)
0(0)
Come through
0(0)
0(0)
0(0)
0(0)
Come up
0(0)
2(0.92)
0(0)
0(0)
Cut off
0(0)
0(0)
1(3.63)
0(0)
End up
0(0)
0(0)
0(0)
0(0)
Figure out
0(0)
2(0.92)
0(0)
0(0)
Fill in
0(0)
1(0.46)
0(0)
0(0)
Fill out
0(0)
0(0)
0(0)
0(0)
Find out
2(1.63)
4(1.83)
0(0)
0(0)
Follow up
0(0)
0(0)
0(0)
0(0)
Get back
0(0)
5(2.29)
0(0)
0(0)
Get down
1(0.82)
4(1.83)
0(0)
1(25.5)
Get in
3(2.45)
16(7.33)
0(0)
0(0)
Get off
1(0.82)
3(1.37)
1(3.63)
0(0)
Get on
4(3.27)
3(1.37)
0(0)
0(0)
Get out
2(1.64)
5(2.29)
0(0)
0(0)
Get through
1(0.82)
0(0)
0(0)
0(0)
Get up
0(0)
2(0.92)
1(3.63)
0(0)
Give back
2(1.64)
5(2.29)
0(0)
0(0)
Give in
0(0)
0(0)
0(0)
0(0)
Give out
1(0.82)
0(0)
0(0)
0(0)
Give up
4(3.27)
8(3.66)
2(7.26)
0(0)
Go ahead
1(0.82)
1(0.46)
0(0)
0(0)
Go along
1(0.82)
2(0.92)
1(3.63)
0(0)
Go around
0(0)
2(0.92)
0(0)
0(0) 37 Yuanyuan Wei Go back
0(0)
4(1.83)
0(0)
0(0)
Go down
0(0)
0(0)
0(0)
0(0)
Go in
0(0)
1(0.46)
0(0)
0(0)
Go off
1(0.82)
1(0.46)
1(3.63)
0(0)
Go on
9(7.36)
18(8.24)
0(0)
0(0)
Go out
32(26.16)
33(15.12)
2(7.26)
0(0)
Go over
1(0.82)
0(0)
0(0)
0(0)
Go through
0(0)
2(0.92)
0(0)
0(0)
Go up
0(0)
2(0.92)
0(0)
0(0)
Grow up
25(20.44)
16(7.33)
1(3.63)
0(0)
Hand over
0(0)
0(0)
0(0)
0(0)
Hang on
0(0)
0(0)
0(0)
0(0)
Hang out
0(0)
0(0)
0(0)
0(0)
Hang up
0(0)
0(0)
0(0)
0(0)
Hold back
0(0)
0(0)
0(0)
0(0)
Hold on
0(0)
0(0)
0(0)
0(0)
Hold out
0(0)
0(0)
0(0)
0(0)
Hold up
0(0)
0(0)
0(0)
0(0)
Keep on
0(0)
0(0)
1(3.63)
0(0)
Keep up
1(0.82)
1(0.46)
0(0)
0(0)
Lay down
2(1.64)
0(0)
0(0)
0(0)
Lay out
0(0)
0(0)
0(0)
0(0)
Line up
0(0)
0(0)
0(0)
0(0)
Look around
2(1.63)
0(0)
0(0)
0(0)
Look back
0(0)
0(0)
0(0)
0(0)
Look down
11(8.99)
11(5.04)
2(7.26)
0(0)
Look out
1(0.82)
3(1.37)
0(0)
0(0)
Look up
0(0)
0(0)
0(0)
0(0)
Make out
0(0)
1(0.46)
0(0)
0(0)
Make up
2(1.63)
5(2.29)
2(7.26)
0(0)
Move back
0(0)
0(0)
0(0)
0(0)
Move in
0(0)
0(0)
0(0)
0(0)
Move on
0(0)
1(0.46)
0(0)
0(0)
Move out
0(0)
2(0.92)
0(0)
0(0)
Move up
0(0)
0(0)
0(0)
0(0)
Open up
0(0)
0(0)
0(0)
0(0)
Pass on
0(0)
0(0)
0(0)
0(0)
Pay off
0(0)
1(0.46)
0(0)
2(50.6)
Pick out
0(0)
1(0.46)
0(0)
0(0)
Pick up
2(1.63)
2(0.92)
1(3.63)
1(25.5)
Play out
0(0)
0(0)
0(0)
0(0)
Point out
0(0)
3(1.37)
0(0)
0(0)
Pull back
0(0)
0(0)
0(0)
0(0)
Pull out
0(0)
1(0.46)
0(0)
0(0)
Pull up
0(0)
0(0)
0(0)
0(0) International Journal of TESOL Studies 3 (4) 38 Put back
0(0)
0(0)
0(0)
0(0)
Put down
0(0)
1(0.46)
0(0)
0(0)
Put in
3(2.45)
9(4.12)
3(10.89)
0(0)
Put off
0(0)
1(0.46)
0(0)
0(0)
Put on
27(22.08)
52(23.81)
8(29.03)
0(0)
Put out
0(0)
0(0)
0(0)
0(0)
Put up
4(3.27)
0(0)
0(0)
0(0)
Reach out
0(0)
0(0)
0(0)
0(0)
Rule out
0(0)
0(0)
0(0)
0(0)
Run out
0(0)
0(0)
0(0)
0(0)
Send out
0(0)
1(0.46)
0(0)
0(0)
Set about
0(0)
0(0)
0(0)
0(0)
Set down
0(0)
0(0)
0(0)
0(0)
Set off
0(0)
0(0)
0(0)
0(0)
Set out
0(0)
0(0)
0(0)
0(0)
Set up
3(2.45)
4(1.83)
0(0)
0(0)
Settle down
0(0)
0(0)
0(0)
0(0)
Show up
0(0)
1(0.46)
0(0)
0(0)
Shut down
0(0)
0(0)
0(0)
0(0)
Shut up
0(0)
1(0.46)
0(0)
0(0)
Sit back
0(0)
0(0)
0(0)
0(0)
Sit down
1(0.82)
0(0)
0(0)
0(0)
Sit up
0(0)
0(0)
0(0)
0(0)
Slow down
0(0)
0(0)
0(0)
0(0)
Sort out
0(0)
0(0)
0(0)
0(0)
Stand out
0(0)
0(0)
0(0)
0(0)
Stand up
1(0.82)
2(0.92)
0(0)
0(0)
Start out
0(0)
0(0)
0(0)
0(0)
Step back
0(0)
0(0)
0(0)
0(0)
Sum up
1(0.82)
0(0)
0(0)
0(0)
Take back
0(0)
2(0.92)
0(0)
0(0)
Take down
0(0)
0(0)
0(0)
0(0)
Take in
2(1.64)
3(1.37)
1(3.63)
0(0)
Take off
2(1.64)
6(2.75)
2(7.26)
1(25.5)
Take on
0(0)
0(0)
0(0)
0(0)
Take out
0(0)
0(0)
1(3.63)
0(0)
Take over
1(0.82)
0(0)
0(0)
0(0)
Take up
2(1.64)
5(2.29)
0(0)
0(0)
Throw out
0(0)
0(0)
0(0)
0(0)
Turn around
0(0)
0(0)
0(0)
0(0)
Turn back
0(0)
0(0)
0(0)
1(25.5)
Turn down
0(0)
0(0)
0(0)
0(0)
Turn off
0(0)
0(0)
0(0)
0(0)
Turn out
0(0)
0(0)
0(0)
0(0)
Turn over
0(0)
0(0)
0(0)
0(0) 39 Yuanyuan Wei Turn up
0(0)
1(0.46)
0(0)
0(0)
Wake up
0(0)
0(0)
0(0)
0(0)
Walk out
0(0)
0(0)
0(0)
0(0)
Wind up
0(0)
0(0)
0(0)
0(0)
Work out
1(0.82)
2(0.92)
1(3.63)
0(0)
Write down
0(0)
0(0)
0(0)
0(0)
Note. Appendix B Frequencies of phrasal verbs and single-word synonyms
Rank1
Rank2
Rank3
Rank4
phrasal verbs synonyms
phrasal verbs synonyms
phrasal verbs synonyms
phrasal verbs synonyms
bring up
7(5.72)
15(12.26)
17(7.78)
40(18.31)
2(7.25)
4(14.52)
0(0)
0(0)
get in
3(2.45)
25(20.44)
16(7.33)
60(27.47)
0(0)
7(25.40)
0(0)
0(0)
go on
9(7.36)
41(33.52)
18(8.24)
136(62.26) 0(0)
16(58.06)
0(0)
7(178.48)
build up
5(4.09)
41(33.52)
2(0.92)
74(33.88)
0(0)
4(14.52)
0(0)
0
get off
1(0.82)
14(11.45)
3(1.37)
24(10.99)
1(3.63)
5(18.14)
0(0)
0
set up
3(2.45)
6(4.91)
4(1.83)
10(4.58)
0(0)
3(10.89)
0(0)
0
take up
2(1.64)
34(27.80)
5(2.29)
70(32.05)
0(0)
10(36.29)
0(0)
0
get down
1(0. 82)
21(17.17)
4(1.83)
9(4.12)
0(0)
2(7.26)
0(0)
1(25.50)
get on
4(3.27)
0(0)
3(1.37)
1(0.46)
0(0)
1(3.63)
0(0)
0
pick up
2(1.63)
303(247.74) 2(0.92)
479(219.29) 1(3.63)
49(177.82) 1(25.5)
3(76.49)
Note. Relative token frequency was calculated through the absolute token frequency divided by word
size of each rank, multiple by one hundred thousand and then rounding it to the nearest hundredth. Both
absolute and relative token frequencies are provided in this table with the latter presented in parentheses. Frequencies of phrasal verbs and single-word synonyms Note. Relative token frequency was calculated through the absolute token frequency divided by word
size of each rank, multiple by one hundred thousand and then rounding it to the nearest hundredth. Both
absolute and relative token frequencies are provided in this table with the latter presented in parentheses. Note. Relative token frequency was calculated through the absolute token frequency divided by word
size of each rank, multiple by one hundred thousand and then rounding it to the nearest hundredth. Both
absolute and relative token frequencies are provided in this table with the latter presented in parentheses. Token frequency of 150 phrasal verbs in the spoken corpus of SWECCL Relative token frequency was calculated through the absolute token frequency divided by word
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absolute and relative token frequencies are provided in this table with the latter presented in parentheses. Note. Relative token frequency was calculated through the absolute token frequency divided by word
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[Research on pattern-based teaching method of English phrasal verbs]. Waiyu Dianhua Jiaoxue
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Quarterly, 41(2), 339-359. https://doi.org/10.1002/j.1545-7249.2007.tb00062.x Gilquin, G. (2015). The use of phrasal verbs by French-speaking EFL learners: a constructional and
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Her research interests are in the fields of language testing and second language acquisition. References Waiyu Jiaoxue [Foreign
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24-30. Zhang, B. (2007). Yingyu xuexizhe dui yingyu duanyu dongci de huibi xianxiang yanjiu [An
investigation of avoidance of English phrasal verbs by Chinese learners of English]. Jiefangjun
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Translation and validation of the vertigo symptom scale into German: A cultural adaption to a wider German-speaking population
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BMC ear, nose and throat disorders
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© 2012 Gloor-Juzi 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. Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7
http://www.biomedcentral.com/1472-6815/12/7 Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7
http://www.biomedcentral.com/1472-6815/12/7 Open Access Abstract Background: Dizziness and comorbid anxiety may cause severe disability of patients with vestibulopathy, but can
be addressed effectively with rehabilitation. For an individually adapted treatment, a structured assessment is
needed. The Vertigo Symptom Scale (VSS) with two subscales assessing vertigo symptoms (VSS-VER) and associated
symptoms (VSS-AA) might be used for this purpose. As there was no validated VSS available in German, the aim of
the study was the translation and cross-cultural adaptation in German (VSS-G) and the investigation of its reliability,
internal and external validity. Methods: The VSS was translated into German according to recognized guidelines. Psychometric properties were
tested on 52 healthy controls and 202 participants with vestibulopathy. Internal validity and reliability were
investigated with factor analysis, Cronbach’s α and ICC estimations. Discriminant validity was analysed with the
Mann–Whitney-U-Test between patients and controls and the ROC-Curve. Convergent validity was estimated with
the correlation with the Hospital Anxiety Subscale (HADS-A), Dizziness Handicap Inventory (DHI) and frequency of
dizziness. Results: Internal validity: factor analysis confirmed the structure of two subscales. Reliability: VSS-G: α = 0.904 and
ICC (CI) =0.926 (0.826, 0.965). Discriminant validity: VSS-VER differentiate patients and controls ROC (CI) =0.99 (0.98,
1.00). Convergent validity: VSS-G correlates with DHI (r = 0.554) and frequency (T = 0.317). HADS-A correlates with
VSS-AA (r = 0.452) but not with VSS-VER (r = 0.186). Conclusions: The VSS-G showed satisfactory psychometric properties to assess the severity of vertigo or vertigo-
related symptoms. The VSS-VER can differentiate between healthy subjects and patients with vestibular disorders. The VSS-AA showed some screening properties with high sensitivity for patients with abnormal anxiety. The severity of symptoms and the perceived limitations
in activities of daily life can be addressed effectively by in-
dividually tailored vestibular rehabilitation based on ves-
tibular
habituation
to
movement
[7-10],
optionally
combined with cognitive behavioural therapy [11-13]. A
requirement for an individually tailored treatment is an
assessment, which has to be carried out as thoroughly as
possible. In order to focus on the symptoms associated
with dizziness, the Vertigo Symptom Scale (VSS) was con-
sidered to be a key instrument. The VSS consists of two
subscales: 1) the Vertigo scale (VSS-VER) which assesses
symptoms mainly associated with disorders of the vestibu-
lar system and 2) the Anxiety and Autonomic symptom
scale (VSS-AA) for the assessment of a group of generic
symptoms which may be associated with autonomic Translation and validation of the vertigo
symptom scale into German: A cultural adaption
to a wider German-speaking population Thomas Gloor-Juzi1, Annette Kurre1, Dominik Straumann2 and Eling D de Bruin3* Background With a lifetime prevalence of 29.3 percent in the general
German adult population, moderate to severe vertigo or
dizziness is a frequent and often recurrent symptom [1]. Furthermore, as 80 percent of the patients reported se-
vere limitations in daily activities [1], a considerable cur-
tailing in health-related quality of life may be expected. Moderate to severe vertigo or dizziness can interrelate
with psychiatric disorders, especially anxiety [2-6], which
may increase disability [4]. * Correspondence: debruin@move.biol.ethz.ch
3Institute of Human Movement Sciences and Sport, ETH Zurich, Zurich,
Switzerland
Full list of author information is available at the end of the article Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7
http://www.biomedcentral.com/1472-6815/12/7 Page 2 of 13 arousal or somatic expressions of anxiety [14]. The ori-
ginal VSS as well as a Spanish and a Swedish version
showed acceptable psychometric properties [5,15]. How-
ever, at the beginning of the study (Spring 2007) there was
no validated VSS available in German. The author of the
original VSS, L. Yardley [14] was contacted and she con-
firmed that she had no knowledge of a currently on-going
validation. Hence, she approved the German translation
and cross-culturally adaptation of the VSS and its valid-
ation. Thereby we intended to apply an approved method-
ology guideline for the translation process [16,17] and to
take in consideration existing cultural and language differ-
ences
between
the
German-speaking
populations
in
Europe (e.g., Austria, Germany, Belgium, Switzerland,
Italy) [18]. A German version of the VSS from Tschan
et al. [19] that in the meantime was published did not spe-
cifically comply with guidelines (e.g. use informed and un-
informed translators and back-translators) and neither
considered the possibility of cross-cultural differences,
which might have biased their results. For this reason the
primary aim of the study was the translation and the
cross-cultural adaptation of the VSS into German for the
German-speaking population of Switzerland and to inves-
tigate its internal validity and reliability for patients with
vestibulopathy. The secondary aim of the study was the
investigation of the discriminant and convergent validity
as parts of external validity determination. merger of both translations and in order to resolve discrep-
ancies. Participants
Th
d The study participants were suffering from vertigo, dizzi-
ness, or unsteadiness associated with a diagnosed ves-
tibular disorder for at least one month. They had to be
between 18 and 75 years old, capable of walking and in-
dependently managing approximately 50% of their daily
tasks and have good German language skills. The exclu-
sion criteria comprised dizziness or unsteadiness exclu-
sively due to cardiopulmonary diseases, musculoskeletal
problems or neurologic disorders like severe paresis,
spasticity, cerebellar ataxia, extrapyramidal diseases, or
sensory loss. Furthermore, patients with diagnosed de-
mentia, psychiatric disorders or blindness were excluded. Recruitment took place between July 2007 and July 2009
through the Interdisciplinary Centre for Vertigo & Bal-
ance Disorders, Departments of ENT, Neurology and
Psychiatry at the University Hospital Zurich. All patients
who were referred to the department were asked to par-
ticipate in this study. Those who met the criteria and
submitted their signed written informed consent were
included in the study. Healthy participants were mainly
family members or friends of the authors and their col-
leagues and were also included after submitting their
signed written informed consent. The approval of the
ethics committee of the Canton of Zurich was obtained
in accordance with the Declaration of Helsinki. Translation and cross cultural adaptation of the VSS
The VSS is a self-completed, 34-item questionnaire (19
items for VSS-VER and 15 for VSS-AA). The frequency
of the symptoms is rated on a Likert-scale: 0 points:
“never”, 1 point: “a few times (1–3 times a year)”, 2
points: “several times (4–12 times a year)”, 3 points:
“quite often (on average, more than once a month)” and
4 points: “very often (on average more than once a
week)”. The total score aims to figure out the latent di-
mension of severity of dizziness and ranges from 0
points (no symptoms) to 136 points (severe vertigo). The
translation
and
cross-cultural
adaptation
into
German was performed in six steps, according to the inter-
national guidelines for self-reported measures published by
the American Association of Orthopaedic Surgeons Out-
come Committee [16] and additional information about
requirements for translators from Wild et al. [17]. First step:
two independent German VSS-translations were requested
in order to reflect possible ambiguous wording. Two native
German-speaking translators with excellent English lan-
guage skills translated independently the VSS into German. Background Third step: back translation of the previously
obtained German version into English by two independent
native English speaking translators with German language
skills, both translators were withheld of the original version
of the VSS, again one person was aware of the concept the
other not. This step was necessary in order to bring out un-
clear wording or cultural peculiarities in symptom descrip-
tion and to help to assure a consistent translation of the
content of each item. Fourth step: an expert committee
consisting of the four translators, the methodologist and
two health and language professionals produced the pre-
final version of the VSS. Prior to this meeting; Lucy Yardley
(author of the original VSS) compared the original VSS
with both back translations. Her comments were also con-
sidered in the discussion. Fifth step: the pre-final VSS-G
was pilot tested according to the recommendations of Wild
[17] in a group of fourteen patients who fulfilled the inclu-
sion/exclusion criteria below. Sixth step: The transcription
of the patient interviews and the documents of the preced-
ing steps were analysed in the final stage and the final ver-
sion of the VSS-G was written. Since cross-cultural
differences exist between German-speaking countries [18]
and both Swiss and German patients frequent our hospital,
special consideration was given to specifically include both
German and Swiss cultural background translators and
physiotherapists in our research team. Results After the translation and pilot testing process of a pref-
inal version, the VSS-G was tested with 202 participants
with vertigo or dizziness and 52 healthy subjects (Table 1:
characteristics of participants). Measures The Hospital Anxiety and Depression Scale (HADS) is a
14-item questionnaire measuring psychological distress. It
is divided into two subscales assessing non-somatic symp-
toms of anxiety and depression [20]. Although the whole
questionnaire was submitted to the participants, only the
results of the anxiety subscale are reported here. The items
are rated from 0 to 3 points and the score ranges from 0
(no sign of anxiety) to 21 (maximum level of anxiety). The
validity of the HADS has been demonstrated by a large
number of patient groups. The psychometric properties of
the German version are good and the recommended cut-
off score for screening abnormal anxiety is eleven [21]. External validity determination Discriminant validity was analysed by comparing the me-
dian scores of VSS-G, VSS-VER and VSS-AA between a
group of 52 healthy subjects and the three diagnostic sub-
groups:
“1=peripheral
vestibular
disorder,
2=central
vestibular disorder and 3= multifactorial/multisensory dis-
orders”. Significant differences between the groups were
investigated with the Mann–Whitney-U-Test for independ-
ent groups. If any significant difference between the groups
was found the ROC curve and the Youden Index [31] were
carried out. The Mann–Whitney-U-Test was further used
to investigate the ability of the VSS-AA median score to dif-
ferentiate abnormal anxiety as defined by the HADS-A
(score≥11). xThree items of the University of California Los
Angeles-Dizziness Questionnaire UCLA-DQ [24] were
used to rate the overall perceived frequency of dizziness
(UCLA-DQ1), its intensity (UCLA-DQ2) and the impact
of dizziness on daily activities (UCLA-DQ3). The pro-
blems were rated on a scale ranging from 1 (lowest level
of the severity of the problem) to 5 (highest level). Fur-
thermore the patients had to rate their level of disability
induced by dizziness as no disability (0 points), mild (1),
moderate (2) or severe (3 points). Convergent validity of the VSS-G and its subscales was
analysed using the Spearman’s correlations to investigate
the association of VSS-G, VSS-VER and VSS-AA with
HADS-A and DHI. Kendall’s Tau rank correlation was
performed to analyse the associations of the VSS with
the estimated level of disability and the 3 items of the
UCLA-DQ: 1) frequency, 2) intensity and 3) impact of
dizziness on daily activities. The postal data collection procedure for the reliability
and validity investigation are summarized in Figure 1. The test-retest reliability analysis was carried out on 40
complete pairs of questionnaires [25]. The data collec-
tion procedure was controlled on a daily basis. The qual-
ity was assured by contacting the patients by phone in
order to clear up ambiguous responses, to fill gaps in
the questionnaires or to remind them to return the
questionnaires. All the analysis was performed on IBM
PASW Statistics (formerly SPSS Statistics) version 18
software. Participants
Th
d Moreover, as recommended by Wild [17], one translator
was aware of the measured concept of the VSS (health pro-
fessional), the other not. Second step: meeting of both of
the translators and an observer to keep record of the Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7
http://www.biomedcentral.com/1472-6815/12/7 Page 3 of 13 statistical analysis, ceiling or floor effects in the VSS-G or
the subscales were checked in the study population. Reliability determination Cronbach's α coefficient was calculated to investigate the
internal consistency of the VSS-G and both subscales. For
psychometric scales Cronbach’s α > 0.8 is generally recom-
mended [26]. The Cronbach's α coefficient was also esti-
mated with each item excluded, where the difference
between α-total and α-with-the-item-deleted should not be
greater than 0.1 [27]. The corrected item-total correlations
(CI-TC) were calculated to investigate the strength of rela-
tionship between a single item and the other items in each
of the scales. CI-TC should range between 0.20 and 0.40
[28], with the minimal recommended value of 0.2 [26]. The intra-tester reliability was calculated with the two-way
random model with single measurement intraclass correl-
ation coefficient (ICC2,1 absolute agreement type) [29,30]. The Dizziness Handicap Inventory (DHI) [22] is a
25-item questionnaire designed to evaluate the precipi-
tating physical factors associated with dizziness and
unsteadiness as well as the functional and emotional
consequences of vestibular disease[22]. The items were
rated with “yes” (4 points), “sometimes” (2 points) and
“no” (0 points). The total score ranges from zero (no
disability) to 100 (severe disability). The original ver-
sion
demonstrated
good
face
validity,
internal
consistency and test-retest reliability in a population
with different aetiologies of dizziness and unsteadiness
[22]. A valid German version is available [23]. Translation and pilot testing of the prefinal translation of
the VSS-G However the expert committee in
the fourth step of the process reached consensus for each
item and the patients (in the pilot testing) were able to
recognise and associated the described symptoms with
their vestibular problems. Compared to the German VSS
version that was published during our data gathering
process [19], we found differences in the way sentences
were constructed. This could be due to cross-cultural dif-
ferences between the German-speaking populations in
Germany and Switzerland [18]. However, the wording
used to describe the symptoms was essentially the same. For the pilot testing (cognitive debriefing) of the pre-
final version, 14 patients (8 male) with vestibular disorders
(8 peripheral (57%), 5 central vestibular disorder (37%)
and 1 multifactorial (7%) causes of dizziness could be
included. The mean age (SD) was 60.5 (14.13) years, the
mean scores of the DHI (SD) = 46 (20.0), the VSS = 49 (25)
and the subscales VSS-VER = 24 (14), VSS-AA = 25 (13). headed, swimmy, giddy or walk properly for the items of
the VSS-VER. There were also some discussion needed
to adapt descriptions of symptoms of the VSS-AA like:
about to black out, tingling, prickling, spots before the
eyes, heart pounding, soreness in muscles, stomach
churning, hot or cold spells (not to confound with symp-
toms of menopause). However the expert committee in
the fourth step of the process reached consensus for each
item and the patients (in the pilot testing) were able to
recognise and associated the described symptoms with
their vestibular problems. Compared to the German VSS
version that was published during our data gathering
process [19], we found differences in the way sentences
were constructed. This could be due to cross-cultural dif-
ferences between the German-speaking populations in
Germany and Switzerland [18]. However, the wording
used to describe the symptoms was essentially the same. The participants estimated the matching of the contents
of the VSS items with their symptoms at 92.2 percent (SD
6.9). One outlier (more than 3 standard deviations differ-
ences to the mean), who could not cite any additional
problem or symptom the VSS was not accounting for, was
excluded from the calculation. Fatigue and weariness were
the most cited problems (3 from 14) which are not
included in the VSS. Translation and pilot testing of the prefinal translation of
the VSS-G According to the preceding studies [14,19], a principal
component analysis (PCA) for two factors with Varimax
rotation was performed, in order to compare the loadings
of the items on both factors representing the structure of
the subscales (VSS-VER and VSS-AA). Prior to the Throughout the translation and cross cultural adaptation
process the translators encountered some difficulties to
adapt definitions like: unsteadiness, actually fall, light- Page 4 of 13 Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7
http://www.biomedcentral.com/1472-6815/12/7 Inclusion:
52 healthy persons
Inclusion:
202 persons with vertigo, dizziness or
imbalance
Baseline set of questionnaires
adapted for healthy persons:
VSS-G/ DHI-G/ HADS-G
Baseline set of questionnaires:
VSS1-G/ DHI-G/ HADS-G/ UCLA-DQ1/
UCLA-DQ2/UCLADQ3 socio-demographic data
Discriminant Validity:
•
Pairwise U-Test between
diagnostic groups and
healthy participants
•
ROC-curve for VSS-VER
After 1 week, a sample of persons
assumed to be in stable health
condition with respect of dizziness
(n=58) received a second
questionnaire (VSS2-G)
Report of
stable health
condition
Exclusion of reliability
analysis
no
n= 18
yes
n= 40
Test-Retest Reliability
•
ICC 2,1
Internal validity:
• PCA with Varimax Rotation (2
factors)
Internal Consistency:
• Cronbach's α / α if item deleted
• CI-TC
Construct Validity:
• Spearman’s Correlation coefficient
• Kendall’s Tau correlation coefficient
Figure 1 Flow-chart of data collection and analysis procedure. Inclusion:
202 persons with vertigo, dizziness or
imbalance Baseline set of questionnaires:
VSS1-G/ DHI-G/ HADS-G/ UCLA-DQ1 After 1 week, a sample of persons
assumed to be in stable health
condition with respect of dizziness
(n=58) received a second
questionnaire (VSS2-G) Internal validity:
• PCA with Varimax Rotation (2
factors)
Internal Consistency:
• Cronbach's α / α if item deleted
• CI-TC
Construct Validity:
• Spearman’s Correlation coefficient
• Kendall’s Tau correlation coefficient Report of
stable health
condition Test-Retest Reliability
•
ICC 2,1 Exclusion of reliability
analysis Figure 1 Flow-chart of data collection and analysis procedure. headed, swimmy, giddy or walk properly for the items of
the VSS-VER. There were also some discussion needed
to adapt descriptions of symptoms of the VSS-AA like:
about to black out, tingling, prickling, spots before the
eyes, heart pounding, soreness in muscles, stomach
churning, hot or cold spells (not to confound with symp-
toms of menopause). Translation and pilot testing of the prefinal translation of
the VSS-G However, to the best of our know-
ledge, even if both symptoms could appear with vestibular
disorders, there is no association of fatigue as a symptom
of dizziness described in literature. Therefore there was
no item added to the VSS-G. The other cited symptoms
were diarrhoea, inflammation of the pharynx (sensation of
burning while swallowing), polyuria and incontinence,
tickling and itching in the ears, hunger and panic. Some
patients missed items with vertigo-causing activities
(e.g. walking, head movements) or emotions like feeling
helpless or alone, fear of becoming dependent or losing
work. These propositions underpin the necessity to assess
limitations in activity, participation and emotional distress
but are beyond the scope of the VSS. Based on these
results, the VSS-G can be considered to be complete. For the pilot testing (cognitive debriefing) of the pre-
final version, 14 patients (8 male) with vestibular disorders
(8 peripheral (57%), 5 central vestibular disorder (37%)
and 1 multifactorial (7%) causes of dizziness could be
included. The mean age (SD) was 60.5 (14.13) years, the
mean scores of the DHI (SD) = 46 (20.0), the VSS = 49 (25)
and the subscales VSS-VER = 24 (14), VSS-AA = 25 (13). Although most patients declared to easily understand
the content of the VSS-G, the interviewers noticed Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7
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Participants with
vestibular disorder (n=202)
Subgroup for
reliability testing (n=40)
Healthy participants (n=52)
Age in years: mean (SD)
50.0
(13.47)
50.7
(13.63)
46.7
(13.12)
Gender: n women/men (% women):
124/78
(61.4)
23/17
(57.5)
28/24
(53.8)
Independence or need of support for ADL/household: n (%)
independent
162
(80.2)
33
(82.5)
52
(100.0)
need of occasional support
27
(13.4)
6
(15.0)
0
need of regular weekly or daily support
13
(6.4)
1
(2.5)
0
Duration of dizziness: n (%)
dizziness since ≥1 month, less than 6 mo
58
(28.7)
8
(20.0)
dizziness since ≥6 mo up to 12 mo
27
(13.4)
7
(17.5)
dizziness since > 12 mo
117
(57.9)
25
(62.5)
Diagnosis: n (%)
Unilateral peripheral vestibular disorder:
73
(36.14)
10
(25.00)
unilat BPPV
26
(12.9)
3
(7.5)
unilat. Morbus Meniere
27
(13.4)
4
(10.0)
unilat. Internal validity determination
Principal component analysis p
p
y
Table 2 contains the results of the two factors extraction
with a PCA and Varimax rotation and, for the sake of com-
parison, the loadings of previous versions and translations
of the VSS were also displayed [5,14,19]. Compared to the
original VSS, 87% (13/15) of the items could be clearly
associated to the VSS-AA scale. Indeed, sixteen items
loaded> 0.5 on factor one and thirteen of these items were
attributed to Yardley’s original VSS-AA subscale. The three
remaining items were vertigo symptoms lasting for less
than 2 minutes (1a, 7a, 18a). Their allocation to the
VSS-VER subscale will be explained below. Two items
(9, 13) showed a clearly higher loading on factor one than
on factor two; these loadings were between 0.4 and 0.5. With high face validity, these two items were integrated in
the VSS-AA subscale of VSS-G, so that the VSS-AA sub-
scale was identical to the AA subscale of the original VSS. The VSS-VER subscale showed not such a clearly item
matching. Only 47% (9/19) of the items were attributed to
the VSS-VER based on their loadings >0.5 on factor two. Four items with loadings from 0.4 to 0.5 (1b, 1e, 7e, 15)
were added to VSS-VER because this loading was clearly
higher on factor two than factor one. Three items (4, 5, 11)
showed unclear loadings either on factor one or two. They
were allocated to VSS-VER for the following reason. Item Table 2 contains the results of the two factors extraction
with a PCA and Varimax rotation and, for the sake of com-
parison, the loadings of previous versions and translations
of the VSS were also displayed [5,14,19]. Compared to the
original VSS, 87% (13/15) of the items could be clearly
associated to the VSS-AA scale. Indeed, sixteen items
loaded> 0.5 on factor one and thirteen of these items were
attributed to Yardley’s original VSS-AA subscale. The three
remaining items were vertigo symptoms lasting for less
than 2 minutes (1a, 7a, 18a). Their allocation to the
VSS-VER subscale will be explained below. Two items
(9, 13) showed a clearly higher loading on factor one than
on factor two; these loadings were between 0.4 and 0.5. With high face validity, these two items were integrated in
the VSS-AA subscale of VSS-G, so that the VSS-AA sub-
scale was identical to the AA subscale of the original VSS. Translation and pilot testing of the prefinal translation of
the VSS-G neuritis vestibularis
4
(2.0)
2
(5.0)
others
16
(7.9)
1
(2.5)
Bilateral peripheral vestibular disorder:
17
(8.4)
4
(10.0)
bilat. BPPV
2
(1.0)
0
bilat. M. Meniere
1
(.5)
0
bilat. neuritis vestibularis
4
(2.0)
3
(7.5)
others
10
(5.0)
1
(2.5)
Central vestibular disorder:
73
(36.1)
18
(45.0)
incomplete central compensation
7
(3.5)
2
(5.0)
psycho-physical vertigo
17
(8.4)
2
(5.0)
others (e.g. vest. Migraine)
49
(24.3)
14
(35.0)
Multifactorial/multisensory vestibular disorder:
39
(19.3)
8
(20.0)
Self percieved level of disability: n (%)
little
56
(27.7)
7
(17.5)
moderate
100
(49.5)
23
(57.5)
severe
46
(22.8)
10
(25.0)
UCLA 1: frequency of dizziness: n (%)
rarely
23
(11.4)
4
(10.0)
sometimes
91
(45.0)
19
(47.5)
approximately half of the time
39
(19.3)
10
(25.0)
usually
34
(16.8)
4
(10.0)
always
15
(7.4)
3
(7.5)
UCLA 2: Intensity of dizziness: n (%)
very mild
10
(5.0)
2
(5.0)
mild
23
(11.4)
4
(10.0)
moderate
78
(38.6)
17
(42.5)
moderately severe
73
(36.1)
13
(32.5)
severe
18
(8.9)
4
(10.0)
UCLA 3: dizziness induced limitations in activities or participation: n (%) Table 1 Characteristics of participants Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7
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no effect at all
15
(7.4)
3
(7.5)
continuing all activities but allowance for dizziness
39
(19.3)
8
(20.0)
continuing most of the activities
79
(39.1)
18
(45.0)
continuing some of the activities
51
(25.2)
9
(22.5)
unable to continue any of the activities
18
(8.9)
2
(5.0) Table 1 Characteristics of participants (Continued) 04 “falling over” showed inconsistent loading across the
studies but was attributed to VSS-VER, due to its face val-
idity as a possible symptom in acute vertigo attacks. Simi-
larly, the items 05 “Nausea” and 11 “Vomiting” are two
generic symptoms of the autonomous system and showed,
as expected, somewhat higher loadings on factor one
(VSS-AA). Nevertheless, because of their association to
vestibular disorder, particularly acute vertigo attack, these
symptoms were attributed to VSS-VER. Another issue was
the clear loading on factor one (VSS-AA) of vestibular
symptoms lasting less than 2 minutes (items 1a, 7a, 18a). Translation and pilot testing of the prefinal translation of
the VSS-G This result seems to confirm a trend observed in other
works (Table 2) [5,14,19] and raises the assumption
whether this result represents the interface between vertigo
and anxiety [4] and thus, could be interpreted as an exacer-
bation of autonomic arousal leading to vertigo. among seven persons (50% of participants) difficulties to
understand how to deal with the two-tiered questions 1,
7 and 18. These items were used to determine the fre-
quency range of a vertigo symptom with different duration. The main problem seemed to be the two time-related con-
cepts of symptom duration and frequency. These symptoms
might appear or increase with activities (e.g. walking, head
movements) and will last through the whole activity, which
possibly is an additional confusing time-related aspect. A
similar problem was also described by Holmberg for a
Swedish version of the VSS [32], in order to counteract this
problem we used a clear structure in the layout of these
items , even so health professionals may take into account,
that patients will need assistance for rating these items. Upon suggestion of some patients, the definitions of the
scale-ranking were reported on top margin of each page of
the questionnaire (Additional file 1). Due to the apparently high stability in face of factor
extraction, particularly for the VSS-AA subscale and the
already documented validity of the structure of the VSS
in general [5,14,19], we suggest to keep Yardley’s original
subscale structure for our VSS-G. Internal validity determination
Principal component analysis The cumulative percentage of the scores of the VSS-G,
VSS-VER and VSS-AA were analysed. There was no
floor effect of the VSS-G and the subscales to be
assumed in our sample because the respective scores of
the 15 percent threshold [33] lays above the minimal
possible score of zero: VSS-G = 16–17, VSS-VER = 6–7,
VSS-AA = 5–6. The same holds true for the ceiling effect
as the scores of the 85 percent threshold [33] lays clearly
under the respective maximal score: VSS-G = 59–60
(maximal score = 136), VSS-VER = 32-33(max = 76), VSS-
AA = 30 (max = 60). Reliability determination All the Cronbach’s α-coefficient estimations showed
good internal consistency on scale-level (Table 3). This
was also the case on item-level, as there were no differ-
ences > 0.1 in Cronbach’s α-coefficient estimation in
case the respective item was deleted (Additional file 2)
and the CI-TC showed no value under 0.2. For test-
retest reliability estimations, the mean time between the
first and the second measurement was 5.5 days (SD
1.97 days). As shown in Table 3, all the ICC coefficients
reached the recommended threshold of 0.75 [29]. The VSS-VER subscale showed not such a clearly item
matching. Only 47% (9/19) of the items were attributed to
the VSS-VER based on their loadings >0.5 on factor two. Four items with loadings from 0.4 to 0.5 (1b, 1e, 7e, 15)
were added to VSS-VER because this loading was clearly
higher on factor two than factor one. Three items (4, 5, 11)
showed unclear loadings either on factor one or two. They
were allocated to VSS-VER for the following reason. Item Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7
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Item
Factor 1
Factor 2
CH
Germany Mexic hosp UK hosp. Prim care (UK) Swiss Germany Mexic hosp UK hosp Prim care (UK
01. Things spinning/moving (V**):
a. less than 2 min
0.55
0.35
0.46*
0.34
0.24
0.02
0.13
−0.07
0.21
−0.06
b. 2 to 20 min
0.27
0.08
0.12
0.13
0.14
0.47*
0.64
0.53
0.71
0.44*
c. 20 min to 1 h
0.17
0.08
−0.03
−0.02
0.13
0.60
0.65
0.54
0.83
0.74
d. several hours
−0.08
−0.01
−0.09
−0.08
−0.08
0.63
0.52
0.59
0.71
0.68
e. more than 12 h
−0.02
0.25
−0.02
0.06
−0.03
0.43*
0.06
0.51
0.52
0.36
02. Heart/chest pain (A***)
0.52
0.52
0.67
0.52
0.52
−0.06
0.04
0.09
−0.14
−0.19
03. Hot or cold spells (A)
0.66
0.61
0.67
0.59
0.65
0.16
0.07
0.25
0.16
0.12
04. Falling over (V)
0.40*
0.42*
0.28
0.27
0.40*
0.24
0.20
0.44*
0.36
0.23
05. Nausea, feeling sick (V)
0.47*
0.50
0.58
0.40*
0.56
0.32
0.14
0.35
0.36
0.23
06. Muscle tension/sore (A)
0.63
0.68
0.81
0.67
0.57
0.08
0.14
0.01
0.01
0.18
07. Light-headed/giddy (V):
a. Reliability determination less than 2 min
0.54
0.28
0.50
0.43*
0.28
0.05
0.17
−0.07
0.11
−0.06
b. 2 to 20 min
0.40
0.18
0.30
0.17
0.11
0.60
0.65
0.46*
0.70
0.53
c. 20 min to 1 h
0.21
0.08
0.13
0.18
0.08
0.73
0.77
0.65
0.70
0.74
d. several hours
0.11
0.10
0.08
0.04
0.12
0.70
0.56
0.71
0.73
0.69
e. more than 12 h
0.05
0.24
0.17
0.02
−0.09
0.45*
0.16
0.50
0.46*
0.55
08. Trembling, shivering (A)
0.56
0.63
0.67
0.56
0.53
0.15
0.08
0.27
0.02
0.16
09. Pressure in the ear (A)
0.41*
0.57
0.60
0.27
0.46*
0.06
0.12
0.16
0.24
0.09
10. Heart pounding (A)
0.54
0.59
0.58
0.66
0.64
0.10
−0.12
0.23
0.02
0.09
11. Vomiting (V)
0.22
0.15
0.30
0.01
0.26
0.12
0.22
0.28
0.32
0.38
12. Heavy feeling arms/legs (A)
0.58
0.74
0.72
0.59
0.70
0.24
0.04
0.04
0.04
0.02
13. Visual disturbances (A)
0.42*
0.71
0.72
0.58
0.61
0.11
0.14
0.16
0.10
0.18
14. Headache/pressure (A)
0.57
0.48*
0.71
0.52
0.58
0.11
0.18
0.23
0.06
0.11
15. Unable to stand/walk (V)
0.16
0.40*
0.38
0.27
0.54
0.42*
0.18
0.40*
0.35
0.35
16. Breathing difficulties (A)
0.63
0.69
0.62
0.53
0.72
0.05
−0.07
0.08
0.06
−0.02
17. Loss of concentration (A)
0.56
0.59
0.64
0.61
0.72
0.28
0.05
−0.03
0.05
0.15
18. Feeling unsteady (V):
a. less than 2 min
0.60
0.27
0.54
0.42
0.24
0.22
0.22
0.13
0.13
−0.04
b. 2 to 20 min
0.39
0.12
0.28
0.13
0.10
0.64
0.64
0.62
0.71
0.58
c. 20 min to 1 h
0.28
0.02
0.17
0.06
0.13
0.75
0.76
0.66
0.74
0.77
d. several hours
−0.00
−0.06
−0.12
−0.03
0.08
0.70
0.60
0.69
0.77
0.68
e. more than 12 h
0.03
0.25
−0.04
0.00
−0.06
0.51
0.29
0.60
0.61
0.49*
19. Tingling, prickling (A)
0.55
0.60
0.75
0.59
0.63
−0.00
0.05
0.00
−0.02
0.06
20. Pain in the lower back (A)
0.59
0.56
0.71
0.56
0.66
−0.04
−0.07
−0.08
−0.20
−0.09
21. Excessive sweating (A)
0.57
0.60
0.19
0.47
0.60
0.21
0.05
0.19
0.12
0.18
22. Feeling faint, black out (A)
0.51
0.61
0.02
0.45
0.60
0.19
0.12
0.26
0.30
0.06
The table is adapted from Tschan (Germany, Mainz) [19] and completed with the results of present study Switzerland, (CH) Interdisciplinary Centre for Vertigo &
Balance Disorders, University Hospital Zurich. * Significant (2 tailed) at a <0.001 level. * Significant (2 tailed) at a <0.001 level. Significant (2 tailed) at a <0.001 level. VSS-G = VSS total score, VSS-VER = VSS vertigo subscale, VSS-AA = VSS somatic anxiety subscale. Mann–Whitney-U-test (U) 1) for comparison of median VSS-VER
scores between healthy and diagnostic groups, 2) median VSS-AA scores between patients without and patients with anxiety (HADS-A ≥11). External validity determination distribution of median scores of VSS-AA showed a sig-
nificant difference between people without and those
with abnormal anxiety as defined by the HADS-A (Score≥
11). However, the ROC-Curve showed very low specificity. Discriminant
validity
The
Mann–Whitney-U-Test
showed significant differences in the distribution of the
median scores of the VSS-G, the VSS-VER (Figure 2)
and VSS-AA between healthy subjects and persons with
vestibular disorders (Table 3). Therefore, the areas under
the
ROC-Curve
(CI)
were
calculated:
VSS-G = 0.95
(0.92, 0.97), VSS-VER = 0.99 (0.98, 1.00) and VSS-AA
= 0.84 (0.78, 0.89) (Figure 3). According to the Youden
Index [31] the best relationship of 0.95 sensitivity and
1.00 specificity could be set at VSS-VER Score = 4.5 (or
0.24 item mean). Particularly the VSS-VER (Figure 2)
was able to discriminate between healthy subjects and
persons with vestibular disorders. But there was no skill
to differentiate between the groups of diagnosis. The Reliability determination Factor loadings ≥0.5 are highlighted in bold face; * highest factor loading in analysis when ≥0.4 (but ≤0.5). As grey
background is not acceptable, it was necessary to add the (V) or (S) definition for ich item; ** (V)= item attributed to VSS-VER; ***(A)= item attributed to VSS-AA. Table 2 PCA with Varimax Rotation: table for two factor loadings comparison Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7
Page 8 of 13
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http://www.biomedcentral.com/1472-6815/12/7 Page 8 of 13 Table 3 Internal validity, reliability, discriminant validity results Table 3 Internal validity, reliability, discriminant validity results
VSS-G
VSS-VER
VSS-AA
Cronbach's α [n = 202]
.904
.859
.864
ICC (CI) [n = 40]
.926 (.826/.965)
.920 (.854/.957)
.913 (.737/.963)
Medians Healthy [n = 52] vs. peripheral vest disorder [n = 90]
U
326.50
43.00
1046.50
Z
−8.529*
−9.782*
−5.486 8*
Medians Healthy [n = 52] vs. central vest. disorder [n = 73]
U
153.500
22.000
467.500
Z
−8.742*
−9.467*
−7.171*
Medians Healthy [n = 52] vs. multifactorial disorder [n = 39]
U
89.50
62.00
210.00
Z
−7.421*
−7.797*
−6.456*
No anxiety [n=166] vs. abnormal anxiety [n = 36]
U
1776.50
2446.00
1427.00
Z
−3.811*
−1.706
−4.912*
* Significant (2 tailed) at a <0.001 level. VSS-G = VSS total score, VSS-VER = VSS vertigo subscale, VSS-AA = VSS somatic anxiety subscale. Mann–Whitney-U-test (U) 1) for comparison of median VSS-VER
scores between healthy and diagnostic groups, 2) median VSS-AA scores between patients without and patients with anxiety (HADS-A ≥11). Convergent validity In order to confirm the structure of the subscale regard-
ing the possible interaction with anxiety, a different cor-
relation of VSS-VER and VSS-AA with measurement of
anxiety (HADS-A) was expected. According to the def-
inition of the thresholds of Gill-Body [34], the VSS-AA
correlated fairly with the HADS-A, while there was only
low correlation between the VSS-VER and the HADS-A
(Table 4). Furthermore, the VSS-G showed moderate
correlation with self-perceived disability as measured by
the DHI (Table 4). The Kendall Tau rank correlation Figure 2 Medians and interquartile range of VSS-VER subscores of the three diagnostic groups and healthy subjects. igure 2 Medians and interquartile range of VSS-VER subscores of the three diagnostic groups and healthy subject Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7
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http://www.biomedcentral.com/1472-6815/12/7 Page 9 of 13 Page 9 of 13 Figure 3 ROC-Curve for discrimination between dizzy patients and healthy subjects. igure 3 ROC-Curve for discrimination between dizzy patients and healthy subjects. vestibular disorders and to integrate the information in
the treatment plan. coefficient (Table 5) showed a significant but only fair
correlation between the VSS-G and the self-rated fre-
quency of dizziness (UCLA-DQ1), this correlation could
partly be displayed by the box plot of the VSS-G median
scores against the UCLA-DQ1 (Figure 4). The VSS-G
total score and both subscores correlated fairly with
self-estimated (mild, moderate, severe) disability. The
impact on daily activities correlated fairly with VSS-G
and VSS-VER; however, it only correlated weakly with
VSS-AA. Discussion The VSS was translated and cross-culturally adapted into
German for a wider German speaking population of
Switzerland. The original’s structure of the subscales
could
be
confirmed
and
acceptable
psychometric
properties were found. The VSS-G may be adopted to
collect the self-perceived symptoms of patients with Table 4 Spearman’s correlation coefficients
Spearman's Rho n = 202
VSS-G
VSS-VER
VSS-AA
HADS-A
VSS-VER
.875**
VSS-AA
.865**
.549**
HADS-A
.369**
.186**
.452**
DHI
.554**
.496**
.464**
.448**
**. The correlation is significant on a 0,01 Level (two sided). VSS-G = VSS total
score, VSS-VER = VSS vertigo subscale, VSS-AA = VSS somatic anxiety subscale,
DHI = Dizziness Handicap Inventory total score, HADS-A = Hospital Anxiety and
Depression Scale - Anxiety subscale). Table 4 Spearman’s correlation coefficients Table 4 Spearman’s correlation coefficients
Spearman's Rho n = 202
VSS-G
VSS-VER
VSS-AA Table 4 Spearman’s correlation coefficients
Spearman's Rho n = 202
VSS-G
VSS-VER
VSS-AA
HADS Internal validity determination
Reliability determination The VSS-G translation exhibits high internal consistency
and acceptable test-retest reliability, similar to the original
VSS and its subscales [14]. This indicates the ability of the
translated instrument to reliably screen groups of patients. The aspect of agreement was not analysed in the study
[35]; however, as the VSS was conceived to screen the fre-
quency of the symptoms rather than to measure change,
this was not deemed a compulsory psychometric character-
istic. However, we admit that measuring change remains an
important topic in vestibular rehabilitation. The short form
of the VSS [36,37], for which acceptable limits of agreement
were found in a Turkish translation [38], seems to be an ap-
propriate instrument for this purpose. The findings of this
study justify a German language translation for the short
form VSS for the purpose of establishing an instrument
capable of measuring change in symptoms. Discriminant validity y
Particularly the VSS-VER subscale was able to discriminate
between healthy subjects and people with dizziness. If the
determined VSS-VER cut-off score would be applied,
eleven participants out of 252 (4.5%) were misclassified as **. The correlation is significant on a 0,01 Level (two sided). VSS-G = VSS total
score, VSS-VER = VSS vertigo subscale, VSS-AA = VSS somatic anxiety subscale,
DHI = Dizziness Handicap Inventory total score, HADS-A = Hospital Anxiety and
Depression Scale - Anxiety subscale). Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7
http://www.biomedcentral.com/1472-6815/12/7 Page 10 of 13 Table 5 Kendall’s Tau rank correlation coefficients of VSS with self-estimated disability and 3 items of UCLA-DQ
Kendall-Tau n = 202
VSS-G
VSS-VER
VSS-AA
frequency
intensity
disability
VSS-VER
.711**
VSS-AA
.703**
.397**
frequency (UCLA-DQ1)
.317**
.262**
.299**
intensity (UCLA-DQ2)
.227**
.275**
.108*
.101
disability
.400**
.385**
.321**
.369**
.469**
impact on daily activities (UCLA-DQ3)
.281**
.309**
.172**
.133*
.590**
.508**
** correlation is significant on a 0,01 Level (two sided). * correlation is significant on a 0,05 Level (two sided). VSS-G = VSS-G total score, VSS-VER = VSS vertigo subscale, VSS-AA = VSS somatic anxiety subscale; UCLA-DQ1 = self-estimated frequency of dizziness, UCLA-DQ2
= self-estimated intensity of dizziness, UCLA-DQ3 = self-estimated impact of dizziness on daily activities; disability = self-rated mild, moderate, severe disability. ble 5 Kendall’s Tau rank correlation coefficients of VSS with self-estimated disability and 3 items o ation coefficients of VSS with self-estimated disability and 3 items of UCLA-DQ correlation is significant on a 0,05 Level (two sided). VSS-G = VSS-G total score, VSS-VER = VSS vertigo subscale, VSS-AA = VSS somatic anxiety subscale; UCLA-DQ1 = self-estimated frequency of dizziness, UCLA-DQ2
= self-estimated intensity of dizziness, UCLA-DQ3 = self-estimated impact of dizziness on daily activities; disability = self-rated mild, moderate, severe disability. false negatives. This may indicate that the vestibular symp-
toms of these patients are very low, which may mislead to
scrutinise the effectiveness of a vestibular rehabilitation. Therefore, particularly for these patients all the aspects of
the health problems like comorbidities and limitations in
activities of daily life and participation should be considered
in the treatment plan. Anyway, further investigations were
necessary to analyse the usefulness of this cut-off score
prior to vestibular rehabilitation. The low discriminant
properties for vestibular disorders of the VSS-AA may
highlight the interface between vestibular dysfunction and
disorders of autonomous system and anxiety. Discriminant validity In contrast
the VSS-AA subscale seemed capable to screen for possible
abnormal anxiety, but it seems fair to state that further re-
search is needed on this discriminative property. research for a tool to measure the severity of vertigo symp-
toms, which is supposed to be uncontaminated by anxiety
[14]. On the other hand, the VSS-AA’s fair association with
HADS-A and its discriminant property for abnormal anx-
iety, may suggest that the symptoms of VSS-AA might be
associated with an anxiety disorder. This may confer to the
subscale a certain ability to screen for possible anxiety pro-
blems without providing any other conclusive information
about anxiety (e.g. aetiology of comorbidity). This property
may be helpful in rehabilitation management to counsel the
patients and refer them to health professionals experienced
in psychological disorders for further assessment. The influence of the severity of the symptoms on limita-
tions of daily activities was showed by correlations of the
VSS-G with measurements of dizziness related disability in
daily activities (DHI, UCLA-DQ3) and the self-estimated
disability due to dizziness. These findings are comparable
to the work of Tamber et al.[39], who showed an associ-
ation between the Norwegian VSS short-form and the DHI; Limitations
h The present study was not without limitations; the capacity
of the VSS-VER subscale to differentiate between the
designed diagnostic groups could not be reproduced like in
other similar studies. Our group allocation hampered com-
parison of the results unlike those in other studies with
somewhat other classifications. However, it showed to be
appropriate when targeting vestibular rehabilitation. Fur-
thermore, this group allocation yielded quite unequal group
sizes which, in turn, could have biased the results. There-
fore, calculations were carried out with random selections
of cases within the groups in order to balance the group
sizes, and the described differences were maintained. The
fact that in the meantime a German translation of the VSS
was published [19] could be seen as a further limitation. However, although this translation by a German team was
published during our data collection process, too late to in-
fluence the generation of our version, our work cannot be
outclassed for several reasons. Firstly, our study method-
ology [16,17] took in consideration cultural and language
differences between the German-speaking population of
Switzerland [18]. Secondly, the broad group allocation in
peripheral, central and multifactorial vestibular disorders
was designed to validate the VSS-G for screening people
with vertigo, dizziness or balance disorders prior to vestibu-
lar rehabilitation. Thirdly, the convergent validity analysis
carried out by Tschan and colleagues did not use any
dizziness-specific measurement of disability. Further than disability, symptoms may also have some
influence on health-related quality of life (HRQoL). This
is an important issue, as HRQoL measurement figures
out the dimension of the impact of dizziness on the
patient’s participation to social life. 1) In order to meas-
ure the outcome of vestibular rehabilitation Morris et al. [40,41] developed the Vestibular Rehabilitation Benefit
Questionnaire (VRBQ) with a HRQoL subscale, which
showed a moderate correlation to the VSS. However, this
result should be interpreted cautiously, because there
were VSS items included in this VRBQ subscale. Never-
theless the scale seems useful for measuring changes in
vestibular rehabilitation. 2) Yanik [38] translated and
validated the VSS short-form and the Vertigo, Dizziness
and Imbalance questionnaire (VDI) [42] (containing a
HRQoL subscale) into Turkish, unfortunately there were
no intercorrelations reported. 3) In her work, Tschan[19]
analysed the correlations of the VSS with generic quality
of life measurement (physical and mental health compo-
nents of the SF-36). Conclusions The present German translation of the VSS shows satisfac-
tory psychometric properties for the assessment of the self-
perceived severity of the symptoms on a patient group level
in a large geographic area of German-speaking countries. The high discriminant validity of the VSS-VER subscale
allows it to be used for screening vestibular dysfunction
and coordinate the treatment. The VSS-AA subscale is able
to screen for symptoms which might refer to an anxiety
disorder, which might be addressed additionally in the con-
text of vestibular rehabilitation. As both scales measure frequency, it may be surprising
that the plot of the VSS-G median scores to the frequency
of dizziness (UCLA-DQ1, Figure 4) is not more distinct. The mismatch seems to appear with increasing frequency
(Figure 4) and is probably due to the different structure of
the instruments. As a matter of fact, in the VSS-G several
item scores were added, while the UCLA-DQ1 is a single
overall estimation of the frequency of dizziness. Possibly
there are some confounders influencing frequency: 1) the
described interaction of intensity [43] and/or 2) the non-
negligible mutual influence between the symptoms of both
subgroups (VSS-VER and VSS-AA). Further research is
needed to illustrate the impact of all these possible
interactions. Competing interests Competing interests
The authors declare that there are no competing interests. Competing interests Convergent validity The low association of the VSS-VER with the HADS-A
seems to imply that our finding might confirm Yardley’s Figure 4 VSS-G median scores plotted to dizziness frequency (UCLA-DQ1). Figure 4 VSS-G median scores plotted to dizziness frequency (UCLA-DQ1). Figure 4 VSS-G median scores plotted to dizziness frequency (UCLA-DQ1). Figure 4 VSS-G median scores plotted to dizziness frequency (UCLA-DQ1). Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7
http://www.biomedcentral.com/1472-6815/12/7 Page 11 of 13 and Yardley who measured disability with the Vertigo
Handicap Questionnaire [5,14]. Limitations
h She found remarkably weaker corre-
lations with HRQoL for the VSS-VER subscale than for
the VSS-AA [19]. However, these results should be inter-
preted cautiously because of a possible lack of relation-
ship between the SF-36 measure and the salient areas of
dizziness. Previously, Prieto et al. [42] recommended the
VDI for more relevant and responsive measurements of
health-related quality of life. However, current in-depth
research on this instrument is lacking, which impedes
the possible discussion about which instrument should
be preferred in future trials. Nevertheless, the findings of
Tschan underpin the influence of emotional or psycho-
logical distress on generally perceived quality of life, which
should also be considered in vestibular rehabilitation. Additional files Additional file 1: Final validated VSS-G version. Additional file 2: Reliability results on item level. Additional file 1: Final validated VSS-G version. Additional file 2: Reliability results on item level. Additional file 1: Final validated VSS-G version. Additional file 2: Reliability results on item level. References Cohen HS, Kimball KT: Increased independence and decreased vertigo after
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http://www.biomedcentral.com/1472-6815/12/7 medical responsibility of the study. EDdB contributed to the interpretation of
the data and revised the article critically for its content. All authors read and
approved the final manuscript. medical responsibility of the study. EDdB contributed to the interpretation of
the data and revised the article critically for its content. All authors read and
approved the final manuscript. 17. Wild D, Grove A, Martin M, Eremenco S, McElroy S, Verjee-Lorenz A, Erikson
P: Principles of Good Practice for the Translation and Cultural Adaptation
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Schmid and D. Hauck, for their accurate work. We also thank S. Hegemann,
E. Buffone, L. Rasi and S. Wittwer for their precious help in patient
recruitment. Many thanks also to M. Schmocker and S. Hanke for preparing
the manuscript. Last but not least, we would like to acknowledge
Physioswiss, the Swiss physiotherapy association, for funding the translation
process. There was no involvement of Physioswiss in the study design, the
collection, analysis and interpretation of data, or in the writing of the report,
as well as in the decision to submit the paper for publication. 19. Tschan R, Wiltink J, Best C, Bense S, Dieterich M, Beutel ME, Eckhardt-Henn
A: Validation of the German version of the Vertigo Symptom Scale (VSS)
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comorbidity in different vestibular vertigo syndromes. Results of a
prospective longitudinal study over one year. J Neurol 2009, 256(1):58–65. 27. Griep RH, Rotenberg L, Vasconcellos AG, Landsbergis P, Comaru CM, Alves
MG: The psychometric properties of demand-control and effort-reward
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2009, 82(10):1163–1172. rospective longitudinal study over one year. J Neurol 2009, 256(1):58 3. Eckhardt-Henn A, Best C, Bense S, Breuer P, Diener G, Tschan R, Dieterich M:
Psychiatric comorbidity in different organic vertigo syndromes. J Neurol
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and factor analysis of the Dutch version of the Dizziness Handicap
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review. Advances in Physiotherapy 2007, 9(3):106–116. 8. Received: 9 January 2012 Accepted: 26 June 2012
Published: 2 July 2012 Received: 9 January 2012 Accepted: 26 June 2012
Published: 2 July 2012 24. Honrubia V, Bell TS, Harris MR, Baloh RW, Fisher LM: Quantitative Evaluation
of Dizziness Characteristics and Impact on Quality of Life. Am J Otol 1996,
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Med 1987, 6(4):441–448. Authors' contributions Further research is also needed to determine the ex-
tent of usefulness of VSS in vestibular rehabilitation
such as the determination of cut-off scores which pro-
duce a relevant predictive evidence for rehabilitation
success on the individual patient level. TG-J contributed to the design of the survey. He conducted the statistical
analysis and wrote the manuscript. AK co-initiated the study and contributed
to the design of the study and to the process of data collection and
interpretation. She reassessed the article. DS contributed to the analysis of
data and revised the article thoroughly for its content. As the co-director of
the interdisciplinary centre for vertigo and balance disorders, he bore the Page 12 of 13 Page 12 of 13 Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7
http://www.biomedcentral.com/1472-6815/12/7 Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7
http://www.biomedcentral.com/1472-6815/12/7 Author details
1Ph
i
h
O Author details
1Physiotherapy Occupational Therapy, University Hospital Zurich, Zurich,
Switzerland. 2Interdisciplinary Centre for Vertigo & Balance Disorders,
Departments of ENT, Neurology & Psychiatry, University Hospital Zurich,
Zurich, Switzerland. 3Institute of Human Movement Sciences and Sport, ETH
Zurich, Zurich, Switzerland. 22. Jacobson GP, Newman CW: The development of the Dizziness Handicap
Inventory. Arch Otolaryngol Head Neck Surg 1990, 116(4):424–427. 1Physiotherapy Occupational Therapy, University Hospital Zurich, Zurich,
Switzerland. 2Interdisciplinary Centre for Vertigo & Balance Disorders,
Departments of ENT, Neurology & Psychiatry, University Hospital Zurich,
3 23. Kurre A, van Gool CJ, Bastiaenen CH, Gloor-Juzi T, Straumann D, de Bruin
ED: Translation, cross-cultural adaptation and reliability of the german
version of the dizziness handicap inventory. Otol Neurotol 2009, 30
(3):359–367. Zurich, Switzerland. 3Institute of Human Movement Sciences and Sport, ETH
Zurich, Zurich, Switzerland. References Clin Otolaryngol Allied Sci 1999, 24(4):286–293. 41. Morris AE, Lutman ME, Yardley L: Measuring outcome from vestibular
rehabilitation, part II: refinement and validation of a new self-report
measure. Int J Audiol 2009, 48(1):24–37. 41. Morris AE, Lutman ME, Yardley L: Measuring outcome from vestibular
rehabilitation, part II: refinement and validation of a new self-report
measure. Int J Audiol 2009, 48(1):24–37. y g
16. Beaton DE, Bombardier C, Guillemin F, Ferraz MB: Guidelines for the
process of cross-cultural adaptation of self-report measures. Spine (Phila
Pa 1976) 2000, 25(24):3186–3191. process of cross-cultural adaptation of self-report measures. Spine (Phila
Pa 1976) 2000, 25(24):3186–3191. Page 13 of 13 Page 13 of 13 Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7
http://www.biomedcentral.com/1472-6815/12/7 Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7
http://www.biomedcentral.com/1472-6815/12/7 Gloor-Juzi et al. BMC Ear, Nose and Throat Disorders 2012, 12:7
http://www.biomedcentral.com/1472-6815/12/7 42. Prieto L, Santed R, Cobo E, Alonso J: A new measure for assessing the
health-related quality of life of patients with vertigo, dizziness or
imbalance: the VDI questionnaire. Qual Life Res 1999, 8(1–2):131–139. 43. Perez N, Martin E, Garcia-Tapia R: Dizziness: relating the severity of vertigo
to the degree of handicap by measuring vestibular impairment. Otolaryngol Head Neck Surg 2003, 128(3):372–381. doi:10.1186/1472-6815-12-7
Cite this article as: Gloor-Juzi et al.: Translation and validation of the
vertigo symptom scale into German: A cultural adaption to a wider
German-speaking population. BMC Ear, Nose and Throat Disorders 2012
12:7. 42. Prieto L, Santed R, Cobo E, Alonso J: A new measure for assessing the
health-related quality of life of patients with vertigo, dizziness or
imbalance: the VDI questionnaire. Qual Life Res 1999, 8(1–2):131–139. 43. Perez N, Martin E, Garcia-Tapia R: Dizziness: relating the severity of vertigo
to the degree of handicap by measuring vestibular impairment. Otolaryngol Head Neck Surg 2003, 128(3):372–381. doi:10.1186/1472-6815-12-7
Cite this article as: Gloor-Juzi et al.: Translation and validation of the
vertigo symptom scale into German: A cultural adaption to a wider
German-speaking population. BMC Ear, Nose and Throat Disorders 2012
12:7. doi:10.1186/1472-6815-12-7
Cite this article as: Gloor-Juzi et al.: Translation and validation of the
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English
| null |
Molecular characterisation of NPM1 and FLT3-ITD mutations in a central South African adult de novo acute myeloid leukaemia cohort
|
African journal of laboratory medicine
| 2,021
|
cc-by
| 5,571
|
African Journal of Laboratory Medicine
ISSN: (Online) 2225-2010, (Print) 2225-2002 Page 1 of 6
Original Research Page 1 of 6 Original Research Page 1 of 6 Page 1 of 6 Dates: Keywords: acute myeloid leukaemia; AML; NPM1; FLT3-ITD; frequency; South Africa. Keywords: acute myeloid leukaemia; AML; NPM1; FLT3-ITD; frequency; South Africa How to cite this article:
Kloppers JF, De Kock A,
Cronjé J, Van Marle A-C. Molecular characterisation of
NPM1 and FLT3-ITD
mutations in a central South
African adult de novo acute
myeloid leukaemia cohort. Afr J Lab Med. 2021;10(1),
a1363. https://doi. org/10.4102/ajlm.v10i1.1363 Molecular characterisation of NPM1 and FLT3-ITD
mutations in a central South African adult de novo
acute myeloid leukaemia cohort Authors:
Jean F. Kloppers1,2
André de Kock1,2
Johané Cronjé1,2
Anne-Cecilia van Marle1,2
Affiliations:
1Department of Haematology
and Cell Biology, School of
Pathology, Faculty of Health
Sciences, University of the
Free State, Bloemfontein,
South Africa Background: Recognition of molecular abnormalities in acute myeloid leukaemia (AML) has
improved our understanding of its biology. NPM1 and FLT3-ITD mutations are recurrent in
AML and clinically significant. NPM1 mutations are associated with a favourable prognosis,
while FLT3-ITD mutations are an independent poor prognostic factor in AML. Objective: This study described the prevalence and molecular characteristics of the NPM1 and
FLT3-ITD mutations in a newly diagnosed AML patient cohort in central South Africa. Objective: This study described the prevalence and molecular characteristics of the NPM1 and
FLT3-ITD mutations in a newly diagnosed AML patient cohort in central South Africa. Read online:
Scan this QR
code with your
smart phone or
mobile device
to read online.
Read online:
Scan this QR
code with your
smart phone or
mobile device
to read online. Affiliations: Affiliations:
1Department of Haematology
and Cell Biology, School of
Pathology, Faculty of Health
Sciences, University of the
Free State, Bloemfontein,
South Africa Methods: The study included 40 de novo AML patients. An NPM1 and FLT3-ITD multiplex
polymerase chain reaction assay was optimised to screen patients for the respective mutations
and were confirmed using Sanger sequencing. The prevalence of the NPM1 and FLT3-ITD
mutations were determined, and mutation-specific characteristics were described in relation
to patients’ demographic information and AML classifications. Methods: The study included 40 de novo AML patients. An NPM1 and FLT3-ITD multiplex
polymerase chain reaction assay was optimised to screen patients for the respective mutations
and were confirmed using Sanger sequencing. The prevalence of the NPM1 and FLT3-ITD
mutations were determined, and mutation-specific characteristics were described in relation
to patients’ demographic information and AML classifications. 2Department of Haematology
and Cell Biology, Universitas
Academic Unit, National
Health Laboratory Services,
Bloemfontein, South Africa Results: The patients’ median age was 38.5 years, with 77.5% (n = 31) of patients being self-
proclaimed Black Africans. AML with recurrent genetic abnormalities was most prevalent
(57.5%; n = 23), of which acute promyelocytic leukaemia (APL) was most common (40.0%;
n = 16). None of the patients had the NPM1 mutation. FLT3-ITD was present in 37.5% (6/16)
of APL patients and in one (20.0%) of five AML patients with a t(8;21) translocation. Most
patients had an FLT3-ITD allele ratio of ≥ 50% and ITD lengths of > 39 bp. Conclusion: FLT3-ITD mutations were mainly found in APL cases at a similar prevalence as
reported in the literature. High FLT3-ITD allele ratios and long ITD lengths predominated. No
NPM1 mutations were detected. Open Access Introduction Over the past decade, the field of acute myeloid leukaemia (AML) diagnostics has shifted from a
primarily clinicopathological assessment to an integrated approach, including morphology,
immunophenotyping, cytogenetics, and molecular genetics.1,2 In an era of precision medicine, the
use of molecular genetic data, in particular, has fundamentally shaped the diagnostic approach to
patients with AML.3 Genetic profiling has led to more accurate diagnostic classification and
improved risk stratification and identification of potential therapeutic targets.1 Acute myeloid
leukaemia is a genetically heterogeneous disease.2 Recurrent structural chromosomal aberrations
detected by conventional cytogenetics were previously considered the most relevant prognostic
variable in addition to the patient’s age and performance status.2,3 Three main cytogenetic risk
groups (low, intermediate, and high) were defined, with approximately 50% of patients having a
normal karyotype and falling into the intermediate cytogenetic risk group.3,4 Copyright:
© 2021. The Authors. Licensee: AOSIS. This work
is licensed under the
Creative Commons
Attribution License. The recognition of additional molecular abnormalities, especially in cytogenetically normal AML
(CN-AML), has helped to improve our understanding of the disease biology and better predict
outcomes.3,4 Mutational analysis performed on diagnostic samples of AML patients enrolled in the
Eastern Cooperative Oncology Group E1900 trial demonstrated somatic mutations in as many as
97.3% of these patients.5 The Cancer Genome Atlas Research Group6 has shown that, on average,
patients with AML have 13 acquired mutations at the time of diagnosis across the entire genome. Two genes in which recurrent mutations, which often co-occur, with known clinical significance
have been identified in patients with AML include the gene encoding nucleophosmin protein-1
(NPM1) and one of the signalling genes, FMS (Feline McDonough Sarcoma gene)-related tyrosine
kinase 3 (FLT3).3,6 NPM1 mutations are associated with a favourable prognosis,3 while the FLT3- Read online:
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to read online. http://www.ajlmonline.org http://www.ajlmonline.org Open Access Original Research Page 2 of 6 Page 2 of 6 internal tandem duplication (ITD) mutation is considered an
independent poor prognostic factor in patients with AML.7
However, the co-occurrence of these two gene mutations
modulates their prognostic impacts.3,7 Study population All adult patients (n = 40; coded FN1–FN40) that were
diagnosed with de novo AML at the Universitas Academic
Hospital in Bloemfontein, South Africa, during the study
period (November 2018 – December 2019), and who were
able to give written informed consent were included in this
study. All AML patients were included regardless of
cytogenetic findings. Patients’ demographic variables (age,
sex, and self-proclaimed ethnic background) and disease-
specific characteristics (AML classification according to the
World Health Organization’s Classification of Tumours of
Haemopoietic and Lymphoid Tissues, revised 4th edition16) were
obtained from patients’ medical records. Based on the
laboratory information records that were reviewed, none of
the patients had any previous clonal myeloid disorder. Blood
samples were obtained from each participant for this study
during routine blood collection by the treating physician. Nucleophosmin is a nucleolar phosphoprotein that shuttles
between the nucleus and the cytoplasm.3 Nucleophosmin
protein-1 is thought to be involved in many cellular functions,
including protein synthesis, DNA replication, and regulation
of the cell cycle.3 The reported frequency of NPM1 mutations
in patients with AML varies between 27% and 30% and is
considered the most frequently identified molecular
abnormality in CN-AML, occurring in 45% – 60% of cases.4,6,8,9
Four common mutations cause mutant NPM1 (one 4 bp
duplication and three separate 4 bp insertions). These
mutations are all restricted to exon 12, the region that encodes
the C-terminus of NPM-1, and result in a frame shift.10 The
FLT3 gene encodes a Class III receptor tyrosine kinase. Mutations in signalling genes such as FLT3, which lead to
constitutive activation of the receptor tyrosine kinase, confer
survival and proliferative advantage to cells.3,9 FMS-related
tyrosine kinase 3 mutations occur in approximately one-third
of patients with AML, and the ITD mutation is considered
the second most common molecular abnormality in CN-
AML (28% – 34% of cases).4,9 FMS-related tyrosine kinase
3-ITD mutations usually occur between exons 14 and 15 and
can range between 3 bp and more than 400 bp in size.11 DNA extraction Genomic DNA was extracted from whole blood samples
using the Wizard® Genomic DNA Purification Kit (Promega,
Madison, Wisconsin, United States). DNA samples were
quantified using the BioDrop™ µLITE instrument (BioDrop,
Cambridge, United Kingdom). NPM1 and FLT3-ITD multiplex PCR optimisation The limited reports on the prevalence of the FLT3-ITD and
NPM1 mutations in African AML populations indicate lower
frequencies than reported internationally. In an Egyptian study
that included 123 AML patients, FLT3-ITD and NPM1
mutations were detected in 17.9% and 19.5% of patients.12
Another study by Sofan et al. (2014) detected NPM1 mutations
in 28% of CN-AML.13 Contrary to these findings, no NPM1
mutations were found in a study of 100 Sudanese AML
patients.14 The frequency of NPM1 and FLT3-ITD mutations in
a South African adult de novo AML cohort was 7.5% and 12%.15 Anonymised samples that were positive and negative for the
NPM1 and FLT3-ITD mutations and formed part of an
external quality control programme (generously donated
by the Department of Haematology at Charlotte Maxeke
Johannesburg
Academic
Hospital,
National
Health
Laboratory Service, Johannesburg, South Africa) were used
to optimise the assay. Published primers, fluorescently
labelled with hexadecimal colour and fluorescein amidites
fluorophores17 were used in the assay (NPM1 Forward: 5’-
GTT TCT TTT TTT TTT TTT CCA GGC TAT TCA AG- 3’;
Reverse: 5’- HEX CAC GGT AGG GAA AGT TCT CAC TCT
GC- 3’; and FLT3-ITD Forward: 5’FAM- AGCA ATT TAG
GTA TGA AAG CCA GCTA- 3’; Reverse: 5’- CTT TCA GCA
TTT TGA CGG CAA CC- 3’). Primers were evaluated for
target-specificity using the Basic Local Alignment Search
Tool (available at https://blast.ncbi.nlm.nih.gov/Blast.cgi)
from the National Center for Biotechnology Information
(NCBI, Bethesda, Maryland, United States). This study investigated the presence and molecular
characteristics of the NPM1 and FLT3-ITD mutations in a
newly diagnosed AML population in central South Africa. The study further aimed to describe the NPM1 and FLT3-ITD
mutations in relation to patient demographics and specific
AML classifications. Sanger sequencing of NPM1 and FLT3-ITD
amplicons NPM1 and FLT3-ITD control samples, as well as the
amplicons from the 40 patient samples, were sequenced to
confirm and validate the PCR results. Sequencing reactions
for the NPM1 and FLT3-ITD mutations were prepared
separately using primers as listed above (primers for
sequencing were not fluorescently labelled). The PCR
products were purified using the ExoSAP-IT® Express PCR
Product Clean-up (Affymetrix, Santa Clara, California,
United States) as per the manufacturer’s instructions. Sanger
sequencing reactions were prepared using the BigDye™
Terminator version 3.1 kit (Applied Biosystems, Foster City,
California, United States) as per the manufacturer’s
instructions. Briefly, the sequencing reaction consisted of
2 µL Sequencing Reaction Mix (BigDye™ Terminator v3.1),
1 µL Sequencing Buffer (BigDye™ Terminator v3.1), 5 µL
nuclease-free water, 1 µL forward primer (FLT3 or NPM1),
1 µL reverse primer (FLT3-ITD or NPM1) and 1 µL PCR
product. The sequencing reaction was subjected to the
following cycling conditions in a 2720 Thermal Cycler
(Applied Biosystems, Foster City, California, United States):
one cycle at 96 °C for 1 min, and 25 cycles at 96 °C for 10 s,
50 °C for 5 s, and 60 °C for 4 min. Sequence reactions were
cleaned up with the Zymo Research DNA Sequencing Clean-
up Kit™ (Promega Madison, Wisconsin, United States) as
per the manufacturer’s instructions. Sequence raw reads Ethical considerations gradient temperatures ranged between 65 °C and 70 °C. The
subsequent PCR cycling conditions were one cycle at 95 °C
for 10 min, 45 cycles of 95 °C for 20 s, 65 °C for 40 s, and 72 °C
for 40 s, and a final step of one cycle at 72 °C for 32 min. Polymerase chain reaction products were subjected to
capillary electrophoresis (3500 Genetic Analyser, Applied
Biosystems, Foster City, California, United States). Briefly,
1 µL of each sample was mixed with 9.5 µL of Hi-Di™
Formamide (Applied Biosystems, Foster City, California,
United States) and 0.5 µL GeneScan™ 600 LIZ™ size standard
(Applied Biosystems, Foster City, California, United States). The optimal primer concentrations (5 μM) and DNA limit of
detection were determined as part of the optimisation
procedure. The NPM1 and FLT3-ITD multiplex PCR assay
had a lower limit of detection of 0.8 ng/µL. Capillary
electrophoretic results were analysed using GeneMapper
version 6 software (Applied Biosystems, Foster City,
California, United States). Ethical considerations A polymerase chain reaction (PCR) mixture was prepared
with the GoTaq® DNA polymerase (Promega Madison,
Wisconsin, United States) reagents. Components of the kit
were manually mixed and the mixture consisted of 5 μl 5Х
GoTaq Buffer, 2 μL of MgCl2, 0.5 μL of dNTPs, and 0.2 μL of
GoTaq DNA polymerase (5 U/µL). Additionally, 1 μL of
genomic DNA (100 ng/μL), 14.3 μL of nuclease-free water,
and 1 μL each of the forward and reverse primers (5 μM) (for
FLT3 and NPM1) were added to the PCR mixture. A
temperature gradient experiment was used to determine
the optimal PCR annealing temperature for the primers. The Approval for the study was obtained from the Health
Sciences Research Ethics Committee from the University
of
the
Free
State
(study
approval
number:
UFS-
HSD2018/1174/2711)
and
the
Free
State
Province
Department
of
Health
(study
approval
number:
FS_201810_016). Written informed consent was obtained
from all participants prior to sample collection. Samples were
allocated study numbers to ensure patient confidentiality,
and patient data were stored on password-protected devices,
which were only accessible to the researchers. http://www.ajlmonline.org http://www.ajlmonline.org Open Access Page 3 of 6 Original Research Page 3 of 6 gradient temperatures ranged between 65 °C and 70 °C. The
subsequent PCR cycling conditions were one cycle at 95 °C
for 10 min, 45 cycles of 95 °C for 20 s, 65 °C for 40 s, and 72 °C
for 40 s, and a final step of one cycle at 72 °C for 32 min. Polymerase chain reaction products were subjected to
capillary electrophoresis (3500 Genetic Analyser, Applied
Biosystems, Foster City, California, United States). Briefly,
1 µL of each sample was mixed with 9.5 µL of Hi-Di™
Formamide (Applied Biosystems, Foster City, California,
United States) and 0.5 µL GeneScan™ 600 LIZ™ size standard
(Applied Biosystems, Foster City, California, United States). The optimal primer concentrations (5 μM) and DNA limit of
detection were determined as part of the optimisation
procedure. The NPM1 and FLT3-ITD multiplex PCR assay
had a lower limit of detection of 0.8 ng/µL. Capillary
electrophoretic results were analysed using GeneMapper
version 6 software (Applied Biosystems, Foster City,
California, United States). calculated by determining the area under the curve for
the mutant to wild-type alleles, expressed as a percentage. The respective positive and negative controls yielded the
expected amplicon sizes upon evaluation of the capillary
electrophoresis results (Figure 1). NPM1 and FLT3-ITD detection in acute myeloid
leukaemia patients The optimised NPM1 and FLT3-ITD multiplex PCR was used
to screen the 40 de novo AML patients for the respective
mutations. Positive and negative controls, as well as a no-
template control, were included in each subsequent run for
quality control purposes. The NPM1 and FLT3-ITD wild-
type amplicon sizes were expected to be 170 base pairs (bp)
and 330 bp. The NPM1 positive samples were expected to
have an amplicon at 170 bp for the wild-type and an
additional amplicon at 174 bp. FMS-related tyrosine kinase
3-ITD positive samples were expected to have an amplicon at
330 bp and an additional amplicon larger than 330 bp. The
allele ratio for the respective mutations in each patient was http://www.ajlmonline.org
Open Access
50 000
25 000
RFU
Fragment size in base pairs (bp)
Fragment size in base pairs (bp)
Fragment size in base pairs (bp)
0
0
120
240
360
480
600
a
b
c
0
120
240
360
480
600
50 000
25 000
0
RFU
RFU
0
120
240
360
480
600
50 000
25 000
0
RFU, relative fluorescence units. Figure 1: Observed capillary electropherograms of the NPM1 and FLT3 control samples (February 2020, Bloemfontein, Free State, South Africa). (a) NPM1 positive control
with an additional amplicon at 174 base pairs representing the 4 base pairs insertion, and wild-type FLT3-ITD at 330 base pairs. (b) FLT3-ITD positive control with an
additional amplicon at 405 base pairs, representing the 75 base pairs internal tandem duplication and NPM1 wild-type at 170 base pairs. (c) NPM1 and FLT3-ITD negative
control with expected amplicons at 170 base pairs and 330 base pairs. a a b RFU, relative fluorescence units. RFU, relative fluorescence units. Figure 1: Observed capillary electropherograms of the NPM1 and FLT3 control samples (February 2020, Bloemfontein, Free State, South Africa). (a) NPM1 positive control
with an additional amplicon at 174 base pairs representing the 4 base pairs insertion, and wild-type FLT3-ITD at 330 base pairs. (b) FLT3-ITD positive control with an
additional amplicon at 405 base pairs, representing the 75 base pairs internal tandem duplication and NPM1 wild-type at 170 base pairs. (c) NPM1 and FLT3-ITD negative
control with expected amplicons at 170 base pairs and 330 base pairs. Data analysis AML, acute myeloid leukaemia; wt, wild-type; Δ, mutant; APL, acute promyelocytic
leukaemia; ITD, internal tandem duplication; CN-AML, cytogenetically normal AML. AML, acute myeloid leukaemia; wt, wild-type; Δ, mutant; APL, acute promyelocytic
leukaemia; ITD, internal tandem duplication; CN-AML, cytogenetically normal AML. Fragment analysis electropherograms (Figure 1) were
generated using the GeneMapper v6 software (Applied
Biosystems, Foster City, California, United States), and
displayed the size of the alleles obtained for the target PCR
fragments (Applied Biosystems, Foster City, California,
United States). FLT3-ITD allelic ratios were determined by
calculating the ratio of the area under the curve of the FLT3-
ITD mutant allele to the FLT3 wild-type allele as displayed
by GeneMapper. Table 2: Acute myeloid leukaemia patients positive for the FLT3-ITD mutation
(August 2020, Bloemfontein, Free State, South Africa). Table 2: Acute myeloid leukaemia patients positive for the FLT3-ITD mutation
(August 2020, Bloemfontein, Free State, South Africa). Patient
AML
classification
Age (years)
Sex
Race
ITD length
(bp)
Allele ratio (%)
FN14
APL t(15; 17)
25
M
ME
39
63.3
FN19
APL t(15; 17)
26
F
B
27
48.4
FN20
APL t(15; 17)
19
M
B
59
50.1
FN21
APL t(15; 17)
33
M
B
44
94.9
FN26
APL t(15; 17)
59
F
W
50
39.6
FN32
APL t(15; 17)
34
F
B
19
79.5
FN36
t(8; 21)
20
M
B
75
17.5
AML, acute myeloid leukaemia; APL, acute promyelocytic leukaemia; M, male; F, female; ME,
mixed ethnicity; B, Black; W, White; ITD, internal tandem duplication; bp, base pairs. The sequence was analysed using the Sequencing Analysis
Program version 5.3.1 (Applied Biosystems, Foster City,
California, United States) and Chromas version 2.6.6
(Technelysium Pty Ltd, Brisbane, Australia). Tables were
generated using Microsoft Word 2016 (Microsoft, Redmond,
Washington, United States). Patients’ demographic data and
AML classification were summarised in table format. Ethnicity, defined as either black, white or mixed race, was
based on patients’ self-proclaimed identity and accordingly
documented. AML, acute myeloid leukaemia; APL, acute promyelocytic leukaemia; M, male; F, female; ME,
mixed ethnicity; B, Black; W, White; ITD, internal tandem duplication; bp, base pairs. duplication lengths of the FLT3 mutations observed in this
study ranged between 19 bp and 75 bp, and the FLT3-ITD
allele ratios ranged between 17.5% and 94.9%. Discussion Acute myeloid leukaemia is a genetically heterogeneous
disease2 with numerous clinical and genetic factors
influencing the final diagnosis. The recognition of
additional molecular abnormalities, such as NPM1 and
FLT3-ITD in AML, has ensured that the disease biology is
better understood and has allowed for improved risk
stratification to be included in the diagnostic algorithms.3,4
This is the first study to investigate the presence of NPM1
and FLT3-ITD in a central South African AML population. The NPM1 was absent in this study population, while the
FLT3-ITD mutation predominated in the APL subtype. Furthermore, the majority of patients that were positive for
the FLT3-ITD mutation had long ITD lengths and allelic
ratios above 50%. NPM1 and FLT3-ITD detection in acute myeloid
leukaemia patients Open Access http://www.ajlmonline.org Page 4 of 6 Page 4 of 6 Original Research Table 1: Classification of acute myeloid leukaemia patients’ NPM1 and FLT3-ITD
genotypes (August 2020, Bloemfontein, Free State, South Africa). AML classification
NPM1wt/
FLT3-ITDwt
NPM1Δ/
FLT3-ITDΔ
NPM1wt/
FLT3-ITDΔ
NPM1Δ/
FLT3-ITDwt
n
%
n
%
n
%
n
%
AML with recurrent genetic
abnormalities
16
40.0
0
0
7
17.5
0
0
Translocation t(8; 21)
4
10.0
0
0
1
2.5
0
0
Inversion 16
1
2.5
0
0
0
0
0
0
Translocation t(9; 22)
1
2.5
0
0
0
0
0
0
APL translocation
t(15;17)
10
25.0
0
0
6
15.0
0
0
AML with myelodysplasia-
related changes
3
7.5
0
0
0
0
0
0
AML not otherwise
specified (CN-AML)
14
35.0
0
0
0
0
0
0
Minimal differentiation
1
2.5
0
0
0
0
0
0
Without maturation
3
7.5
0
0
0
0
0
0
With maturation
6
15.0
0
0
0
0
0
0
Myelomonocytic
4
10.0
0
0
0
0
0
0
AML, acute myeloid leukaemia; wt, wild-type; Δ, mutant; APL, acute promyelocytic
leukaemia; ITD, internal tandem duplication; CN-AML, cytogenetically normal AML. Table 1: Classification of acute myeloid leukaemia patients’ NPM1 and FLT3-ITD
genotypes (August 2020, Bloemfontein, Free State, South Africa). were loaded onto the 3500 Genetic Analyser (Applied
Biosystems, Foster City, California, United States). For
comparison, sample sequences were aligned against NPM1
(GenBank accession: NG_016018.1) and FLT3-ITD (GenBank
accession: NG_007066.1) reference sequences retrieved
from the National Center for Biotechnology Information
using the Local Pairwise Sequence Alignment Algorithm
software (Available at https://embnet.vital-it.ch/software/
LALIGN_form.html). Sanger sequence analysis confirmed
that the NPM1 positive control had a 4 bp insertion (bases:
CATG), and the FLT3-ITD positive control had a 75 bp ITD. The mutation-negative controls were compared to the
respective reference sequences, and were found to be 100%
similar. Conclusion The FLT3-ITD mutation was mainly found in APL cases at a
similar prevalence as reported in the literature. High FLT3-
ITD allele ratios and long ITD lengths predominated. No
NPM1 mutations were detected. The absence and lower
frequency of NPM1 and FLT3-ITD mutations could possibly
be attributed to the low median age at presentation and the
majority of patients presenting with AML with recurrent
genetic abnormalities. Acknowledgements The authors acknowledge the National Health Laboratory
Service for funding the project, Ms Leandi du Plessis for
technical assistance and Dr Daleen Struwig for assistance
with the technical and editorial preparation of the
manuscript. An interesting observation was the unusually high number
of APL cases among our AML cohort, which, according to
the revised 4th edition of the Classification of Tumours of
Haemopoietic and Lymphoid Tissues, only accounts for 5% – 8%
of AML cases.16 Notably, eight of our APL patients presented
within a single month. Several studies, including a South
African study, have alluded to the association between APL
clustering and seasonality.25,26,27 With such a small study
cohort, one can only speculate that seasonality may have
contributed and warrants further investigation. Competing interests The authors declare that they have no financial or personal
relationships that may have inappropriately influenced them
in writing this article. Limitations This study was limited by a small sample population. In
addition, the predominance of APL cases in our cohort may
have contributed to a biased prevalence of the FLT3-ITD
mutation. An association between phenotype and genotype
was also not determined. Internal tandem duplication lengths of the FLT3 mutations
observed in this study ranged between 19 bp and 75 bp and
were found in the juxtamembrane domain of the gene. According to Liu et al.,23 longer ITD lengths (more than
39 bp) have been associated with a worse prognosis, the
possible reason being that longer insertions in the
juxtamembrane domain may cause more significant
disruption of the domain’s auto-inhibitory function.23 In our
study, 71.4% (5/7) of the patients with FLT3-ITD mutations
had an ITD length of 39 bp or longer. Further investigations
are needed to determine whether the high prevalence of
FLT3-ITD mutations in our APL cohort confers a worse
prognosis. This investigation would be justified considering
that the majority (4 of 6) of APL patients with FLT3-ITD
mutations had allele ratios above 50% and in other AML
subgroups, allelic ratios above 50% are stratified as an
adverse prognostic risk.24 Original Research considering that the frequency of NPM1 mutations increases
proportionally with age.28 In addition, NPM1 mutations are
more
commonly
associated
with
CN-AML,16
which
accounted for only 35% (n = 14) of all cases in this study.4,9
The Cancer Genome Atlas Research Network reported that
nearly 50% of all AML patients are CN-AML cases, and the
observed prevalence of CN-AML in the current study was
lower.6 The lower prevalence of CN-AML relative to AML
with recurrent genetic abnormalities in our study, and, by
association, the lower prevalence of these two mutations
commonly linked with CN-AML, might be attributed to
race, as Black patients were previously found to be more
likely
to
have
AML
associated
with
cytogenetic
abnormalities.18,29 self-proclaimed
Black
Africans,
and
there
was
no
predominant disease presentation based on sex. Two other
African studies found a similar age distribution and sex
ratio.19,20 The FLT3-ITD mutation was only detected in patients with
AML with recurrent genetic abnormalities, of which APL
predominated. Compared to a reported frequency of FLT3-
ITD mutations in APL of up to 40%,16 our results were not
surprising. However, FLT3-ITD mutations in AML with a
t(8;21) translocation, detected in one of our patients, is
thought to be uncommon, occurring in less than 10% of AML
cases.21 The reported frequency of FLT3-ITD mutations in
CN-AML in African populations varies significantly from
11% in South African patients to up to 34.6% in an Egyptian
cohort, which is more in line with data from high-income
countries.9,15,22 The absence of FLT3-ITD in CN-AML patients
in our study was unexpected, but could likely be attributed
to the small study cohort. We recommend that the FLT3-ITD
frequency should be investigated in a larger central South
African CN-AML population. Results The AML patients (FN1 to FN40) were all successfully
screened with the NPM1 and FLT3-ITD multiplex PCR assay. The AML patient cohort had a median age of 38.5 years
(range 18–85 years) at presentation and the ratio of women to
men was 1:1.2. The cohort included 31 black African patients,
four white patients, and five patients of mixed ethnicity. Acute myeloid leukaemia subtypes in our patient cohort
comprised of AML with recurrent genetic abnormalities,
AML with myelodysplasia-related changes, and AML not
otherwise specified (also referred to as CN-AML). Acute
myeloid leukaemia with recurrent genetic abnormalities was
most prevalent (57.5%; n = 23), of which acute promyelocytic
leukaemia (APL) was most common (40.0%; n = 16). None of
the patients had the NPM1 mutation. FMS-related tyrosine
kinase 3-ITD was present in 37.5% (6/16) of APL patients and
in one (20.0%) of five AML patients with a t(8; 21) translocation
(Table 1). Patients with an FLT3-ITD mutation had a median
age of 30.8 years at presentation, and the majority of patients
were black Africans (n = 5/7) (Table 2). Internal tandem It was notable that the median age of our patient cohort was
38.5 years. The global median age at presentation for AML is
67 years18 and, in comparison, the current patient cohort
presented with the disease at a considerably younger age. This finding was similar to a previous South African study,
where adult AML patients presented with the disease at a
median age of 41 years.15 Most patients in this study were http://www.ajlmonline.org Open Access Page 5 of 6 Page 5 of 6 Original Research References 19. Gmidène A, Sennana H, Wahchi I, et al. Cytogenetic profile of a large cohort of
Tunisian de novo acute myeloid leukemia. Hematology. 2012;17(1):9–14. https://
doi.org/10.1179/102453312X13221316477417 1. Shanmugam V, Kim AS. Molecular versus morphological classifications of
myeloproliferative neoplasms: You don’t know JAK! The Hematologist: ASH News
and Reports [serial online]. 2019;16(1):12–13. Available from: https://pdfs. semanticscholar.org/5ac1/b04d4eed355331e2c6910a351efdc79b6c39.pdf?_
ga=2.24175993.2125433055.1594648374-906043641.1594648374 1. Shanmugam V, Kim AS. Molecular versus morphological classifications of
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ga=2.24175993.2125433055.1594648374-906043641.1594648374 20. Khoubila N, Bendari M, Hda N, et al. Cytogenetic profile of a representative cohort
of young adults with de novo acute myéloblastic leukaemia in Morocco. Cancer
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bjh.16360 21. Lin P, Chen L, Luthra R, Konoplev SN, Wang X, Medeiros J. Acute myeloid leukemia
harboring t(8;21)(q22;q22): A heterogeneous disease with poor outcome in a
subset of patients unrelated to secondary cytogenetic aberrations. Mod Pathol. 2008;21(8):1029–1036. https://doi.org/10.1038/modpathol.2008.92 3. Kansal R. Acute myeloid leukemia in the era of precision medicine: Recent
advances in diagnostic classification and risk stratification. Cancer Biol Med. 2016;13(1):41–54. https://doi.org/10.20892/j.issn.2095-3941.2016.0001 22. Shamaa S, Laimon N, Aladle DA, et al. Prognostic implications of NPM1 mutations
and FLT3 internal tandem duplications in Egyptian patients with cytogenetically
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10.1179/1607845413Y.0000000085 4. Roloff GW, Griffiths EA. When to obtain genomic data in acute myeloid leukemia
(AML) and which mutations matter. Blood Adv. 2018;2(21):3070–3080. https://
doi.org/10.1182/bloodadvances.2018020206 23. Liu SB, Dong HJ, Bao XB, et al. Impact of FLT3-ITD length on prognosis of acute
myeloid leukemia. Haematologica. 2019;104(1):e9–e12. https://doi.org/10.3324/
haematol.2018.191809 5. Patel JP, Gönen M, Figueroa ME, et al. Prognostic relevance of integrated genetic
profiling in acute myeloid leukemia. N Engl J Med. 2012;366(12):1079–1089. https://doi.org/10.1056/NEJMoa1112304 24. Tallman MS, Wang ES, Altman JK, et al. Acute myeloid leukemia, version 3.2019,
NCCN clinical practice guidelines in oncology. J Natl Compr Canc Netw. 2019;17(6):721–749. https://doi.org/10.6004/jnccn.2019.0028 6. Cancer Genome Atlas Research Network, Ley TJ, Miller C, et al. Genomic and
epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med. 2013;368(22):2059–2074. https://doi.org/10.1056/NEJMoa1301689 25. Nunes Barroso KS, Lorand-Metze I, Pagnano KB, et al. Authors’ contributions J.F.K. was involved in the study design, assisted in
performing the study, performed data analysis and
contributed to writing the manuscript. A.-C.v.M. analysed
clinical data and contributed to writing the manuscript. J.C. assisted in performing the study and critically evaluated the
manuscript. A.d.K. was involved in the study design,
supervised the study and critically evaluated the manuscript. All the authors approved the final version of the article prior
to submission. Similar to a Sudanese study involving 100 AML patients,14
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Egyptian CN-AML patients and 7.5% of South African AML
patients.13,15 The complete absence of the NPM1 mutation in
this study could be attributed to the low median age at
disease presentation in the study cohort, especially http://www.ajlmonline.org Open Access Page 6 of 6 Original Research Disclaimer 16. Arber DA, Brunning RD, Le Beau MM, et al. Acute myeloid leukaemia with
recurrent genetic abnormalities. In: Swerdlow SH, Campo E, Harris NL, et al. editors. WHO classification of tumours of haematopoietic and lymphoid tissues. Revised 4th ed. Lyon: IARC, 2017; p. 129–170. The views and opinions expressed in this article are those of
the authors and do not necessarily reflect the official policy or
position of any affiliated agency of the authors. 17. Huang Q, Chen W, Gaal KK, et al. A rapid, one step assay for simultaneous detection
of FLT3/ITD and NPM1 mutations in AML with normal cytogenetics Br J Haematol. 2008;142(3):489–492. https://doi.org/10.1111/j.1365-2141.2008.07205.x 18. National Cancer Institute. SEER Program. Cancer stat facts: Leukemia – Acute
myeloid leukemia (AML) [homepage on the Internet]. Available from: https://seer. cancer.gov/statfacts/html/amyl.html Sources of support 12. El Gammal MM, Ebid GT, Madney YM, et al. Clinical effect of combined mutations
in DNMT3A, FLT3-ITD, and NPM1 among Egyptian acute myeloid leukemia
patients. Clin Lymphoma Myeloma Leuk. 2019;19(6):e281–e290. https://doi. org/10.1016/j.clml.2019.02.001 Funding was received from the National Health Laboratory
Service Research Development Grant (GRANT004_94767). 13. Sofan MA, Elmasry SA, Salem DA, et al. NPM1 gene mutation in Egyptian patients
with cytogenetically normal acute myeloid leukemia. Clin Lab. 2014;60(11):
1813–1822. https://doi.org/10.7754/clin.lab.2014.140121 Data availability 14. Elzain E, Khalil HB. Frequency and prognostic value of NPM1 mutations in
Sudanese acute myeloid leukemia patients. bioRxiv. 2020 (pre-print). https://doi. org/10.1101/2020.05.31.126334 Data are available from the corresponding author upon
reasonable request. 15. Marshall RC, Tlagadi A, Bronze M, Kana V, Wiggil TM, Carmona SC. Lower
frequency of NPM1 and FLT3-ITD mutations in a South African de novo AML
cohort. Int J Lab Hematol. 2014;36(6):656–664. https://doi.org/10.1111/
ijlh.12204 References Evaluation of seasonality in
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Transplant. 2020;55(4):740–748. https://doi. org/10.1038/s41409-019-0721-z 26. Mohammed AS, Ali TH, Alwan AF. Seasonality in acute promyelocytic leukemia: Fact
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s12288-017-0821-0 27. Gilbert RD, Karabus CD, Mills AE. Acute promyelocytic leukemia. A childhood
cluster. Cancer. 1987;59(5):933–935. https://doi.org/10.1002/1097-
0142(19870301)59:5%3C933::AID-CNCR2820590513%3E3.0.CO;2-R 9. Döhner H, Weisdorf DJ, Bloomfield CD. Acute myeloid leukemia. N Engl J Med. 2015;373(12):1136–1152. https://doi.org/10.1056/NEJMra1406184 28. Verhaak RG, Goudswaard CS, Van Putten W, et al. Mutations in nucleophosmin
(NPM1) in acute myeloid leukemia (AML): Association with other gene
abnormalities and previously established gene expression signatures and their
favorable prognostic expression significance. Blood. 2005;106(12):3747–3754. https://doi.org/10.1182/blood-2005-05-2168 10. Falini B, Mecucci C, Tiacci E, et al. Cytoplasmic nucleophosmin in acute
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352(3):254–266. https://doi.org/10.1056/NEJMoa041974 29. Sekeres MA, Peterson B, Dodge RK, et al. Differences in prognostic factors and
outcomes in African Americans and whites with acute myeloid leukemia. Blood. 2004;103(11):4036–4042. https://doi.org/10.1182/blood-2003-09-3118 29. Sekeres MA, Peterson B, Dodge RK, et al. Differences in prognostic factors and
outcomes in African Americans and whites with acute myeloid leukemia. Blood. 2004;103(11):4036–4042. https://doi.org/10.1182/blood-2003-09-3118 11. Bacher U, Kohlmann A, Haferlach C, Haferlach T. Gene expression profiling in
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169–180. https://doi.org/10.1016/j.beha.2009.04.003 http://www.ajlmonline.org
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Development of a core outcome set for congenital pulmonary airway malformations: study protocol of an international Delphi survey
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To cite: Hermelijn S,
Kersten C, Mullassery D, et al.
Development of a core outcome
set for congenital pulmonary
airway malformations: study
protocol of an international
Delphi survey. BMJ Open
2021;11:e044544. doi:10.1136/
bmjopen-2020-044544 Protocol Protocol Open access Development of a core outcome set for
congenital pulmonary airway
malformations: study protocol of an
international Delphi survey Strength and limitations of this study After the core outcome set has been defined
we intend to design an international randomised controlled
trial: the COllaborative Neonatal NEtwork for the first CPAM
Trial, which will be aimed at determining the optimal
management of patients with asymptomatic CPAM ►
►Existing comprehensive literature reviews shall be
used to inform initial outcome parameters instead
of a systematic literature search as no randomised
trials and very few prospective studies have yet
been published regarding the outcome of patients
with CPAM. ►
►Parents’ and patients’ views are not included in the
protocol as the final core outcome set is specifical-
ly intended for patients with asymptomatic CPAM,
however their input will play an important role when
designing future studies. ►
►Parents’ and patients’ views are not included in the
protocol as the final core outcome set is specifical-
ly intended for patients with asymptomatic CPAM,
however their input will play an important role when
designing future studies. Ethics and dissemination Electronic informed consent
will be obtained from all participants. Ethical approval is
not required. After the core outcome set has been defined,
we intend to design an international randomised controlled
trial: the COllaborative Neonatal NEtwork for the first CPAM
Trial, which will be aimed at determining the optimal
management of patients with asymptomatic CPAM. For numbered affiliations see
end of article. Correspondence to
Dr Marco Schnater;
j.schnater@erasmusmc.nl
© Author(s) (or their
employer(s)) 2021. Re-use
permitted under CC BY. Published by BMJ. born with a CPAM are asymptomatic but
others may show symptoms such as neonatal
respiratory distress, persistent cough or
recurrent lung infections in the first years
of life.3 A worldwide lack of consensus exists
on the optimal management and follow-up
of infants with an asymptomatic CPAM.4 5
Prospective studies on postnatal management
are lacking, and cohort studies vary widely in
the outcome measures they report.2 6 Development of a core outcome set for
congenital pulmonary airway
malformations: study protocol of an
international Delphi survey Sergei Hermelijn ,1 Casper Kersten,1 Dhanya Mullassery,2 Nagarajan Muthialu,2
Nazan Cobanoglu,3 Silvia Gartner,4 Pietro Bagolan,5 Carmen Mesas Burgos,6
Alberto Sgro,7 Stijn Heyman,8 Holger Till,9 Janne Suominen,10 Maarten Schurink,11
Liesbeth Desender,12 Paul Losty,13 Kjetil Ertresvag,14 Harm A W M Tiddens,15
Rene M H Wijnen,1 Marco Schnater,1 On behalf of the CONNECT study consortium
COS development group, On behalf of the CONNECT study consortium COS
development group ►
►Prepublication history for
this paper is available online.
To view these files, please visit
the journal online (http://dx.doi.
org/10.1136/bmjopen-2020-
044544). Strength and limitations of this study Introduction A worldwide lack of consensus exists on
the optimal management of asymptomatic congenital
pulmonary airway malformation (CPAM) even though
the incidence is increasing. Either a surgical resection
is performed or a wait-and-see policy is employed,
depending on the treating physician. Management is
largely based on expert opinion and scientific evidence
is scarce. Wide variations in outcome measures are
seen between studies making comparison difficult thus
highlighting the lack of universal consensus in outcome
measures as well. We aim to define a core outcome set
which will include the most important core outcome
parameters for paediatric patients with an asymptomatic
CPAM. ►
►The core outcome set is a disease-specific collec-
tion of the most important outcomes that will be es-
tablished by consensus between key stakeholders. Participants will be an international group of special-
ists with experience in the treatment of patients with
congenital pulmonary airway malformation (CPAM),
which will result in a universally approved synthesis
of expert opinion. ►
►Prepublication history for
this paper is available online. To view these files, please visit
the journal online (http://dx.doi.
org/10.1136/bmjopen-2020-
044544). ►
►This protocol describes an international online
Delphi survey that should identify the most import-
ant core outcome parameters including optimal tim-
ing and modality to monitor paediatric patients with
an asymptomatic CPAM. Received 09 September 2020
Revised 17 March 2021
Accepted 23 March 2021 Methods and analysis This study will include a critical
appraisal of the current literature followed by a three-
stage Delphi process with two stakeholder groups. One
surgical group including paediatric as well as thoracic
surgeons, and a non-surgeon group including paediatric
pulmonologists, intensive care and neonatal specialists. All participants will score outcome parameters according
to their level of importance and the most important
parameters will be determined by consensus. Methods and analysis This study will include a critical
appraisal of the current literature followed by a three-
stage Delphi process with two stakeholder groups. One
surgical group including paediatric as well as thoracic
surgeons, and a non-surgeon group including paediatric
pulmonologists, intensive care and neonatal specialists. All participants will score outcome parameters according
to their level of importance and the most important
parameters will be determined by consensus. Ethics and dissemination Electronic informed consent
will be obtained from all participants. Ethical approval is
not required. Received 09 September 2020
Revised 17 March 2021
Accepted 23 March 2021 Study design Study design
The COS will be developed in an online, three-round,
Delphi process, preceded by an appraisal of previously
published literature. METHODS The COS development will follow the Core Outcome Set-
STAndards for Development
Recommendations12 and the Core Outcome Measures
in Effectiveness Trials (COMET) handbook.11 This COS
development study was registered with the COMET
Initiative in May 2020 (http://www.comet-initiative.org/
studies/details/1570). The COS development will follow the Core Outcome Set-
STAndards for Development Stakeholders and recruitment Paediatric surgeons and thoracic surgeons are the health-
care professionals who are most frequently involved in
the operative management of patients with CPAM. Initial
consultation or follow-up is variably done by paediatric
surgeons, maternal-fetal medicine specialists, neonatol-
ogists, paediatricians or paediatric pulmonologists. We
therefore decided to form two stakeholder groups: (1)
surgeons (ie, paediatric and thoracic surgeons) and
(2) non-surgeons (eg, maternal-fetal medicine special-
ists, paediatricians, paediatric pulmonologists and
neonatologists). Recommendations12 and the Core Outcome Measures
in Effectiveness Trials (COMET) handbook.11 This COS
development study was registered with the COMET
Initiative in May 2020 (http://www.comet-initiative.org/
studies/details/1570). Open access up until the age of 18 years and will not include fetal
outcome. Outcome parameters as well as their measure-
ment instruments, and age at assessment will be included. An asymptomatic CPAM is either surgically resected or
a wait-and-see policy is employed depending on physi-
cian preference or local guidelines. Either way, cases are
ideally discussed in a multidisciplinary team in which
parental preferences are taken into account as well. To
date, no definitive evidence exists on the optimal manage-
ment, long-term outcomes are still unknown and factors
for predicting symptoms are still being investigated.6 The
arguments for a wait-and-see policy are that the malfor-
mation is originally benign, potentially regresses and in
most cases remains asymptomatic.7 The arguments for
surgical management include the risk of recurrent lung
infections (which could make subsequent surgery more
difficult), risk of acute respiratory distress, potential
malignant transformation, parental anxiety and allowing
for compensatory lung growth.8 9 Literature review A systematic literature review is recommended to yield
an initial outcome set for the first round in the Delphi
process.11 To date, no randomised studies have yet been
done and other studies examining the management of
asymptomatic CPAMs report a large variety in outcome
parameters.6 A literature review will be done by the study
management coordinators (SH, CK) informed by two
literature reviews, previously published in a special issue
of a paediatric surgery scientific journal, each covering
opposing thoughts on the arguments for surgical manage-
ment8 or a wait-and-see policy.7 In addition, we will scru-
tinise a recent systematic review and meta-analysis, which
covers the risks associated with either surgical resection
or a wait-and-see policy of asymptomatic CPAM.3 Existing
definitions, measurement tools and common measure-
ment time-points for outcomes will be extracted, and
formatted into appropriately phrased questions for use
in the first round of the Delphi process. The indepen-
dent coordinators will be blinded for participant identity
during the process by means of a unique identification
number and will ensure the Delphi process is performed
according to the protocol. Consensus needs to be reached on outcome measures
and their timing that can be applied in international
studies aimed at identifying the optimal management
of asymptomatic CPAM. A core outcome set (COS) is a
disease-specific collection of outcomes that have been
identified by consensus between key stakeholders as
being the most important in determining success of a
treatment.10 Such consensus is often reached through a
Delphi method in which stakeholders anonymously rate
outcome measures according to their importance, in
one or multiple rounds.11 We aim to develop a COS for
patients with CPAM using the Delphi method as a tool
for reaching consensus and present the study protocol for
this. INTRODUCTION Congenital pulmonary airway malforma-
tion (CPAM), formerly known as congenital
cystic adenomatoid malformation, is the most
common congenital lung abnormality.1 The
incidence of CPAM has increased up to 4 per
10 000 births over the last years.2 Most infants Correspondence to
Dr Marco Schnater;
j.schnater@erasmusmc.nl Hermelijn S, et al. BMJ Open 2021;11:e044544. doi:10.1136/bmjopen-2020-044544 1 Open access Delphi round 3 analysis and final COS development Delphi round 3 analysis and final COS development All outcome parameters meeting the criteria for consensus
of inclusion by all participants will be included in the final
COS. All other outcome parameters will be excluded. To
achieve a COS that is feasible for clinical use in trials, we
aim to include a maximum of 10 outcome parameters
in the final COS. If the number of outcome parameters
meeting the criteria for consensus of inclusion greatly
exceeds this maximum number, we will only include the
10 outcomes with the highest level of consensus for the Delphi round 2 All participants who completed the first round will auto-
matically be invited to participate in round 2. Per stakeholder group, the median scores assigned in
the first round will be made known. This will allow partic-
ipants to consider the views of the other participants in
the stakeholder group. They will be invited to look at
all items again and consider adjusting their own scores. Furthermore, they will be asked to score the newly added
outcome parameters suggested in the first round. Sample size There is no consensus on the optimal sample size for a
Delphi study11; recruitment will therefore be based on
the prospective study for which it is primarily intended
(CONNECT). For the CONNECT trial, we aim to include
at least 12 international centres, and therefore set the
minimum of participants in each stakeholder group in
the final round at 12. To reduce bias, no more than two
participants from a single centre can participate in a
stakeholder group. To minimise attrition bias in consec-
utive rounds, we aim to achieve that 75% of participants
complete a round.11 13 Therefore, the minimum number
of participants in each stakeholder group for round 1 will
be 21, and 16 for round 2. We believe that these minimum
numbers constitute a representative sample, consid-
ering the rarity of the disease and the limited number of
professionals with experience in managing this disease. Previous Delphi COS studies in paediatric surgery used
similar numbers for investigating a more common disease
such as appendicitis.14 15 Participants are invited to suggest additional outcome
parameters stating: (1) the outcome parameter, (2) the
measurement instrument and (3) the age at assessment. These additional outcome parameters will be scored in
the second round. Delphi round 3 All participants who completed the first and second
rounds will be invited to participate in the third round. The median score of their own stakeholder group and the
score of the other stakeholder group will be presented to
participants. This will allow participants to consider the
views of the other stakeholder group before rescoring the
outcomes. They will be invited to look at all remaining
items again and consider adjusting their own scores. In
addition, participants will be asked to identify a single
outcome parameter, which is the most important for
determining the treatment choice in patients with asymp-
tomatic CPAM according to them. Delphi round 1 analysis Outcome parameters will be analysed separately for each
stakeholder group (surgeons and non-surgeons) and all
parameters will be included in the second round of the
Delphi process. The additional outcomes provided by
participants will be reviewed to confirm they represent
new outcomes. If confirmed, the item in question will be
included in the second round as well. Delphi study
Consensus All outcome parameters meeting consensus criteria for
exclusion by all participants will be excluded from the
third round. All other parameters will be included in the
third round. Participants will be asked to score each outcome param-
eter using the Grading of Recommendations, Assessment,
Development and Evaluations scale.16 The 9-point Likert
scale will label 1–3 as ‘not important’, 4–6 as ‘important
but not critical’ and 7–9 as ‘critical’. Consensus for inclu-
sion is reached if ≥70% of participants rate the outcome
parameter 7–9 and <15% rate it 1–3. Consensus for exclu-
sion is reached if >70% participants rate the outcome 1–3
and <15% rate it 7–9. Outcomes not meeting these defi-
nitions will be classified as ‘no consensus’.11 Open access “How important would you rate the following outcome parameter
including measurement instrument and age in determining the
best management of asymptomatic CPAM patients?” “How important would you rate the following outcome parameter
including measurement instrument and age in determining the
best management of asymptomatic CPAM patients?” Timeline Participants will be asked to complete each round of
the Delphi process within 4 weeks. A weekly reminder
email will be sent to those who have not yet completed
the survey. Those failing to complete the questionnaire
within the allocated 4 weeks will be excluded from next
rounds. The deadline shall be extended if the projected
minimum sample size has not been reached and those
failing to complete the questionnaire shall be approached
individually. Attrition bias Attrition bias will be assessed separately for each round
of the Delphi process, and separately in each stakeholder
group. In each group, the median score of every outcome
will be compared using the Wilcoxon rank-sum test,
between those only completing the previous round and
those completing the consecutive round as well.11 Delphi round 2 analysis Scope This protocol describes the Delphi method which shall
define a COS for all asymptomatic patients who are either
prenatally or postnatally diagnosed with a CPAM. Asymp-
tomatic patients are defined as those who have no need
for prolonged respiratory support (>24 hours) including
supplemental oxygen and ventilation. The COS will
include the most important outcome measures for
patients with CPAM, regardless of the management. The
COS may be used as a guideline for clinical follow-up or in
future research studies. After the COS has been defined,
we intend to design a randomised controlled trial: the
COllaborative Neonatal NEtwork for the first European
CPAM Trial (CONNECT). This trial will be performed
by the CONNECT study consortium and aims to iden-
tify the optimal management of patients with asymptom-
atic CPAM. The COS is intended for paediatric patients We will recruit study participants through an existing
international network of paediatric surgeons and pulmo-
nologists who have expressed interest in collaborating on
the CONNECT trial. We will inform potential participants
on the aims and procedures of the Delphi process and
encourage them to enrol other specialists involved in the
care of patients with CPAM in their own centres. Prior
to the first round, those who have been found willing to
participate will be sent an email explaining the aims and
procedures of the Delphi process, and emphasising the
importance of finishing each round within the allocated
time. 2 Hermelijn S, et al. BMJ Open 2021;11:e044544. doi:10.1136/bmjopen-2020-044544 ORCID iD ORCID iD
Sergei Hermelijn http://orcid.org/0000-0002-7296-9932 Ethics and dissemination Electronic informed consent will be obtained from all
participants. Prior ethical approval for the Delphi study
is not required. The final COS will be published in an
international peer-reviewed scientific journal and on the
COMET Initiative website (https://www.comet-initiative.
org/). Contributors All authors contributed to the design of this protocol. SH, DM, NC, SG,
PB, CMB, AS, SH, JS, MS, LD, KE, HT, RW and JMS initiated the project. The protocol
was drafted by SH and CK. The protocol was critically reviewed by DM, NM, NC,
SG, PB, CMB, AS, SH, HT, JS, MS, LD, PL, KE, HAWMT, RMHW and JMS. All authors
contributed to the manuscript and read and approved the final manuscript. The
CONNECT study consortium COS development group consists of all participants of
the Delphi process. They have all read, refined and approved the final manuscript. Funding The authors have not declared a specific grant for this research from any
funding agency in the public, commercial or not-for-profit sectors. Open access Open access COS and report those excluded in this stage. The level
of consensus will be determined by the median score of
each outcome parameter in round 3. C. Wallis (Great Ormond Street Hospital, London, UK); D. Cox (Children’s Health
Ireland at Crumlin, Dublin, Ireland); P. Bagolan, F. Morini (Bambino Gesu Pediatric
Hospital, Rome, Italy); C. Mesas Burgos, P. Conner, E. Caffrey Osvald, C. Bitkover
(Karolinska Institutet, Stockholm, Sweden); H. Decaluwe, M. Proesmans, M. Boon,
J. Deprest (University Hospital Leuven, Leuven, Belgium); S. Gartner, A. Lain
(Hospital Universitari Vall d Hebron, Barcelona, Spain); P.D. Losty, I. Sinha (Alder
Hey Children’s Hospital, School Of Health and Life Science, University of Liverpool,
Liverpool, UK); I. Yardley (Evelina London Children’s Hospital, London, UK); M. Singh
(Birmingham Children’s Hospital, Birmingham, UK); L. Wessel, K. Zahn, T. Schaible
(University Hospital Mannheim, Mannheim, Germany); N. Qvist (Odense Univeristy
Hospital, Odense, Denmark); M. Zampoli (Red Cross Children’s Hospital, Cape Town,
South Africa); G. Aksnes (Oslo University Hospital, Oslo, Norway); C.K. van der Ent,
K.M. Winter-de Groot (University Medical Centre Utrecht, Utrecht, The Netherlands);
R. Peters (Royal Manchester Children’s Hospital, Manchester, UK); E. Hannon (Leeds
Children’s Hospital, Leeds, UK); Q. Jöbsis, M. Bannier (Maastricht University Medical
Centre, Maastricht, The Netherlands). The final COS will be a collection of the most important
outcome parameters in patients with CPAM. The final
COS will be annotated according to the outcome
taxonomy, which was constructed to maximise future
data harmonisation. Additionally, the final COS will be
divided into the four core areas of the OMERACT filter:
death, life impact, pathophysiological manifestations and
resource use.10 REFERENCES 10Pediatric Surgery, University of Helsinki Children's Hospital, Helsinki, Finland 1 EUROCAT. Prevalence tables. 2007-2018. Available: http://www.
eurocat-network.eu/ACCESSPREVALENCEDATA/PrevalenceTables
[Accessed Jan 2019]. 11Pediatric Surgery, Radboud University Medical Centre Amalia Children's Hospital,
Nijmegen, The Netherlands 12Pediatric Surgery, Ghent University Faculty of Medicine and Health Sciences, Gent,
Belgium 2 Stocker LJ, Wellesley DG, Stanton MP, et al. The increasing
incidence of foetal echogenic congenital lung malformations: an
observational study. Prenat Diagn 2015;35:148–53. 2 Stocker LJ, Wellesley DG, Stanton MP, et al. The increasing
incidence of foetal echogenic congenital lung malformations: an
observational study. Prenat Diagn 2015;35:148–53. 13Pediatric Surgery, University of Liverpool, Liverpool, UK 14Pediatric Surgery, Oslo University Hospital, Oslo, Norway 3 Stanton M, Njere I, Ade-Ajayi N, et al. Systematic review and meta-
analysis of the postnatal management of congenital cystic lung
lesions. J Pediatr Surg 2009;44:1027–33. 15Pediatric Pulmonology, Erasmus MC Sophia Children's Hospital, Rotterdam, The
Netherlands 15Pediatric Pulmonology, Erasmus MC Sophia Children's Hospital, Rotterdam, The
Netherlands 4 Lo AY-S, Jones S. Lack of consensus among Canadian
pediatric surgeons regarding the management of congenital
cystic adenomatoid malformation of the lung. J Pediatr Surg
2008;43:797–9. Collaborators The following people form the CONNECT study consortium
COS development group: S.M. Hermelijn, C.M. Kersten, J.M. Schnater, R.M.H. Wijnen, H.A.W.M. Tiddens, S.C.M. Cochius-den Otter (Sophia Children’s Hospital,
Rotterdam, The Netherlands); J. Suominen, M. Pakarinen, L. Martelius (Helsinki
Children’s Hospital, Helsinki, Finland); S. Heyman, D. Vervloessem (ZNA-GZA Paola
Children’s Hospital, Antwerp, Belgium); H. Steyaert (Queen Fabiola Children’s
University Hospital, Brussels, Belgium); A. Sgro, P. Gamba (University Hospital
Padua, Padua, Italy); M. Schurink, S. van der Heide, J. Roukema (Amalia Children’s
Hospital, Nijmegen, The Netherlands); N. Rikkers-Mutsaerts (Leiden University
Medical Centre, Leiden, The Netherlands); S. Terheggen-Lagro, S. de Beer, E. Haarman (Amsterdam University Medical Centre, Amsterdam, The Netherlands);
H. Till, G. Singer (Medical University of Graz, Graz, Austria); M. Metzelder, P. Sezen
(Medizinische Universität Wien, Vienna, Austria); L. Desender, H. Schaballie (Ghent
University Hospital, Ghent, Belgium); N. Cobanoglu, G. Gollu (Ankara University,
School of Medicine, Ankara, Turkey); M. Stanton (University Hospital Southampton,
Southampton, UK); A. Bonnard (Hopital Universitaire Robert Debré, Paris, France);
R. Sfeir (Hopital Jeanne de Flandre, Lille, France); N. Muthialu, D. Mullassery, 5 Morini F, Zani A, Conforti A, et al. Current Management of Congenital
Pulmonary Airway Malformations: A “European Pediatric Surgeons’
Association” Survey. Eur J Pediatr Surg 2018;28:001–5. 5 Morini F, Zani A, Conforti A, et al. Delphi round 1 The three-round Delphi process shall be performed
using ‘Welphi’, an online data system specifically devel-
oped for this use.17 All participants shall be approached
simultaneously and asked to rate each of the previously
identified outcome parameters on importance as follows: 3 Hermelijn S, et al. BMJ Open 2021;11:e044544. doi:10.1136/bmjopen-2020-044544 Hermelijn S, et al. BMJ Open 2021;11:e044544. doi:10.1136/bmjopen-2020-044544 Provenance and peer review Not commissioned; externally peer reviewed. Author affiliations
1Pediatric Surgery, Erasmus MC Sophia Children's Hospital, Rotterdam, The
Netherlands
2Pediatric Surgery, Great Ormond Street Hospital for Children, London, UK
3Pediatric Pulmonology, Ankara University Faculty of Medicine, Ankara, Turkey
4Pediatric Pulmonology, Hospital Universitari Vall d'Hebron, Barcelona, Spain
5Department of Medical and Surgical Neonatology, Ospedale Pediatrico Bambino
Gesu, Roma, Italy
6Pediatric Surgery, Karolinska Institutet, Stockholm, Sweden
7Pediatric Surgery, Padua University Hospital, Padova, Italy
8Pediatric Surgery, ZNA-GZA Paola Children's Hospital, Antwerp, Belgium
9Pediatric Surgery, Medical University of Graz, Graz, Austria
10Pediatric Surgery, University of Helsinki Children's Hospital, Helsinki, Finland
11Pediatric Surgery, Radboud University Medical Centre Amalia Children's Hospital,
Nijmegen, The Netherlands
12Pediatric Surgery, Ghent University Faculty of Medicine and Health Sciences, Gent
Belgium
13Pediatric Surgery, University of Liverpool, Liverpool, UK
14Pediatric Surgery, Oslo University Hospital, Oslo, Norway
15Pediatric Pulmonology, Erasmus MC Sophia Children's Hospital, Rotterdam, The
Netherlands Data collection and confidentiality Participants will complete questionnaires using the
‘Welphi’ survey tool.17 Anonymised data will be stored on
a secure online server and will be managed according to
the European General Data Protection Regulation.18 Competing interests None declared. Patient and public involvement Patients and/or the public were not involved in
the design, or conduct, or reporting, or dissemination plans of this research. Patient consent for publication Not required. Patient consent for publication Not required. Provenance and peer review Not commissioned; externally peer reviewed. Provenance and peer review Not commissioned; externally peer reviewed. Author affiliations
1 Open access This is an open access article distributed in accordance with the
Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits
others to copy, redistribute, remix, transform and build upon this work for any
purpose, provided the original work is properly cited, a link to the licence is given,
and indication of whether changes were made. See: https://creativecommons.org/
licenses/by/4.0/. ORCID iD ORCID iD
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(General data protection regulation), OJ 2016 L 119/1, 2018. 18 (GDPR) Gdpr. Regulation (EU) 2016/679 of the European Parliament
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Phospholipid analysis in sera of horses with allergic dermatitis and in matched healthy controls
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Lipids in health and disease
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cc-by
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* Correspondence: raija.hallamaa@elisanet.fi
1Veterinary Clinic, Nummela, Finland
2University of Helsinki, Faculty of Veterinary Medicine, Helsinki, Finland
Full list of author information is available at the end of the article © 2016 Hallamaa and Batchu. 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. Phospholipid analysis in sera of horses with
allergic dermatitis and in matched healthy
controls Raija Hallamaa1,2* and Krishna Batchu3 Hallamaa and Batchu Lipids in Health and Disease (2016) 15:45
DOI 10.1186/s12944-016-0209-4 Hallamaa and Batchu Lipids in Health and Disease (2016) 15:45
DOI 10.1186/s12944-016-0209-4 Open Access Results The main phospholipid classes analysed were phosphat-
idylcholine (PC), phosphatidylethanolamine (PE), phos-
phatidylserine (PS), phosphatidylinositol (PI), phosphatidic
acid (PA) and sphingomyelin (SM). Of these, all except for
PS were detected across all the samples. Relative contents
of the major phospholipid classes before (stage 0) and
after the 4-week therapy (stage 4) of eczema horses are
listed in Table 1. In addition to the major phospholipid
classes, all the samples also contained PA 36:2 and lysoPC
species of 16:0, 18:2 and 18:0. Insect bite hypersensitivity (Fig. 1a) is the most com-
mon allergic skin disease of the horse [24] displaying
many features similar to atopic dermatitis (AD) of
humans [25, 26]. Intense and recurrent pruritus, IgE-
mediated responses, perivascular mast cell, eosinophil
and T-lymphocyte infiltrations are typical findings of
both disorders [25, 27–32]. In addition, horses with in-
sect hypersensitivity show IgE-mediated sensitization
also to various environmental allergens [27], as do patients
with atopic dermatitis [31, 32]. However, disturbed skin
barrier function due to inherited defects of filaggrin has
not been studied on horses [26]. Treatment of equine IBH
is challenging, since total isolation from biting insects is
impossible. Antihistamines or allergen-specific immuno-
therapy have not been beneficial [33, 34]. Therefore there
is a need for comprehensive understanding of this harmful
disease. In our previous study [35], we analysed phospho-
lipid contents of autoserum preparations that have been
recently used in the therapy of insect hypersensitivity in
horses. The idea is to collect remnant lipid particles in
these preparations after serial washings [35]. Concentra-
tions of phosphatidylcholine (PC) and sphingomyelin
(SM) were significantly more abundant in the preparations Phosphatidylcholine was the most abundant of all the
phospholipid classes detected (Table 1). Its major mo-
lecular species in both eczema and control horses at
stages 0 and 4 are presented in Figs. 2 and 3, respect-
ively. Of the molecular species, PC 36:2 was the most
abundant. Additionally, trace amounts of PC 26:0, 28:0,
31:1, 33:1, 33:2, 33:3, 36:2, 36:3, 36:6 and 38:8 were de-
tected. In the samples collected later in the summer
(Table 2), PC concentrations were found to be more abun-
dant than in the samples taken earlier (Figs. 2 and 3). Horses in the eczema group had a significantly lower total
concentration of PC than healthy horses before the start
of the therapy (Table 3). Abstract Background: Lipids have become an important target for searching new biomarkers typical of different
autoimmune and allergic diseases. The most common allergic dermatitis of the horse is related to stings of insects
and is known as insect bite hypersensitivity (IBH) or summer eczema, referring to its recurrence during the summer
months. This intense pruritus has certain similarities with atopic dermatitis of humans. The treatment of IBH is
difficult and therefore new strategies for therapy are needed. Autoserum therapy based on the use of serum
phospholipids has recently been introduced for horses. So far, serum lipids relating to these allergic disorders have
been poorly determined. The main aim of this study was to analyse phospholipid profiles in the sera of horses with
allergic dermatitis and in their healthy controls and to further assess whether these lipid profiles change according
to the clinical status after therapy. Methods: Sera were collected from 10 horses with allergic dermatitis and from 10 matched healthy controls both
before and 4 weeks after the therapy of the affected horses. Eczema horses were treated with an autogenous
preparation made from a horse’s own serum and used for oral medication. Samples were analysed for their
phospholipid content by liquid chromatography coupled to a triple-quadrupole mass spectrometer (LC-MS). Data
of phospholipid concentrations between the groups and over the time were analysed by using the Friedman test. Correlations between the change of concentrations and the clinical status were assessed by Spearman’s rank
correlation test. Results: The major phospholipid classes detected were phosphatidylcholine (PC), sphingomyelin (SM),
phosphatidylinositol (PI) and phosphatidylethanolamine (PE). Eczema horses had significantly lower total
concentrations of PC (p < 0.0001) and SM (p = 0.0115) than their healthy controls. After a 4-week therapy, no
significant differences were found between the groups. Changes in SM concentrations correlated significantly
with alterations in clinical signs (p = 0.0047). Conclusions: Horses with allergic dermatitis have an altered phospholipid profile in their sera as compared
with healthy horses and these profiles seem to change according to their clinical status. Sphingomyelin seems
to have an active role in the course of equine insect bite hypersensitivity. Keywords: Horse, Allergy, Allergic dermatitis, Lipids, Phospholipids, Insect bite hypersensitivity, Summer eczema,
Autoserum therapy Page 2 of 9 Hallamaa and Batchu Lipids in Health and Disease (2016) 15:45 Page 2 of 9 Background made from the sera of the affected than from healthy
horses and the amounts of these phospholipids showed
significant associations with the severity of prevailing clin-
ical status [35]. Various new and important insights into the roles of lipids
have been demonstrated over the past few decades. Most
of these findings are related to inflammatory and immune
responses including studies on pro-resolving lipid media-
tors [1, 2], lipid antigens [3, 4] and lipid presenting CD1
molecules [5–7]. Mass spectrometry with specific software
applications has made it possible to identify and quantify a
multitude of lipid species both in mammalian cells and
also of those existing in circulation [8–12]. Although
phospholipids are commonly present in mammalian sera
[13, 14], changes in their concentrations under various
pathological conditions are so far poorly understood
[9, 15, 16]. Presently, there is an increasing need to
find new biomarkers feasible for diagnosis, treatment and
prognosis – not only in allergic diseases, but also in other
immune mediated and neurological disorders [2, 16–23]. The purpose of the current study was to analyse by li-
quid chromatography-mass spectrometry (LC-MS) phos-
pholipids in the sera of horses with insect bite allergic
dermatitis and in their matched healthy controls. We
hypothesize that phospholipid profiles may differ be-
tween the groups. The additional aim was to assess
whether these profiles change according to the clinical
status after autoserum therapy. Results After the 4-week therapy, no sig-
nificant difference could be found between the groups
(Table 3, Fig. 3). In the eczema group, the total PC con-
centration had increased significantly, while in the control Fig. 1 Typical clinical signs in the tail of a horse with insect bite allergic dermatitis (a) and the same horse after a 4-week therapy (b) Fig. 1 Typical clinical signs in the tail of a horse with insect bite allergic dermatitis (a) and the same horse after a 4-week therapy (b) Hallamaa and Batchu Lipids in Health and Disease (2016) 15:45 Page 3 of 9 Page 3 of 9 Table 1 Relative contents of the major phospholipids detected
in sera of the horses
Phospholipids
Relative content (mean ± sd/%)
Eczema group
Control group
Stage 0
PC
76.0 ± 4.8
83.3 ± 9.8
SM
21.9 ± 3.8
14.8 ± 8.5
PI
1.9 ± 1.4
1.7 ± 1.5
PE
0.2 ± 0.2
0.2 ± 0.2
Stage 4
PC
87.4 ± 1.7
85.6 ± 2.7
SM
11.0 ± 1.6
12.4 ± 1.9
PI
1.4 ± 0.7
1.8 ± 1.3
PE
0.2 ± 0.2
0.2 ± 0.2
Samples of horses with allergic dermatitis (n = 10) and their matched healthy
controls (n = 10) are collected before treatment (stage 0) and when affected
horses have been on autoserum therapy for 4 weeks (stage 4). Phospholipid
classes: phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylinositol
(PI), phosphatidylethanolamine (PE). Table 1 Relative contents of the major phospholipids detected
in sera of the horses the stages in both eczema and healthy horses. Concentra-
tions of SM, like PC, were more abundant in the samples
collected later in the summer (Table 2, Figs. 4 and 5). Phosphatidylinositol and PE displayed small amounts
of the phospholipids detected (Table 1). Of PI, molecular
species of 32:0, 34:1, 34:2, 36:1, 36:2, 36:3, 36:4 and 38:4
were found. The pairwise comparisons showed no sig-
nificant differences between the two groups at any stages
(Table 3). The detected molecular species of PE were
that of 32:0, 36:1, 36:2, 36:3, 36:4, 38:4 and 38:5. Unlike
PI, the amounts of PE increased significantly from stage
0 to 4 in both groups (Table 3). However, its relative
content did not change due to its small total amount
(Table 1). No significant differences between the groups
were observed at any of the stages. Results Five of the horses had mild and 5 moderate clinical
signs at the beginning of the study and the total score of
signs was 15, according to the 3-graded scale used. After
a 4-week autoserum therapy, the signs had relieved in 6
horses (Fig. 1b), remained unchanged in 3, while one
horse suffered from aggravated clinical signs, the total
score being 10. Horses with positive clinical outcome
showed a milder decrease in SM concentrations than
horses with poorer response or controls (Fig. 6). The
change in total SM concentrations correlated signifi-
cantly with alterations in the clinical signs (P = 0.0047)
and of the major molecular species, SM 15:0 exhibited a
significant correlation (P = 0.0268). None of the other
phospholipid classes showed significant correlations with
the change of clinical signs. Samples of horses with allergic dermatitis (n = 10) and their matched healthy
controls (n = 10) are collected before treatment (stage 0) and when affected
horses have been on autoserum therapy for 4 weeks (stage 4). Phospholipid
classes: phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylinositol
(PI), phosphatidylethanolamine (PE). Samples of horses with allergic dermatitis (n = 10) and their matched healthy
controls (n = 10) are collected before treatment (stage 0) and when affected
horses have been on autoserum therapy for 4 weeks (stage 4). Phospholipid
classes: phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylinositol
(PI), phosphatidylethanolamine (PE). group there was a small decrease (Figs. 2 and 3). Concen-
trations of all the major molecular species presented in
Figs. 2 and 3 increased significantly (at level p < 0.05) in
the affected horses between stages 0 and 4, while in the
healthy horses, the only significant change observed was a
decrease in the concentration of PC 40:8 (P = 0.0007). Concentrations of SM were found to be significantly
lower in eczema horses in comparison with their healthy
controls at stage 0 (Table 3), although the relative con-
tent of SM was higher in the affected horses (Table 1). The total SM concentrations decreased in both groups
during the study period (Table 3, Figs. 4 and 5) and at
the second sampling there was no significant difference
observed between the two groups (Table 3). The major
SM species detected and their changes between the
stages 0 and 4 are shown in Figs. 4 and 5. Results The most
abundant species was SM 20:0 in both groups and its
concentration differed neither between the groups nor
the stages. Concentrations of SM 16:0, 18:0, 24:0 and
24:1 decreased significantly (at level p <0.05) between No side effects relating to the therapy were observed. Control horses did not develop signs typical of IBH dur-
ing the study period. Discussion our previous study [35], we found that autoserum prepa-
rations made from the sera of healthy horses and from
horses with allergic eczema showed significantly different
phospholipid profiles. Additionally, lipid concentrations
displayed significant relationship with the severity of clin-
ical signs. Therefore, the present study was focused on
serum and changes observed in its lipid profiles in re-
sponse to autoserum therapy, but not on the clinical effi-
cacy of this therapy per se. Horses with insect hypersensitivity showed signifi-
cantly lower total concentrations of PC and SM than
their matched healthy controls. After a 4-week therapy,
those differences faded. The cause of these originally
lower concentrations in eczema horses is unclear. Obser-
vations from earlier studies indicated that small children
with elimination diets or horses fed with oil supplements
show altered lipid compositions in their sera [36, 37]. In
addition,
fasting
influences
lipid
concentrations
[9]
which stresses the relevance of matching and equal tim-
ing when blood is collected from non-fasting horses and
their controls. Horses suffering from severe clinical signs
may spend time for scratching and thus eat differently
than healthy horses. However, none of the horses were
so severely affected. Eczema horses were otherwise in a Phosphatidylcholine, the major phospholipid present in
mammalian sera [13, 14], was found to be the most abun-
dant also in the present study. In humans, alterations in
its concentrations have been linked to various pathological
conditions, such as cancer, atopic dermatitis and type-1
diabetes, where in altered levels of both PC and SM in
sera have been observed [17, 22, 23, 38]. Fuchs et al. showed that in rheumatoid arthritis, PC/lysoPC ratio
acted as an indicator of this autoimmune disease and add-
itionally, an increased ratio seemed to be associated with
the success of the therapy [39]. Discussion The purpose of this study was to analyse the major phos-
pholipids typical of mammals in the sera of healthy horses
and horses with allergic dermatitis. The additional aim
was to assess whether profiles of the detected phospho-
lipids change according to clinical status when horses
were treated with an autogenous serum preparation. In Fig. 2 Concentrations of the most abundant phosphatidylcholine (PC) species at the start of therapy. Eczema horses (n = 10) and their matched
healthy controls (n = 10) are numbered similarly. All species showed significantly lower concentrations in eczema horses, at level P < 0.0001 i
h
f h
E
h
(
0)
d h i
h d Fig. 2 Concentrations of the most abundant phosphatidylcholine (PC) species at the start of therapy. Eczema horses (n = 10) and their matched
healthy controls (n = 10) are numbered similarly. All species showed significantly lower concentrations in eczema horses, at level P < 0.0001 Hallamaa and Batchu Lipids in Health and Disease (2016) 15:45 Page 4 of 9 Fig. 3 Concentrations of the most abundant phosphatidylcholine (PC) species after a 4-week therapy. Eczema horses (n = 10) and their matched
healthy controls (n = 10) are numbered similarly. Concentrations of PC 34:1 and 36:1 were significantly lower in eczema horses, while 40:8 was in
the controls, at level P < 0.05 Fig. 3 Concentrations of the most abundant phosphatidylcholine (PC) species after a 4-week therapy. Eczema horses (n = 10) and their matched
healthy controls (n = 10) are numbered similarly. Concentrations of PC 34:1 and 36:1 were significantly lower in eczema horses, while 40:8 was in
the controls, at level P < 0.05 normal physical condition without signs of emaciation
and their feeding regimen and daily activities were similar
to their controls, neither had the owners observed any
changes in their appetite. Although the feeding regimen
was similar between the horses, there could be a possibil-
ity that some unknown factors might have influenced the
lipid concentrations over time, since sera collected in the
late summer showed more abundant concentrations of PC
and SM than did sera collected earlier in the summer and
this trend was detectable in both affected and healthy
horses. This emphasizes the importance of matching ac-
companied by simultaneous samplings in these kinds of
studies that are prone to environmental impacts. 0 horses with allergic dermatitis and B) their 10 matched healthy controls. The horse and its matched control are numbered similarly Discussion LysoPC is a derivative of Table 2 Matching of the horses
A
Breed
Gender
Age (years)
Feed
B
Breed
Gender
Age (years)
Feed
Date of 1stsample
1
Finnhorse
♀
3
same fodder
1
Finnhorse
♀
9
same fodder
24 April
2
Icelandic horse
♀
22
same fodder
2
Shetland pony
♀
22
same fodder
21 May
3
Icelandic horse
♀
23
same fodder
3
Icelandic horse
♂
9
same fodder
21 May
4
Finnhorse
♂
8
same fodder
4
Finnhorse
♂
7
same fodder
12 June
5
Finnhorse
♂
8
same fodder
5
Finnhorse
♂
7
same fodder
12 June
6
Finnhorse
♀
8
same pasture
6
Finnhorse
♀
8
same pasture
12 June
7
Finnhorse
♀
4
same pasture
7
Finnhorse
♀
7
same pasture
17 July
8
Finnhorse
♀
4
same pasture
8
Finnhorse
♀
6
same pasture
11 Aug. 9
Icelandic horse
♀
10
same fodder
9
Icelandic horse
♀
29
same fodder
7 Sept. 10
Finnhorse
♀
11
same pasture
10
Finnhorse
♀
19
same pasture
12 Sept. A) 10 horses with allergic dermatitis and B) their 10 matched healthy controls. The horse and its matched control are numbered similarly A) 10 horses with allergic dermatitis and B) their 10 matched healthy controls. Discussion The horse and its matched control are numbered similarly Hallamaa and Batchu Lipids in Health and Disease (2016) 15:45 Page 5 of 9 Page 5 of 9 Table 3 Pairwise comparisons of serum phospholipid
concentrations
PC 0
PC 4
PC 0 c
PC 4 c
PC 0
p = 0.0007
p < 0.0001
p < 0.0001
PC 4
p = 0.0007
ns
ns
PC 0 c
p < 0.0001
ns
ns
PC 4 c
p < 0.0001
ns
ns
SM 0
SM 4
SM 0 c
SM 4 c
SM 0
ns
p = 0.0115
ns
SM 4
ns
p = 0.0005
ns
SM 0 c
p = 0.0115
p = 0.0005
p = 0.0186
SM 4 c
ns
ns
p = 0.0186
PI 0
PI 4
PI 0 c
PI 4 c
PI 0
ns
ns
ns
PI 4
ns
ns
ns
PI 0 c
ns
ns
ns
PI 4 c
ns
ns
ns
PE 0
PE 4
PE 0 c
PE 4 c
PE 0
p = 0.0058
ns
p < 0.0001
PE 4
p = 0.0058
ns
ns
PE 0 c
ns
ns
p = 0.0018
PE 4 c
p < 0.0001
ns
p = 0.0018
Phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylinositol (PI) and
phosphatidylethanolamine (PE) detected from horses with allergic dermatitis
and from their matched healthy controls (c); samples collected before (0) and
4 weeks after (4) the therapy of the affected horses Table 3 Pairwise comparisons of serum phospholipid
concentrations clinical signs, neither was there any prognostic association
between the first PC concentrations and the response to
therapy. In contrast to our horse study, higher levels of
PC predicted better outcome in patients with atopic ec-
zema after monoclonal antibody therapy [38]. y
y
Concentrations of SM were significantly lower in the
affected than in healthy horses before the treatment, but
not afterwards. Therefore, the elevated relative content
of SM in eczema horses at the first sampling is in par-
ticular due to the lower levels of PC. Surprisingly, the
amounts of SM decreased significantly among the con-
trol horses during the study period, which demonstrates
that concentrations of certain lipids may fluctuate in
healthy horses, at least to some extent without largely
undermining the relative lipid content. This fluctuation
possibly resulted from environmental changes, since SM
concentrations decreased also in eczema horses. Discussion However,
the decrease was evident only in those eczema horses that
showed a poorer outcome in response to therapy. This
suggests that affected horses have a specific need for SM
during recovery. Sphingomyelins are important lipids in-
volved in multiple biochemical reactions, especially in the
skin and nervous tissue [15, 41–43]. These phospho-
lipids are necessary not only as structural components,
but also as bioactive regulators and messengers in cuta-
neous homeostasis and immune mediated responses,
particularly via the main derivative, sphingosine-1-
phosphate [41, 42, 44, 45]. Sphingosine-1-phosphate
(S1P) plays a crucial part in mast cell regulation by af-
fecting mast cell degranulation and by activating other
immune cells involved [44, 45]. The engagement of S1P
to its receptors on the plasma membrane seems to be
the most critical point in the pathway of mast cell acti-
vation [44–46]. In addition to S1P, ceramide is another
important metabolite of SM and is one of the main lipid
components in skin barrier [41]. In the affected skin,
ceramides are continuously utilised for repairing [41]. Although various lipid combinations and their changes Phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylinositol (PI) and
phosphatidylethanolamine (PE) detected from horses with allergic dermatitis
and from their matched healthy controls (c); samples collected before (0) and
4 weeks after (4) the therapy of the affected horses PC and it is easily formed from PC during storage at room
temperature [40]. In horse sera, the relative content of
lysoPC is small [14] and therefore it is not an applicable
marker in horses as it is in humans. In the present study,
lysoPC was detected in minor concentrations and conse-
quently was not evaluated further. Although there was a
highly significant difference in PC concentrations between
the healthy and the eczema horses at the first sampling,
PC did not show any correlation with the change in Fig. 4 Concentrations of the most abundant sphingomyelin (SM) species at the start of therapy. Eczema horses (n = 10) and their matched
healthy controls (n = 10) are numbered similarly. Species of SM 15:0 and 24:1 showed significantly lower concentrations in eczema horses, at
level P < 0.05 Fig. 4 Concentrations of the most abundant sphingomyelin (SM) species at the start of therapy. Eczema horses (n = 10) and their matched
healthy controls (n = 10) are numbered similarly. Discussion Species of SM 15:0 and 24:1 showed significantly lower concentrations in eczema horses, at
level P < 0.05 Hallamaa and Batchu Lipids in Health and Disease (2016) 15:45 Page 6 of 9 Fig. 5 Concentrations of the most abundant sphingomyelin (SM) species after a 4-week therapy. Eczema horses (n = 10) and their matched
healthy controls (n = 10) are numbered similarly. Concentrations of SM 18:0 and 21:0 were significantly lower in eczema horses, at level P < 0.05 Fig. 5 Concentrations of the most abundant sphingomyelin (SM) species after a 4-week therapy. Eczema horses (n = 10) and their matched
healthy controls (n = 10) are numbered similarly. Concentrations of SM 18:0 and 21:0 were significantly lower in eczema horses, at level P < 0.05 in response to therapy have been linked to atopic
dermatitis in humans [38, 47], SM seems to be a poten-
tial player in insect bite allergy of the horse, since alter-
ations in its concentrations were significantly associated
with changes in clinical signs. Similarly, sphingolipids
have been shown to act as biomarkers in delayed type
hypersensitivity of mice [16]. Horses in the present study were treated with an auto-
serum preparation and phospholipids were analysed in
response to this treatment. However, we had no control
group for the therapy; therefore it is not relevant to con-
clude that the observed changes could be specific for the
current treatment. In fact, further studies are needed to
evaluate a relationship between phospholipid concentra-
tions and responses to therapy, comprising placebo or
other therapeutic interventions and a greater number of
horses than those enrolled in this study. In our study, both PI and PE were detected in minor
amounts, which is consistent with the previous studies
[14]. These phospholipids are probably not involved in
insect bite hypersensitivity, since there was no difference
between the groups in the first sampling and the con-
centrations of PE changed similarly in both groups. Traces of PA were found in all samples and as an inner
leaflet phospholipid, it is not normally detected from the
horse serum [14]. Presence of PA is probably related to
blood cells that remained when the samples were har-
vested without centrifugation [35]. Blood sampling that make lipids less water-soluble and enable them to
concentrate into autoserum preparations, although ac-
cording to the current study the total concentrations of
PC and SM were lower in these horses. This interpret-
ation could explain with caution the underlying mechan-
ism of this therapy, possibly culminating in the ability of
signalling molecules to engage their corresponding re-
ceptors which has been recognized to be a critical event
for the following mast cell responses [44–46]. The first sample (marked as stage 0 in text, tables and
figures) was taken when the horse had shown typical
clinical signs of IBH for at least 2 weeks. Blood sample
was first collected using 10 ml plain vacuum tubes that
were filled in full and kept at room temperature for at
least 3 hours before harvesting. The serum was har-
vested without using a centrifuge, thus avoiding destruc-
tion of serum lipids. With a calibrated pipette, 0.065 ml
of the serum was taken from the superficial layer and
stored in 4.2 ml of 48 % ethanol. The second sample
(marked as stage 4 in text, tables and figures) was col-
lected after a 4-week therapy of the affected horse and
handled equally. Samples were drawn from the counter-
part horse similarly and simultaneously. Therapy of the eczema horses All horses in the eczema group were treated with an oral
administration of an autoserum preparation for 4 weeks. Sera used for the preparations were drawn at the time of
the first blood sampling. Sera were harvested without
using a centrifuge and 0.065 ml of the serum was taken
from the superficial, lipid-containing layer of the tube. Lipid particles were washed twice with sterile water
(1:100), accordingly 0.065 ml collected from the superfi-
cial layer of the previous dilution. After the second
washing, 0.065 ml was taken from the superficial layer of
the latter solution and added finally in 48 % ethanol
(1:100) and absorbed in sugar granules [49]. A dose of
granules was given orally once a day for 2 weeks,
followed by a 1-week pause, after which horses were
medicated for one further week. Horses in the control
group received no therapy. Discussion y
The lower concentration of PC and SM detected in
the sera of affected horses was an unexpected finding,
since our earlier study demonstrated that autoserum
preparations made from the sera of horses with allergic
dermatitis showed significantly higher concentrations of
PC and SM than the preparations made from the sera of
healthy horses [35]. This contradictory result may be as-
sociated with the hydrophobic/hydrophilic interactions
of the lipid particles. The serum for autoserum prepara-
tions was collected from the superficial layer of the
blood sample, washed twice with water and finally added
in ethanol; all consecutive transfers were collected from
the superficial layer of the previous solution [35]. Thus
the most hydrophobic and water-insoluble lipid mole-
cules were presumably concentrated in such autoserum
preparations. Instead of washings, samples of the present
work were mixed directly with alcohol. Therefore, these
samples included not only the most hydrophobic but
also the less hydrophobic lipid particles, such as PC 26:0
with a short acyl chain or PC 36:6, 38:8 and 40:8 with
several double bonds; species that were not detected in
autoserum preparations [35]. The discrepancy with the
amounts of PC and SM in the serum versus autoserum
preparations may be interpreted as so far unknown,
hydrophilic/hydrophobic interactions possibly linked to
mineral salts that have been found to affect peculiarly
the distribution of lipids after successive washings [48]. Sera of affected horses may contain those mineral salts Fig. 6 Difference of sphingomyelin (SM) concentrations (pmol in total of
0.5 ml) after therapy. The change between the pre (stage 0-week) and
the post (stage 4-week) concentrations in eczema horses with (A, n = 6)
and without (B, n = 4) a positive clinical response and in their matched
healthy controls (Ac and Bc, respectively) Fig. 6 Difference of sphingomyelin (SM) concentrations (pmol in total of
0.5 ml) after therapy. The change between the pre (stage 0-week) and
the post (stage 4-week) concentrations in eczema horses with (A, n = 6)
and without (B, n = 4) a positive clinical response and in their matched
healthy controls (Ac and Bc, respectively) Page 7 of 9 Hallamaa and Batchu Lipids in Health and Disease (2016) 15:45 Page 7 of 9 Methods
Horses A total of 20 horses entered in this study in 2014, be-
tween the seasons of spring and autumn, the period
when horses are exposed to bites of insects. The horses
comprised 10 animals with clinical signs of insect bite
hypersensitivity (eczema group) and 10 matched healthy
controls (control group). The matching is presented in
Table 2. Clinical signs of eczema were categorised as
mild, moderate or severe at the time of blood sampling
and scored 1–3, respectively. Signs were mild, if the
horse had only pruritus without skin lesions. Pruritus
with mild skin affections in the mane, tail (Fig. 1a) and/
or body was graded as moderate, while pruritus over
large skin lesions was regarded as a severe sign. Clinical
signs were evaluated after the 4-week therapy and con-
sidered as relieved when signs became milder on this
scale (Fig. 1a and b). The changes in clinical signs were
recorded as relieved, no change or aggravated and re-
spectively scored as −1, 0 or +1. Five of the horses had
mild and 5 moderate clinical signs in the beginning of
the study. The matched healthy controls without a his-
tory of IBH lived in the same farms and were fed with
the similar fodder as their affected counterparts. Horses
were also matched with breed, gender and age as closely
as possible (Table 2). The eczema horse and its matched
control are numbered similarly in the tables and figures
throughout this article. This study has been approved by
the Regional State Administrative Agency of Southern
Finland (ESAVI/1016/04.10.07/2014) and owners’ signed
permission for inclusion was obtained. Conclusions Horses with an allergic dermatitis have an altered lipid
profile in their sera as compared with healthy controls and
these differences seem to change according to the clinical
status of the horses. Sphingomyelin shows a functional
role in equine insect bite hypersensitivity. Lipid profiling
may become an important target for further studies,
since relevant fingerprints delineating different patho-
logical disorders are needed – in both human and veter-
inary medicine. Acknowledgements We thank Doc Marja Raekallio for her valuable advice, suggestions and
supervision and Prof Pentti Somerharju for the cooperation with this study. We are grateful to Mrs Tarja Grundstrom and Mrs Leena Wathen for their
excellent laboratory assistance. We also wish to thank the horse owners for
their cooperation. 19. Quehenberger O, Armando AM, Brown AH, Milne SB, Myers DS, Merrill AH,
et al. Lipidomics reveals a remarkable diversity of lipids in human plasma. J Lipid Res. 2010;51:3299–305. doi:10.1194/jlr.M009449. 20. Fischer K, Kettunen J, Würtz P, Haller T, Havulinna AS, Kangas AJ, et al. Biomarker profiling by nuclear magnetic resonance spectroscopy for the
prediction of all-cause mortality: an observational study of 17,345 persons. PLoS Med. 2014;11(2):e1001606. doi:10.1371/journal.pmed.1001606. Author details
1V
i
Cli i 1Veterinary Clinic, Nummela, Finland. 2University of Helsinki, Faculty of
Veterinary Medicine, Helsinki, Finland. 3Department of Medical Biochemistry
and Developmental Biology, Institute of Biomedicine, University of Helsinki,
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y
Phospholipid classes analysed were PC, PE, PS, PI, PA
and SM. All the samples stored in ethanol were sub-
jected to Folch’s method [48] for lipid extraction, dried
under a N2 stream, reconstituted in 500 μl chloroform/
methanol (1:2) and further spiked with the following la-
belled standardsa corresponding to each head group:
D9(di-44:2) and D9(di-40:2) for PC, D4(di-40:2) and
D4(di-20:0) for PE, D3(di-40:2) and D3(di-44:2) for PS,
D6(di-36:2) and D6(di-28:0) for PI and finally unlabelled
25:0-SM. Liquid
chromatography-mass
spectrometry
(LC-MS) with selective reaction monitoring (SRM) was
used for the analyses. Waters ACQUITY Ultra Perform-
ance LC systemb equipped with a Waters ACQUITY BEH
C18 column (1.0 × 100 mm) was used to separate the mo-
lecular species using gradient elution. Solvent A was aceto-
nitrile/H2O (60:40) with 10 mM ammonium formate and
1 % NH4OH, while solvent B was isopropanol/acetonitrile
(90:10) containing 10 mM ammonium formate and 1 %
NH4OH. The flow rate was 0.13 ml/min and the column Hallamaa and Batchu Lipids in Health and Disease (2016) 15:45 Hallamaa and Batchu Lipids in Health and Disease (2016) 15:45 Page 8 of 9 Page 8 of 9 Page 8 of 9 temperature 60 °C. Solvent B was set to 40 % at in-
jection and increased linearly to 100 % in 14 min,
remained at this value for 3 min, decreased back to
40 % in 1 min and then remained there until the end
of the gradient at 20 min. The eluent was directed to
the electrospray ionization (ESI) source of Waters
Quattro Premier triple-quadrupole mass spectrome-
terb operated in the positive ion mode. For SRM tran-
sitions, proton adducts of the PC, PE, PS and PI
species were selected as the precursors, while the
product ion was either the head group (PC, SM, PI)
or the diacylglycerol fragment (PE, PS). For quantifi-
cation
purposes,
the
SRM
chromatograms
were
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Analysis of biological organic extract of plants in suppressing aphid (Myzus persicae) infestations
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World Journal of Biology Pharmacy and Health Sciences
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Abstract Myzus persicae commonly known as the peach-potato aphid, poses a significant threat to agricultural productivity,
particularly in potato cultivation, where infestations lead to substantial yield losses and quality degradation. In this
study, we investigated the potential of botanical extracts from neem (Azadirachta indica), clove (Syzygium aromaticum),
black pepper (Piper nigrum), and bay leaf (Laurus nobilis) in controlling Myzus persicae infestations. Experimental trials
were conducted to assess the mortality effects of these botanical extracts on Myzus persicae populations. Results
revealed that neem extract exhibited the highest mortality effect, with an impressive 97.7% mortality rate. Clove and
black pepper extracts followed closely, demonstrating mortality rates of 92.2% and 77.7%, respectively. In contrast,
bay leaf extract exhibited the least mortality effect, with only 48.8% mortality observed. Keywords: Myzus persicae; Natural pesticides; Eco-friendly pest control; Botanical extracts; Toxicity; Mortality. Analysis of biological organic extract of plants in suppressing aphid (Myzus persicae)
infestations Rahmah Khan and Newton Paul * Department of Zoology, Isabella Thoburn college, Lucknow, India. World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 ublication history: Received on 15 March 2024; revised on 27 April 2024; accepted on 29 April 2024 Article DOI: https://doi.org/10.30574/wjbphs.2024.18.2.0235 Analysis of biological organic extract of plants in suppressing aphid (Myzus persicae)
infestations * Corresponding author: Newton Paul. Copyright © 2024 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons or(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. Copyright © 2024 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. 2.1. Rearing of insect Myzus persicae specimens were reared under controlled conditions using non-infested fresh daisy twigs as nutritive
substrates to establish and maintain a standardized insect culture for experimental purposes. Plastic rearing containers,
equipped with perforated cloth to ensure adequate ventilation are utilized. Uninfected fresh daisy twigs sourced from
pesticide-free environments serve as nutritive substrates maintaining experimental integrity. Myzus persicae specimens
are introduced onto these twigs with precision using a brush ensuring controlled inoculation. Environmental
parameters were maintained. This includes maintaining a lower temperature range of 18-20°C and humidity levels
sustained at 50-60% to simulate more temperate conditions conducive to experimental requirements. Additionally, a
photoperiod of 16 hours simulates daylight conditions. Continuous twig replenishment ensures consistent aphid
nutrition. After a period of 25-30 days, new instances of M. persicae were observed on the twigs indicating the
completion of their developmental cycle. These aphids were carefully separated from the twigs to ensure accurate and
controlled experimental conditions (Plate-1: Figure-5). 2.3. Preparation of extracts: The prepared extracts were inspired by the research paper cited (Erdogan et. al., 2020), (Khalid et. al., 2020), (Madanat
et. al., 2016) though few changes were made. The process involved mixing 10 g of fine powder of spices or leaves with
100 ml of water. Boiling the mixture for 30 minutes on medium-high flame with constant stirring, followed by cooling
and filtration using Whatman filter paper no. 1 yielded an aqueous solution. To this solution 100 ml of ethanol was
added. The resulting solution was kept in the dark for 48-72 hours before being used in spray bottles for it to ferment
(Plate-1: Figure-4). The Neem extract was combined with the other spice extracts in a 1:1 w/w ratio to create three distinct mixtures for
demonstrating their joint efficacy against Myzus persicae thereby facilitating the assessment of potential synergistic
effects. World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 Botanical pesticides, derived from natural sources like plants, animals, microbes, and specific minerals, are known for
their low toxicity towards non-target organisms such as humans, animals, beneficial insects, and the environment
(Adusei & Azupio, 2022) (Adusei & Azupio, 2022) 2. Materials and methods The experiment was conducted at 15°±5°C and 94 ± 5% humidity. The experimental design comprised 4 treatments,
each replicated 5 times. The experimental setup involved the deployment of plastic containers as controlled
environments, with oasis serving as the substrate for experimental conditions, and nylon synthetic fabric employed for
precise filtration purposes. 2.4. Experimental setup 2.4.1. BIOASSAY 1: Analysis of toxic effect of plants extracts on Myzus persicae 2.2. Preparation of powder and extracts Four extract treatments were prepared from two leaf specimens, Azadirachta indica (Neem) and Laurus nobilis (Bay
Leaf), as well as two spice specimens, Syzygium aromaticum (Clove) and Piper nigrum (Black Pepper). Neem leaves
(Azadirachta indica) were collected from the campus of Isabella Thoburn College, Lucknow, while fruits of black pepper
(Piper nigrum), flower buds of clove (Syzygium aromaticum), and leaves of bay laurel (Laurus nobilis) were obtained
from domestic sources. After air-drying for 5 days, Neem leaves were desiccated until crisp, while all specimens were then ground to a fine
powder using an electric grinder. The resulting powders, each weighing 10 grams, underwent sieving for uniformity,
yielding final samples weighing 8-10 grams for precise experimental conditions. 1. Introduction Potato holds significant agricultural importance both in India and across Asia (Scott & Saurez, 2011). In Central India,
the cultivation of potatoes occurs during the short winter season, typically from October to March. Within this
agricultural context, diverse species of aphids assume a significant role as vectors for viral diseases affecting potato
crops, consequently contributing to seed degeneration (Bhatnagar et. al., 2012). In India, numerous researchers have previously conducted extensive studies on the appearance and population
dynamics of aphids on potatoes, as well as their correlation with virus transmission. This exploration stands as a crucial
prerequisite in enhancing our comprehension of aphids and their ecosystem (Bhatnagar et. al., 2012). Aphids, belonging
to the family Aphididae within the order Hemiptera, are notable agricultural nuisances present worldwide. They cause
considerable financial harm through their direct consumption of plant tissues, transmission of plant viruses, and
secretion of honeydew (Kareem Ullah et al., 2023). A wealth of research has consistently emphasized the economic
importance of aphids as agricultural pests (Emden & Harrington, 2017). The swift reproductive rate of aphids coupled
with their capacity to transmit plant pathogens presents a formidable menace to crop yields on a global scale, thus
demanding the implementation of efficient control strategies (Emden & Harrington, 2017). The transmission of over
110 plant viruses is attributed solely to Myzus persicae (Sulzer) (Singh & Agarwal, 2022). Wingless parthenogenetic
adult females are oval-shaped, 1.2 to 2.1 mm long, displaying colours such as whitish green, pale yellow-green, grey-
green, mid-green, dark green, pink, or red (CABI, 2021). The tobacco variant (nicotianae) ranges from bright yellow to
nearly black. Genetic colour diversity exists, with some intensifying green or magenta pigmentation in colder climates. Immature stages have a noticeable sheen, while adults are less shiny. Winged variants feature a black dorsal patch on
the abdomen (CABI, 2021). M. persicae has the potential to diminish the photosynthetic capacity of plants through the
secretion of sugary honeydew, consequently impacting both the quality and quantity of crops (Frantz, et. al., 2004). 2.5. Statistical analysis The collected data were analysed in a completely randomized design to find significant difference between Neem, Black
pepper, Clove and Bay leaf extract treatment. The analysis of variance (ANOVA) was applied. The test significant value
is 𝑃< 0 ∙05, the calculation was performed by using IMB SPSS statistical ver. 27 software and graphs are plotted using
the GraphPad prism 9 software for Microsoft. 2.4.3. BIOASSAY 3: Analysis of repellent action of different plants extract on Myzus persicae The repellency assay involved using a petri dish with filter paper divided into treated and untreated halves. Each half
was subjected to extract sprayed. Ten adults Myzus persicae were introduced onto each filter paper, covered to prevent
escape. After approximately 12 hours, the number of insects in treated and untreated areas was recorded. Three
replications were conducted for each spice. Repellence percentage was calculated using the following formula (Plate-1:
Figure-3). 𝑟𝑒𝑝𝑒𝑙𝑙𝑒𝑛𝑐𝑦% = (𝑁𝐶−𝑁𝑇)
(𝑁𝐶+ 𝑁𝑇) × 100 𝑟𝑒𝑝𝑒𝑙𝑙𝑒𝑛𝑐𝑦% = ( 𝐶
)
(𝑁𝐶+ 𝑁𝑇) × 1 Where, NC number of insects in control (untreated) area, And NT number of insects in treated area Where, NC number of insects in control (untreated) area, And NT number of insects in treated area 2.4.1. BIOASSAY 1: Analysis of toxic effect of plants extracts on Myzus persicae Plastic containers 3mm in diameter and 56mm in height, underwent thorough cleaning and drying procedures to ensure
sterility. Oasis, a hydrophilic foam was hydrated in water for 20-25 minutes to facilitate water absorption. Employing a
smaller container with a 4mm diameter, Oasis was precisely cut to fit the container with a 1cm height to support the
twigs. Uninfected twigs measuring 5cm in length were meticulously positioned within the Oasis substrate. 30 Myzus
persicae aphids were introduced onto the twigs under controlled conditions. To maintain a pest and contaminant free
environment, synthetic nylon fiber was used to cover the container opening secured with rubber bands. This
methodology ensures a controlled and sterile experimental environment conducive to scientific investigation. Each 25 World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 container was diligently labelled with the corresponding extract identifier to ensure precise investigation and treatment
allocation, adhering to rigorous scientific protocols. The experiment involves counting the mortality rate by counting
the number of dead Myzus persicae after 6hr, 12hr, 18hr, 24hr, 30hr, 36hr, 42hr, 48hr, 54hr, 60, 66hr, 72hr. In order to
enhance result accuracy and facilitate comprehensive data analysis, each aphid-extract container was duplicated across
five distinct sets. Additionally, to establish a comparative baseline a control group consisting of 30 untreated Myzus
persicae within 5cm stems was also replicated across five separate sets, conducted concurrently (Plate-1: Figure-1). container was diligently labelled with the corresponding extract identifier to ensure precise investigation and treatment
allocation, adhering to rigorous scientific protocols. The experiment involves counting the mortality rate by counting
the number of dead Myzus persicae after 6hr, 12hr, 18hr, 24hr, 30hr, 36hr, 42hr, 48hr, 54hr, 60, 66hr, 72hr. In order to
enhance result accuracy and facilitate comprehensive data analysis, each aphid-extract container was duplicated across
five distinct sets. Additionally, to establish a comparative baseline a control group consisting of 30 untreated Myzus
persicae within 5cm stems was also replicated across five separate sets, conducted concurrently (Plate-1: Figure-1). Neem + black pepper; Neem + clove and Neem + bay leaf Plastic containers were individually prepared, each containing a 5cm twig submerged in oasis substratum. Subsequently, 20 Myzus persicae were introduced into each container. A specific combination of extracts in a 1:1 v/v
ratio was applied via spraying. Containers were carefully labelled to facilitate proper identification of the extract
combinations. After 12 and 24 hours, the containers were examined, and the number of surviving and deceased Myzus
persicae was recorded. The mortality percentage was computed using a designated formula. An analysis of the results
was conducted to ascertain the efficacy of various spice combinations in repelling the insects (Plate-1: Figure-2). 𝑚𝑜𝑟𝑡𝑎𝑙𝑖𝑡𝑦% = 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑒𝑎𝑑 𝑖𝑛𝑠𝑒𝑐𝑡𝑠
𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑖𝑛𝑠𝑒𝑐𝑡𝑠 × 100 2.4.3. BIOASSAY 3: Analysis of repellent action of different plants extract on Myzus persica mortality% = number of dead insects
total number of insects × 100 mortality% = number of dead insects
total number of insects × 100 2.4.2. BIOASSAY 2: Analysis of mixed plants extract toxicity effect on Myzus persicae Another aspect of the experiment involved investigating synergistic and antagonistic interactions by combining
different extracts to assess their combined effects. Synergism, also known as potentiation, occurs when the combined
effect of two or more extracts exceeds the sum of their individual effects. Antagonism, on the other hand occurs when
the combined effect is less than the cumulative sum of their individual effects. The experiment was done by using a mixture of neem extract with other extracts. Prepared different combinations of
extracts in equal parts. The mixtures were: Neem + black pepper; Neem + clove and Neem + bay leaf 3. Result During analysis it was found that the efficacy of specific extracts against Myzus persicae, a common agricultural pest,
was evaluated. The study focused on assessing the mortality rates, combined effects and repellent actions of four distinct
extracts. Through rigorous observation and analysis, it was determined that Azadirachta indica (neem) and Syzygium
aromaticum (clove) exhibited the most notable insecticidal properties, demonstrating significant effectiveness against
Myzus persicae in Piper nigrum (black pepper) displayed comparatively lower potency than clove. Conversely, Laurus
nobilis was identified as the least efficacious among the extracts tested. These findings contribute to the understanding 26 World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 of natural insecticidal agents and their potential applications in pest management strategies (Table1 and Figure6, in
Table2 and Figure7 and in Table3 and Figure8). The highest mortality rates observed within 72 hours post-application. Neem powder treatments exhibited the most
substantial impact, achieving a mortality rate of 97.7%. Following closely behind, clove and black pepper treatments
recorded mortality rates of 92.2% and 77.76% respectively. Conversely, other treatments displayed comparatively
lower levels of mortality, with bay leaf treatment resulting in a 48.8% mortality rate (Neem>Clove>Black pepper>Bay
leaves) in Table1 and Figure6. In a combined action study, mortality counts were assessed 24 hours post-treatment. Neem extract served as the standard, mixed in a 1:1 v/v ratio with various prepared extracts in Table2 and Figure7. Depict the outcomes, with the Azadirachta indica and Syzygium aromaticum mixture demonstrating the highest toxicity,
resulting in a mortality rate of 64.43%. Following this, the combination of Azadirachta indica and Piper nigrum exhibited
a mortality rate of 35.53%. Conversely, the combination of Azadirachta indica and Laurus nobilis displayed the lowest
toxicity, with a mortality rate of 24.4%. (Neem + Clove > Neem + Black pepper> Neem + Bay leaves) (Table 2 and Figure
7). In the analysis of variance (ANOVA), it was observed that neem extract demonstrated the highest repellency efficacy,
registering a significant percentage of 83.3%. Following neem, clove and black pepper exhibited repellency percentages
of 66.6% and 56.6% respectively. Bay leaf demonstrated a repellency effect of 23.3% against Myzus persicae, as
illustrated in Table3 and Figure8. Figure 1 Experimental set up for mortality effect of
prepared extract. Figure 2 Experimental set up for joint effect of prepared
extracts
Figure 3 Experimental setup for repellency
Figure 4 Prepared extracts Figure 2 Experimental set up for joint effect of prepared
extracts Figure 1 Experimental set up for mortality effect of
prepared extract. Figure 2 Experimental set up for joint effect of prepared
extracts Figure 1 Experimental set up for mortality effect of
prepared extract. Figure 3 Experimental setup for repellency Figure 4 Prepared extracts 27 27 World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 Figure 5 Rearing of aphids Figure 5 Rearing of aphids Table 1 Mean mortality effect of different extracts on Myzus persicae
S. No. Extract No. Three of the five replicas were considered; Test significance is 𝒑> 𝟎. 𝟎𝟓 World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 of
Myzus
persicae
(N)
Mortality (Mean±S.E)
6hr
12hr 18hr 24hr 30hr
36hr
42hr
48hr
54hr
60hr
66hr
72hr
1. Control 30
0.33±
0.33
0.33±
0.33
1.00±
0.00
1.33±
0.33
1.33±
0.33
1.33±
0.33
1.66±
0.33
1.66±
0.33
1.66±
0.33
2.00±
0.33
2.33±
0.33
2.33±
0.33
2. Neem
30
2.33±
0.33
4.33±
0.33
6.33±
0.33
8.33±
0.33
12.66±
0.33
14.66±
0.66
17.33±
0.33
19.33±
0.33
21.66±
0.88
25.00±
0.57
26.00±
0.57
29.33±
0.33
3. Black
pepper
30
1.66±
0.33
2.33±
0.66
5.33±
0.33
7.66±
0.33
8.66±
0.33
10.00±
0.57
11.33±
0.88
12.66±
0.66
14.66±
0.66
16.33±
0.88
18.33±
1.20
23.33±
0.57
4. Clove
30
2.00±
0.57
4.33±
0.88
6.00±
0.57
7.00±
0.57
8.33±
0.33
12.66±
0.33
14.33±
0.33
16.66±
0.33
18.33±
0.33
21.33±
0.88
24.33±
0.33
27.66±
0.33
5. Bay leaf 30
0.33±
0.33
1.33±
0.33
2.66±
0.33
4.33±
0.33
6.00±
0.57
7.33±
0.33
8.66±
0.33
10.00±
0.57
11.33±
0.33
12.66±
0.88
13.33±
0.33
14.66±
0.88
Data represented in the table are the mean of three out of five replications; Test significance is 𝑷˂𝟎. 𝟓 Table 1 Mean mortality effect of different extracts on Myzus persicae Table 2 Mean mortality effect of different extracts mixed with Neem extract on Myzus persicae
S.No
Extract
Number of aphids
Mortality (Mean ±S.E)
Type of action
12hr
24hr
1
Control
30
1.33±0.33
1.66±0.33
2
Neem + black pepper
30
7.33±
0.66
10.66±
0.33
Synergistic
3
Neem +clove
30
10.66±
0.66
19.33±
0.88
Synergistic
4
Neem+ bay leaf
30
4.66±
0.33
7.33±
0.33
Synergistic
Three of the five replicas were considered; Test significance is 𝒑> 𝟎. 𝟎𝟓 Table 2 Mean mortality effect of different extracts mixed with Neem extract on Myzus persicae 28 World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032
Table 3 Repellency effect of different extracts on Myzus persicae
S. No
Plants used for
Extract
Number
of
aphids
Number
of
aphids
on
untreated area
Number of aphids on
treated area
Mean±S.E
A
B
C
a
b
c
1
Neem
10
9
8
8
1
2
2
8.33±0.33
2
Black pepper
10
5
6
6
5
4
4
5.66±0.33
3
Clove
10
7
7
6
3
3
4
6.66±0.33
4
Bay leaf
10
2
2
3
8
8
7
2.33±0.33
Three out of five replications are considered; Test significance is𝑷< 𝟎. 4. Discussion Parallel work found out that, during the nymphal period of Aphid, mortality rates reached 100.0% at the highest
concentration, indicating significant and potentially lethal effects when treated with neem bio-pesticide (Santos et. al.,
2004), which stands true for this experiment as the mortality rate of replica treated with Neem extract reaches 97.76%
(Table1 and Figure6). Another study showed, Neem seed oil (NSO) at a 1.0% concentration induced mortality ranging
from 94% to 100% in second instar nymphs of the green peach aphid, Myzus persicae (Sulzer) (Murray, 1994). Another
showed that the Hydroethanolic extract derived from neem leaves exhibited mortality rates exceeding 95% on Myzus
persicae, indicating significant efficacy against this species (Déla, et. al., 2014). Neem leaf extract contains saponins,
meliantriol, and azadirachtin, acting as vegetable insecticides against aphids. Saponins are stomach and contact poisons,
meliantriol works as a repellent, while azadirachtin inhibits aphids' hormone function, crucial for their development. (Tobing, et. al., 2023), which stands true with this experiment (Table1 and Figure6). The estimated lethal concentration (LC95) of clove essential oil, designed to terminate 95% of aphids, in the work of
(Pedro, et al., 2020), which stands true for this experiment as the mortality rate of replica treated with Neem extract
reaches 97.7% (Table1 and Figure6). Neem oil exhibited the highest nymph mortality rates at 96.67%, 100%, and 100%
in concentirations of 1%, 3%, and 5% respectively, after 72 hours. Clove oil followed with 80.00% mortality of Myzus
persicae seen in (Sonowal, et al., 2023). The methanol extracts from Azadirachta indica leaves, at concentrations of both
5% and 10%, provided complete protection (100%) to lentil and chickpea seeds from aphids by (Bhuiyah, et al., 2003). Therefore, this study indicates that neem exhibits the highest mortality rates, followed by clove, attributed to the
presence of Azadirachta and eugenol respectively. Our findings align closely with (Haddi, et al., 2015) studies indicating
that clove oil possesses insecticidal toxicity against Sitophilus zeamais Motschulsky, attributed to eugenol and the
sesquiterpene β-caryophyllene. (Table1 and Figure6) Another study of (Ahmed, et. al., 2021) states that, Black pepper essential oil has been identified as a potent insecticide,
demonstrating significant efficacy with an impressive 80% mortality rate upon contact application. The bay leaf extract
demonstrated minimal efficacy, with less than 50 percent mortality recorded during the shortest exposure time by
(Kaur, et. al., 2018) (Table1 and Figure6). World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 𝟎𝟓
Figure 6 Mean mortality effect of different extracts on Myzus persicae Table 3 Repellency effect of different extracts on Myzus persicae
S. No
Plants used for
Extract
Number
of
aphids
Number
of
aphids
on
untreated area
Number of aphids on
treated area
Mean±S.E
A
B
C
a
b
c
1
Neem
10
9
8
8
1
2
2
8.33±0.33
2
Black pepper
10
5
6
6
5
4
4
5.66±0.33
3
Clove
10
7
7
6
3
3
4
6.66±0.33
4
Bay leaf
10
2
2
3
8
8
7
2.33±0.33
Three out of five replications are considered; Test significance is𝑷< 𝟎. 𝟎𝟓 Table 3 Repellency effect of different extracts on Myzus persicae Figure 6 Mean mortality effect of different extracts on Myzus persicae Figure 6 Mean mortality effect of different extracts on Myzus persicae Figure 7 Mean mortality effect of different extracts mixed with Neem extract on Myzus persicae Figure 7 Mean mortality effect of different extracts mixed with Neem extract on Myzus persicae Figure 7 Mean mortality effect of different extracts mixed with Neem extract on Myzus persicae 29 29 World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 Figure 8 Mean of repellency effect of different extracts on Myzus persicae Figure 8 Mean of repellency effect of different extracts on Myzus persicae References [1]
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(Hemiptera; Aphididae), Journal of Bioresource Management, 9 (2). ISSN: 2309-3854 online [5]
Baig, B. & Yousaf, S., (2021). Phytochemical Screening of Neem and Black Pepper for Bio efficacy against Insect
Pests of Okra and Potato. Sarhad Journal of Agriculture, 37(2), pp. 697-705. [6]
Bhatnagar, A., Somi, A. K. & Chander, S., (2012). Buildup of Myzus persicae and development of mosaics on potato
in central India. Potato Journal 39(2):208-210, p. 3. [7]
Bhuiyah, M., Karim, A., Islam, B. & Alam, M., (2003). Control of pulse beetle in stored chickpea and lentil by
treating sacks with methanol extract of some selected botanicals. Bangl J Entomol., Volume 13, pp. 59-69. [8]
CABI, (2021). Myzus persicae (green peach aphid). CABI Campendium. [8]
CABI, (2021). Myzus persicae (green peach aphid). CABI Campendium. [9]
Déla, M. A., Ketoh, K. G., Amevoin, K. & Ameline, A., (2014). Evaluation of neem leaves-based preparations as
insecticidal agents against the green peach aphid, Myzus persicae. African Journal of Agricultural Research, 9(2),
pp. 1344-1352. [10] Emden, H. v. & Harrington, R., (2017). Aphids as crop pest. CABI. [10] Emden, H. v. & Harrington, R., (2017). Aphids as crop pest. CABI. [11] Erdogan P, Aksu P, Esin Kilinc G, Kahyaoglu Murat, Numan E Babaroglu (2020). Formulation of Allium sativum L. and Melia azedarach L. plant extracts and their effects on Myzus persicae Sulzer, 1776 (Hemiptera: Aphididae). J
Bacteriol Mycol Open Access. 2020;8(3):72‒76. DOI: 10.15406/jbmoa.2020.08.00277. [12] Frantz, J. D., Gardner, J., Hoffmann, M. P. 5. Conclusion In conclusion, the conducted experiment confirmed the high effectiveness of neem against Myzus persicae, with a
repellency rate exceeding that of clove and black pepper extracts. However, while clove and black pepper demonstrated
notable repellent properties, bay leaf extract emerged as the least effective bio-pesticide in combating aphids. These
findings underscore the potential of neem as a natural and potent solution for aphid control, highlighting the importance
of continued research into botanical alternatives for pest management in agriculture. 4. Discussion In a recent study, the efficacy of neem against aphids was examined, revealing a repellency rate of 68.9%. This finding
underscores the potential of neem as a natural and effective means of aphid control in agricultural settings. In another
recent study (Hartati, 2020), the synergistic effect of a blend containing clove and neem extracts was assessed, resulting
in an observed repellency rate of more than 30.33% against pest. The combined utilization of biopesticides, specifically
A. indica and E. globulus, at a concentration of 50%, has proven highly efficacious in combating wheat aphids (Baig &
Yousaf, 2021). Furthermore, the synergistic potency of these botanicals surpasses their individual effectiveness when
applied alone (Table2 and Figure7). In the recent scientific investigation, (Adhikari, et. al., 2020) empirical data
indicated that neem exhibits an 83% repellent efficacy, thereby providing further validation for the results obtained in
the conducted experiment. In (Lacotte, et. al., 2023) it was demonstrated that the repellent efficacy of clove, black
pepper and bay leaves is measured at 40%, 35% and 23.3% respectively, thus providing empirical validation for the
outcomes of the conducted experiment (Table3 and Figure8). 30 World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 Statement of informed consent Informed consent was obtained from all individual participants included in the study. Disclosure of conflict of interest Disclosure of conflict of interest Disclosure of conflict of interest The authors have no any conflict of interest for publishing this article. The authors have no any conflict of interest for publishing this article. Acknowledgments The author is thankful to the in-charge Prof. (Mrs.) Chitra Singh, Department of Zoology and Principal Prof. (Mrs.) Panzy
Singh of Isabella Thoburn College, Lucknow all the necessary laboratory facilities and support. World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 References Jahn, M. M., Frantz JD, Gardner J, Hoffmann MP, Jahn M.M (2004). Greenhouse screening of Capsicum accessions for resistance to green peach Aphid (Myzus persicae). hortscience,
39(6), pp. 1332-1335. 31 31 World Journal of Biology Pharmacy and Health Sciences, 2024, 18(02), 024–032 [13] Khalid Haldi, Eugenio E Oliveria, Leda R A Faroni, Daniela C Guedes, Natalie N S Miranda (2015). Sublethal
exposure to clove and cinnamon essential oils induces hormetic-like responses and disturbs behavioral and
respiratory responses in Sitophilus zeamais (Coleoptera: Curculionidae) Journal of eco entomology, 108(6). [14] Hartati, S., (2020). Effectiveness of citronella, clove, and neem essential oil mix formulas against budok disease
of patchouli plant. IOP Conference Series: Earth and Environmental Science, Volume 458, pp. 012-013. [15] Kareem Ullah, R. M., Gao, F., Sikandara, A. & Haiyan, W., (2023). Insights into the Effects of Insecticides on Aphids
(Hemiptera:Aphididae):Resistance Mechanisms and Molecular Basis. international journal of molecular science,
p. 18. [16] Kaur, M. KK Chahal, Amit Kumar, Ramandeep Kaur and Urvashi. (2018). Nematicidal activity of bay leaf (Laurus
nobilis L.) essential oil and its components against Meloidogyne incognita. Journal of Entomology and Zoology
Studies, 6(2), pp. 1057-1064. [17] Khalid, M. R., Aqueel, M. A., Majeed, M. Z. & Raza, A. B. M., (2020). In-vitro Response of Cereal Aphid Sitobion
avenae (Homoptera: Aphididae) against some Selected Botanical Extract. Journal of Agriculture and Food, 1(2),
pp. 1-12. [18] LacotteV., Marjolaine Rey, Sergio Peignier, PierreEdouard Mercier, Isabelle Rahioui, Catherine Sivignon, Lionel
Razy, Sylvain Benhamou, Sébastien Livi, Pedro da Silva. (2023). Bioactivity and chemical composition of forty
plant essential oils against the pea aphid Acyrthosiphon pisum revealed peppermint oil as a promising
biorepellent. Industrial Crops and Products, Volume 197. [19] Madanat, H. M., Al Antary, T. M. & Abu Zarqa, M. H., (2016). Toxicity of six Ethanol plant extracts against the green
peach aphid Myzus persicae Sulzer (Homoptera: Aphididae). Fresenius Environmental Bulletin, 25(3), pp. 706-
718. [20] Murray, B. I., (1994). Insect growth regulating effects of neem extract and azadirachtin on aphids. Entomologia
Experimentalis et Applicata, Volume 72, pp. 77-84. [21] Santos, T. M. d., Costa, N. P., Torres, A. L. & Arlindo Júnior, A. L., (2004). Effect of neem extract on the cotton aphid. Core, Volume 39, pp. 1071-1076. [22] Scott, G. & Saurez, V., (2011). Growth rates for potato in India 1961-2009 and their implications for industry. potato J. [23] Singh, R. & Agarwal, R., (2022). References Association of the aphids (Insecta: Homoptera: Aphididae) with monocots
flowering plants in India. International Journal of Biological Innovations, 4(2), pp. 252-281. [24] Sonowal, S., Rajkumari, P., Borah, N., Bora, B., Neog, P. P., & Borah, B. K. (2023). Efficacy of Different Essential Oils
Against Nymphal Stage of Aphid (Aphis craccivora Koch) in Green Gram (L.). International Journal of
Environment and Climate Change, 13(12), 473–478. https://doi.org/10.9734/ijecc/2023/v13i123704 [25] Tobing, O. L., Mulyaningsih, Y. & Safitri, A. D., (2023). The effect of concentration and frequency of neem leaf
extract on aphid attacks on chili plants. Indonesian Journal of applied research, 4(2), pp. 146-158. [26] Pedro F.S. Toledo, Luis O. Viteri Jumbo, Sarah M. Rezende, Khalid Haddi, Bruno A. Silva, Tarcísio S. Mello,
Terezinha M.C. Della Lucia, Raimundo W.S. Aguiar, Guy Smagghe, Eugenio E. Oliveira. (2020). Disentangling the
ecotoxicological selectivity of clove essential oil against aphids and non-target ladybeetles, Science of The Total
Environment, Volume 718,137328, ISSN 0048-9697, https://doi.org/10.1016/j.scitotenv.2020.137328. 32 32
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И.М. Микайлов, П.В. Григорьев, Д.А. Пташников, С.В. Майков ФГБУ «Российский научно-исследовательский институт травматологии
и ортопедии им. Р.Р. Вредена» Минздрава России
Санкт-Петербург, Россия Цель исследования – оценить результаты эндопротезирования плечевого сустава после радикальной резекции опу-
холи проксимального отдела плечевой кости, а также сравнить полученные функциональные результаты с исходами
эндопротезирования плечевого сустава у пациентов с обширным повреждением проксимального отдела плечевой кости
неопухолевого генеза. у
Материал и методы. Основную группу составили 38 пациентов, прооперированных в РНИИТО им. Р.Р. Вредена в
период с 2001 по 2013 г., из них замещение пострезекционного дефекта однополюсными эндопротезами проводилось у 26
(68%) человек, ревизионными и модульными системами с реверсивными головками – у 12 (32%). Контрольную группу составили 46 пациентов с обширным поражением проксимального отдела плечевой кости не-
опухолевого генеза. Сроки наблюдения составили от 7 месяцев до 9 лет. Оценка функциональных результатов прово-
дилась в сроки от 6 месяцев до года. Результаты. Анализ семилетней выживаемости по методу Каплана – Мейера, проведённый для пациентов с первич-
но злокачественными опухолями проксимального отдела плеча (25 больных), составил 77%. В основной группе среднее
значение функциональных результатов по шкале MSTS составило 77,7%. Однополюсное эндопротезирование показало
плохие результаты как в контрольной (61,3% по шкале MSTS, 60,7 по шкале Neer), так и в основной (67,7% – MSTS,
61,1 – Neer) группах. ) ру
Заключение. На сегодняшний день методом выбора, не ухудшающим онкологическую составляющую лечения паци-
ентов с опухолевым поражением плечевого сустава, является его эндопротезирование модульными реверсивными систе-
мами в сочетании с дополнительными средствами фиксации мягких тканей. Ключевые слова: опухоли плечевого сустава, эндопротезирование плечевого сустава, модульные эндопротезы. оригинальные статьи оригинальные статьи т р а в м а т о л о г и я и о р т о п е д и я р о с с и и оригинальные статьи ограничения амплитуды движения (особенно
отведения и наружной ротации) встречалось
в 95% случаев и было связано с болевым син-
дромом либо механическим препятствием. Патологические переломы и литическое разру-
шение кости произошли у 14 (36,9%) пациен-
тов: 9 (23,7%) – с патологическим переломом
хирургической шейки плеча, 5 (13,2%) –
с тотальной литической деструкцией прокси-
мального отдела плечевой кости. ограничения амплитуды движения (особенно
отведения и наружной ротации) встречалось
в 95% случаев и было связано с болевым син-
дромом либо механическим препятствием. Патологические переломы и литическое разру-
шение кости произошли у 14 (36,9%) пациен-
тов: 9 (23,7%) – с патологическим переломом
хирургической шейки плеча, 5 (13,2%) –
с тотальной литической деструкцией прокси-
мального отдела плечевой кости. Так, по данным литературы, использование
аллотрасплантата с целью рефиксации ротатор-
ной манжеты обеспечивает лучшую функцию
оперированной конечности, однако сопряжено
с такими осложнениями, как перелом аллотран-
сплантата и инфекции [5, 10, 17]. Использование аутотрансплантата из мало-
берцовой кости – технически сложная опера-
ция, требующая соответствующего уровня от
оперирующего хирурга и дающая далеко не
лучшую функцию. Самым частым осложнени-
ем при этом виде оперативного лечения явля-
ется перелом трансплантата [10]. Перед операцией проводились следующие
виды обследования: – онкологическое, когда при отсутствии на
предоперационном этапе результатов гистоло-
гии и данных о распространении опухоли вы-
полняли детальный онкологический поиск, в
том числе и пункционную биопсию; – онкологическое, когда при отсутствии на
предоперационном этапе результатов гистоло-
гии и данных о распространении опухоли вы-
полняли детальный онкологический поиск, в
том числе и пункционную биопсию; Мы в своей практике для замещения постре-
зекционного дефекта у данной категории паци-
ентов используем ревизионные и модульные
онкологические системы со стандартными и ре-
версивными головками. у
у
– общесоматическое – традиционное пред
операционное исследование соматического ста-
туса пациента, выявление сопутствующей пато-
логии и подготовка его к операции; Цель исследования – оценить результаты
эндопротезирования плечевого сустава после
радикальной резекции опухоли проксимального
отдела плечевой кости, а также сравнить полу-
ченные функциональные результаты с исходами
эндопротезирования плечевого сустава у пациен-
тов с обширным повреждением проксимального
отдела плечевой кости неопухолевого генеза. р
– местное, целью которого являлось опреде-
ление локализации процесса, степени поражения
кости, распространенности на окружающие ткани
для определения объема операции, ее прогноза и
подбора эндопротеза. Для этого выполняли рент-
генологическое обследование пораженного плече-
вого сустава в двух стандартных проекциях с за-
хватом плеча до локтевого сустава. оригинальные статьи При обширных
распространенных процессах выполняли КТ по-
раженной области, дополняя это исследование ан-
гиографией для выяснения характера кровоснаб-
жения опухолевого очага и его взаимоотношения
с магистральными сосудами. С целью уточнения
степени распространенности процесса, взаимо-
отношения опухолевых масс с мягкими тканями,
окружающими сустав, и протяженности распро-
странения опухоли по костному каналу выполня-
ли МРТ данной области [1, 3, 4, 13, 16, 17]. – местное, целью которого являлось опреде-
ление локализации процесса, степени поражения
кости, распространенности на окружающие ткани
для определения объема операции, ее прогноза и
подбора эндопротеза. Для этого выполняли рент-
генологическое обследование пораженного плече-
вого сустава в двух стандартных проекциях с за-
хватом плеча до локтевого сустава. При обширных
распространенных процессах выполняли КТ по-
раженной области, дополняя это исследование ан-
гиографией для выяснения характера кровоснаб-
жения опухолевого очага и его взаимоотношения
с магистральными сосудами. С целью уточнения
степени распространенности процесса, взаимо-
отношения опухолевых масс с мягкими тканями,
окружающими сустав, и протяженности распро-
странения опухоли по костному каналу выполня-
ли МРТ данной области [1, 3, 4, 13, 16, 17]. Введение ния, за последний период значительно увеличи-
лись [9, 11, 17, 18]. Это привело к росту неудачных
исходов малоинвазивных операций, таких как
внутрикостный остеосинтез. Повысилось коли-
чество ревизий на фоне прогрессирования ло-
кального литического опухолевого процесса и,
как следствие, нестабильности конструкции. В
связи с этим выросла актуальность более ради-
кальных первичных операций [16–18]. ния, за последний период значительно увеличи-
лись [9, 11, 17, 18]. Это привело к росту неудачных
исходов малоинвазивных операций, таких как
внутрикостный остеосинтез. Повысилось коли-
чество ревизий на фоне прогрессирования ло-
кального литического опухолевого процесса и,
как следствие, нестабильности конструкции. В
связи с этим выросла актуальность более ради-
кальных первичных операций [16–18]. Проксимальный отдел плечевой кости явля-
ется достаточно частой локализацией первич-
ных опухолей и метастатических поражений
костей [1, 3, 9, 11, 17, 18]. Органосохраняющие операции при опухоле-
вом поражении проксимального отдела плечевой
кости у взрослых пациентов всегда сопряжены
со значительными хирургическими трудностя-
ми. Чаще всего это связано с распространением
опухолевого процесса за пределы кости, что при-
водит к необратимому поражению ротаторной
манжеты (надостная, подостная, малая круглая
и подлопаточная мышцы) и капсулы плечевого
сустава, в результате чего при выполнении резек-
ции опухоли теряются основные стабилизаторы
плечевого сустава. В то же время современные
исследования показывают, что при использова-
нии реверсивного эндопротеза существует воз-
можность восстановления функции плечевого
сустава в тех случаях, когда удается сохранить
дельтовидную мышцу и ее иннервацию без ущер-
ба для онкологического результата (сохраняя от-
рицательный хирургический край) [1, 4, 15, 16]. р
р
[
]
На сегодняшний день существует целый ряд
органосохраняющих операций, используемых
при опухолевом поражении проксимального
отдела плечевой кости: аллопластика прокси-
мального отдела плеча в сочетании с накостным
остеосинтезом пластиной; использование алло-
трансплантата в сочетании с эндопротезом пле-
чевой кости; изолированное использование эндо-
протезов плечевого сустава, как однополюсных,
так и реверсивных; аутопластика дефекта васку-
ляризированным трансплантатом малоберцовой
кости в сочетании с накостным остеосинтезом. Все эти методики широко используются онко-
ортопедами, и каждая имеет ряд преимуществ и
недостатков [5, 10, 17]. б р
рур
р
) [
]
Средние сроки выживаемости пациентов с
вторичным поражением костей, вызванным раз-
витием комплексного противоопухолевого лече- Выбор того или иного вида операции зависит
от предпочтений хирурга, его опыта, наличия
банка донорской кости. т р а в м а т о л о г и я и о р т о п е д и я р о с с и и
2014 – 4 (74) 27 2014 – 4 (74) оригинальные статьи Материал и методы В клинике РНИИТО им. Р.Р. Вредена в пе-
риод с 2001 по 2013 г. было выполнено 38 ор-
ганосохраняющих операций пациентам с опу-
холевым поражением проксимального отдела
плечевой кости. Возраст пациентов составил от 15 лет до
71 года (38,5±3,34). Мужчин было 10 (26,3%),
женщин – 28 (73,7%). Распределение по нозоло-
гии: хондросаркома – 5 (13,2%) случаев; ГКО – 10
(26,3%); остеосаркома – 3 (7,9%); саркома Юинга –
1 (2,6%); плазмацитома – 1 (2,6%); лимфо-
ма кости – 1 (2,6%); гемангиоэндотелиома – 1
(2,6%); метастатическое поражение кости – 10
(26,3%), из них рак молочной железы (РМЖ) –
5 (13,2); рак почки – 3 (2,6%), рак легких –
2 (5,2%); аневризмальная костная киста –
3 (7,9%), хондробластома – 3 (7,9%). [
]
Все пациенты были условно разделены на
три группы: 1-я группа. Пациенты с опухолями прокси-
мального отдела плечевой кости, не выходящи-
ми за пределы кости и не распространяющимися
по костному каналу ниже уровня прикрепления
дельтовидной мышцы (рис. 1). Все пациенты получали соответствующую
неоадъювантную, химио- и/или лучевую тера-
пию согласно стандартным протоколам лече-
ния. Хондросаркомы и доброкачественные но-
вообразования плечевой кости лечили только
хирургическим методом. 2-я группа. Пациенты с опухолями, поражаю-
щими проксимальный отдел плеча, с мягкоткан-
ным компонентом, выходящим за пределы кости,
с вовлечением в опухолевый процесс ротаторной
манжеты, но не распространяющимися ниже при-
крепления дельтовидной мышцы (рис. 2). Сроки наблюдения за пациентами составили
от 7 месяцев до 9 лет, средний срок – 52 месяца. 3-я группа. Пациенты с опухолями, поража-
ющими проксимальный отдел плеча, с мягко
тканным компонентом, выходящим за пределы
кости, с вовлечением в опухолевый процесс ро-
таторной манжеты, распространяющимися ниже
прикрепления дельтовидной мышцы (рис. 3). Клинически опухолевое поражение прок-
симального отдела плечевой кости у всех
пациентов
характеризовалось
постоянным
болевым синдромом, усиливающимся при дви-
жении. Нарушение функции сустава в виде 2014 – 4 (74) т р а в м а т о л о г и я и о р т о п е д и я р о с с и и 28 оригинальные статьи Рис. 1. Рентгенограммы пациента Л., 62 лет, с метастатическим поражением головки
плечевой кости, в анамнезе рак почки: а, б – до операции; в, г – после операции
а
б
в
г в г б г в а Рис. 1. Рентгенограммы пациента Л., 62 лет, с метастатическим поражением головки
плечевой кости, в анамнезе рак почки: а, б – до операции; в, г – после операции а
б
в
Рис. 2. т р а в м а т о л о г и я и о р т о п е д и я р о с с и и
2014 – 4 (74) Рис. 3. Пациентка Т., 48 лет, с гемангиоэндотелиомой проксимального отдела левой плечевой кости:
а – ренгенограмма до операции; б – КТ с ангиографией до операции; в – рентгенограмма после операции т р а в м а т о л о г и я и о р т о п е д и я р о с с и и Материал и методы Рентгенограммы пациентки Ф., 56 лет, в анамнезе рак молочной железы, метастатическое поражение
проксимального отдела левой плечевой кости: а, б – до операции; в – через 1,5 месяца после операции а б в Рис. 2. Рентгенограммы пациентки Ф., 56 лет, в анамнезе рак молочной железы, метастатическое поражение
проксимального отдела левой плечевой кости: а, б – до операции; в – через 1,5 месяца после операции б
а
в б
а
в б в а Рис. 3. Пациентка Т., 48 лет, с гемангиоэндотелиомой проксимального отдела левой плечевой кости:
а – ренгенограмма до операции; б – КТ с ангиографией до операции; в – рентгенограмма после операции а в м а т о л о г и я и о р т о п е д и я р о с с и и
2014 – 4 (74) 2014 – 4 (74) 29 оригинальные статьи Пациентам 1-й группы выполнялась резек-
ция пораженного проксимального отдела пле-
чевой кости с сохранением вращательной ман-
жеты (16 человек). Пациетам 2-й и 3-й групп
выполнялась объемная резекция очага ново-
образования с соблюдением онкологических
принципов (22 человека). Величина резекции
составила от 5 до 17 см. женное дегенеративное поражение ротаторной
манжеты плеча. 30 пациентам был установлен
однополюсный протез, 16 пациентам – ревер-
сивный эндопротез. Особенности
техники
операции. Имплантацию эндопротеза осуществляли в
положении больного на спине в положении
«пляжного кресла» с полностью свободной опе-
рируемой конечностью. Для замещения пострезекционных дефек-
тов проксимального отдела плечевой кости у 26
(68%) пациентов использовались ревизионные
ножки цементной фиксации с однополюсными
головками, у 12 (32%) – ревизионные и стан-
дартные ножки цементной фиксации с ревер-
сивными головками. р ру
Мы использовали два варианта доступа,
применяемых при стандартном эндопротези-
ровании: трансдельтовидный или дельтовидно-
пекторальный [2, 11, 14, 15, 18]. При наличии
большого мягкотканного компонента опухоли
для увеличения обзора и облегчения выделе-
ния опухоли мы выполняли акромионтомию. Резекцию проксимального отдела плечевой кос
ти производили в пределах здоровых тканей. В
случаях, когда операция проводилась по поводу
злокачественного новообразования, остеотомию
плеча выполняли из расчета захвата 2–3 см ви-
зуально здорового участка кости [2, 9, 14, 17]. Выполнив остеотомию, отправляли содержимое
костномозгового канала с края опила на срочное
цитологическое исследование. Удостоверившись
в достаточности уровня резекции, абластично, в
пределах здоровых тканей, проводили выделе-
ние проксимального отдела плеча с полным его
удалением единым блоком вместе с головкой и
капсулой сустава. Образовавшийся пострезек-
ционный дефект кости замещали длинной реви-
зионной ножкой цементной фиксации или мо-
дульным эндопротезом. Материал и методы Глубина посадки ножки
эндопротеза составляла от 7 до 12 см (в среднем
9 см). При использовании ревизионной ножки
эндопротеза выполняли пластику ее не погру-
женной в кость части костным цементом с анти-
биотиком. С целью рефиксации отсечённых со-
храненных мышц в цемент внедряли лавсановые
нити. Использование этой методики способство-
вало уменьшению полости, формирующейся
после удаления опухоли, и стабилизации плече-
вого сустава (рис. 4). Во всех случаях суставной отросток ло-
патки не был вовлечен в опухолевый процесс. Пациентам выполнялся первый тип резекции
по М.М. Малауэру [11, 13, 17]. Край опила плечевой кости не содержал опу-
холевых клеток (R-0), подмышечный нерв не
был вовлечен в опухолевый процесс либо по-
врежден в ходе операции. Оценка результатов лечения больных про-
водилась с учетом как ортопедических (ана-
лиз динамики клинико-рентгенологической
картины и функции сустава), так и онко-
логических критериев (число местных ре-
цидивов новообразований и выживаемость
пациентов). Функциональный результат лечения оцени-
вали по шкалам Musculoskeletal Tumour Society
(MSTS) и Neer [7, 8, 12]. В клиническом раз-
деле шкалы MSTS учитываются 6 параметров:
боль, функция конечности, позиционирование
руки, ловкость, подъем тяжести и эмоциональ-
ное восприятие результата лечения. Каждый
параметр оценивается по 5-балльной шкале,
после чего по сумме набранных баллов вычис-
ляется процент от максимально возможного
количества баллов 30. Шкала Neer учитывает
как восприятие пациентом функции опериро-
ванной конечности, так и оценку амплитуды
всех возможных движений. Максимальный
результат – 100 баллов, хороший – более 89
баллов, удовлетворительный 80–89 баллов,
неудовлетворительный 70–79 баллов, плохой
– менее 70 баллов. Рис. 4. Использование цементной пластики и лавсановых
нитей с целью рефиксации отсеченных мышц Контрольную группу составили 46 пациен-
тов с обширным поражением проксимального
отдела плечевой кости неопухолевого гене-
за (асептический некроз, последствия много
оскольчатых застарелых переломов головки
плечевой кости), которым было выполнено эн-
допротезирование в РНИИТО им. Р.Р. Вредена
в период с 2006 по 2012 г. Дефект кости составил
от 3 до 5 см, во всех случаях наблюдалось выра- Рис. 4. Материал и методы Использование цементной пластики и лавсановых
нитей с целью рефиксации отсеченных мышц 2014 – 4 (74) т р а в м а т о л о г и я и о р т о п е д и я р о с с и и
2014 – 4 (74) т р а в м а т о л о г и я и о р т о п е д и я р о с с и и 30 оригинальные статьи У шести пациентов для рефиксации отсечён-
ных мышц мы использовали тубы из полимер-
ного волокна. После операции пациенты получали профи-
лактическую антибактериальную и антикоагу-
лянтную терапию. Дренажи удаляли на 2–5-е
сутки после операции. Во всех случаях удалось достигнуть хороше-
го натяжения мягких тканей, восстановить дли-
ну конечности. Активизация больного начиналась на 2-е
сутки после операции с участием специалистов
по лечебной физкультуре. Рентгенологический
контроль осуществлялся ежеквартально. В первой группе пациентов мы пытались
максимально сохранить ротаторную манжету,
что способствовало лучшей стабилизации сус
тава и раннему восстановлению функции ко-
нечности. После удаления опухолевого очага
выполняли установку эндопротеза с ножкой
цементной фиксации в положении головки
20–25° ретроверсии. Результаты Онкологический результат. Cроки наблюде-
ния составили от 7 месяцев до 9 лет, в среднем
3,4 ± 0,7 года. Местный рецидив опухоли после
резекции проксимального отдела плечевой кос
ти и замещения дефекта эндопротезом отмечен
в одном случае, что составляет 2,6% от обще-
го числа больных. У данного пациента через 8
месяцев выявлено распространение новообра-
зования (хондросаркомы) по мягким тканям в
области оперативного вмешательства. Больной
направлен в онкологический стационар для
выполнения ампутации. В течение 18 месяцев
пациент умер от прогрессирования онкологи-
ческого заболевания. На момент контрольного
осмотра в разные сроки (3 и 7 лет) умерли еще
2 пациента из-за прогрессирования основного
заболевания (метастатического поражения лег-
ких), из них один случай остеосаркомы и один
случай саркомы Юинга. Анализ семилетней вы-
живаемости по методу Каплана – Мейера, про-
ведённый для пациентов с первично злокаче-
ственными опухолями проксимального отдела
плеча (25 больных), составил 77% (рис. 5). Во второй группе больных ввиду вовлечения
в опухолевый процесс ротаторной манжеты в
ряде случаев мы использовали эндопротезы с
реверсивными головками. В третьей группе пациентов принципиаль-
ным моментом было восстановление места
прикрепления дельтовидной мышцы, по воз-
можности большой грудной мышцы, что спо-
собствовало стабилизации эндопротезапроте-
за, а также в значительной степени влияло на
функциональный результат. Имплантацию метаглена и гленосферы осу-
ществляли по стандартной технологии с допол-
нительной фиксацией 3–4 винтами. Послеоперационное
ведение
больных. Конечность фиксировали на отводящей шине,
что облегчало уход за больным в раннем после-
операционном периоде, а также способствовало
формированию первичных рубцов в правиль-
ном положении конечности. Рис. 5. Выживаемость пациентов со злокачественными опухолями
проксимального отдела плечевой кости Рис. 5. Выживаемость пациентов со злокачественными опухолями
проксимального отдела плечевой кости т р а в м а т о л о г и я и о р т о п е д и я р о с с и и т р а в м а т о л о г и я и о р т о п е д и я р о с с и и
2014 – 4 (74) 31 31 2014 – 4 (74) оригинальные статьи Оценка функциональных результатов по
MSTS проводилась в сроки от 6 месяцев до года
после операции у 38 пациентов. Результаты сравнительной оценки функци-
ональных результатов, полученные в основной
(онкологической) и контрольной (ортопедиче-
ской) группах, представлены в таблице 3. Распределение функциональных результа-
тов по группам отражено в таблице 1. ) ру
,
р
Осложнения при эндопротезировании прок-
симального отдела плечевой кости. Обсуждение Вывих (подвывих) эндопротеза. Профилак
тика вывиха обеспечивается за счет фиксации
сохраненных мышц (ротаторный манжеты) непо-
средственно к протезу, что дает сбалансированное
натяжение мягких тканей, центрацию головки эн-
допротеза, объем движений, а также способствует
стабилизации сустава. Для этого желательно ис-
пользовать толстые нерезорбируемые нити. Мы
в ряде случаев использовали полимерные тубы,
что значительно упрощало восстановление точек
прикрепления мышечного аппарата, ускоряло
восстановление функции конечности. Еще одним
фактором стабилизации является восстановление
длины конечности, что дает хорошее натяжение
как мышц, так и фасции и способствует равно-
мерному распределению нагрузки. С целью про-
филактики вывиха эндопротеза головка должна
быть установлена в ретроверсии от 15 до 30° [2,
11, 13, 14, 16]. Функциональные результаты. Как показал
анализ литературы, оценка функциональных ре-
зультатов у данной категории пациентов прово-
дится исключительно по шкале MSTS. Для более
детального изучения данного вопроса мы реши-
ли ввести в исследование контрольную группу
пациентов с поражением плечевого сустава не-
опухолевого генеза и оценить функциональный
результат при помощи шкал MSTS и Neer. р
у
р
По данным различных авторов, при использо-
вании модульных онкологических эндопротезов
плечевого сустава среднее значение функцио-
нальных результатов по MSTS колеблется от 65%
до 88% [1, 2, 6, 17, 18, 19]. По результатам нашего
исследования, этот показатель составил 87,6%
при использовании реверсивных эндопротезов
и 67,7% при использовании однополюсных эн-
допротезов, средний показатель – 77,7%. Однако
стоит отметить, что на функцию реверсивного
эндопротеза значительно влияет сохранность
естественного места прикрепления дельтовид-
ной мышцы и ее целостность. Как показало наше
исследование, суммарный результат в 1-й и 2-й
группах (пациенты с сохраненной дельтовидной
мышцей) составил 93,1% по MSTS. В нашем исследовании вывих протеза отме-
чен у 1 (2,6%) пациента через 6 месяцев после
операции. Пациенту было выполнено ревизи-
онное оперативное вмешательство с заменой
эндопротеза на реверсивный. Контрольный ос-
мотр через 6 месяцев показал хорошую функ-
цию – 83% по MSTS. )
В контрольной группе функциональные по-
казатели для реверсивных эндопротезов соста-
вили 95% по MSTS и 89,25 по шкале Neer. Для
пациентов основной группы при использовании
реверсивных эндопротезов функциональный ре-
зультат составил 87,6% по MSTS и 83,5 по Neer. Разницу в показателях можно объяснить резким
снижением функции в 3-й группе «онкологи-
ческих» пациентов, которым выполнялась ре-
фиксация дельтовидной мышцы к эндопротезу. Суммарный результат в 1-й и 2-й группах сопо-
ставим с результатами в контрольной группе. Инфекционные осложнения. По данным раз-
личных авторов, инфекционные осложнения
являются достаточно распространенной при-
чиной, приводящей к ревизионным операциям
и удалению эндопротеза. Результаты Общее ко-
личество осложнений, приведших к реэндо-
протезированию, составило 3 (7,9%) случаев. Инфекционные осложнения развились у од-
ного пациента на фоне нарушения двигатель-
ной активности на 14-е сутки после операции. Пациенту было выполнено ревизионное опе-
ративное вмешательство, протез сохранен. Мы
наблюдали один случай вывиха однополюс-
ного эндопротеза, развившегося через 6 ме-
сяцев после первичного эндопротезирования. Также был выявлен один случай асептической
нестабильности однополюсного эндопротеза,
развившейся через 3,5 года после первичного
оперативного вмешательства, потребовавший
ревизионного эндопротезирования с заменой
протеза на реверсивный. Разрушения конструк-
ции мы не наблюдали. Таблица 1
Средние функциональные результаты
онкологического эндопротезирования
по шкале MSTS
Группа
Однополюсный
эндопротез
Реверсивный
эндопротез
n
оценка, %
n
оценка, %
1
13
70,4
3
95,6
2
8
68,5
6
90,6
3
5
64,2
3
76,6
В среднем
по группам
–
67,7
–
87,6
n – количество пациентов. Таблица 1
Средние функциональные результаты
онкологического эндопротезирования
по шкале MSTS n – количество пациентов. По шкале Neer результаты однополюсного
эндопротезирования у 23 пациентов были оце-
нены в разные сроки наблюдения (в среднем 3
года), а результаты применения реверсивных
эндопротезов оценивались у 12 пациентов в
сроки от 6 месяцев до года (табл. 2). При использовании однополюсных эндо-
протезов в отдаленном периоде (через 2–3 года
с момента операции) мы наблюдали подвывих
головки эндопротеза у 8 (35%) больных, что не
повлияло на результат. Таблица 2
NEER Таблица 2
Средняя оценка функциональных результатов онкологического эндопротезирования по шкале NEER
Группа
Однополюсный эндопротез
Реверсивный эндопротез
количество
пациентов
средний результат,
баллы
количество
пациентов
средний результат,
баллы
1
13
68,1 (плохой)
3
90,6 (хороший)
2
7
63 (плохой)
6
87,5 (удовлетворительный)
3
3
52,2 (плохой)
3
72,6 (неудовлетворительный)
В среднем
по группам
–
61,1 (плохой)
12
83,5 (удовлетворительный) Таблица 3
Средняя оценка функциональных результатов эндопротезирования по шкалам Neer и MSTS
в основной и контрольной группах
Группа
Шкала MSTS, %
Шкала Neer, баллы
Однополюсное
эндопротезирование
Реверсивное
эндопротезирование
Однополюсное
эндопротезирование
Реверсивное
эндопротезирование
Контрольная
61,3
95
60,7 плохой
результат
89,25 хороший результат
Основная
67,7
87,6
61,1 плохой
результат
83,5 удовлетворительный
результат т р а в м а т о л о г и я и о р т о п е д и я р о с с и и
2014 – 4 (74) т р а в м а т о л о г и я и о р т о п е д и я р о с с и и 32 оригинальные статьи Обсуждение Потери онкологиче-
ских эндопротезов плечевого сустава вслед-
ствие развития местного инфекционного вос-
палительного процесса составляют от 5 до 15%
[9, 11, 13, 14, 17, 18]. В нашем исследовании парапротезная ин-
фекция наблюдалось у 1 (2,6%) пациента. Срок
развития осложнения составил 14 дней. Нам
удалось купировать инфекционный процесс
путем хирургической обработки раны без уда-
ления эндопротеза. Однополюсное эндопротезирование показа-
ло плохой результат как в контрольной (61,3%
MSTS, 60,7 Neer), так и в основной (67,7%
MSTS, 61,1 Neer) группах. Асептическая нестабильность эндопротеза. Асептическая нестабильность эндопротеза. Нестабильность эндопротеза отмечена у 1
(2,6%) пациента через 3,5 года после операции. Данному пациенту первоначально был уста-
новлен однополюсный эндопротез на ножке
цементной фиксации. Нами было выполнено
ревизионное эндопротезирование с установкой
реверсивного эндопротеза на длинной ревизи-
онной цементной ножке. По данным различных
авторов, асептическая нестабильность у этой
категории пациентов встречается в пределах
1,5–5,5% [11, 13, 14, 16–18]. О Это дает возможность с уверенностью говорить
о том, что использование реверсивных систем у
пациентов с опухолевым поражением прокси-
мального отдела плеча – перспективная методи-
ка, которая не только сохраняет конечность па-
циенту, но и восстанавливает хорошую функцию,
соизмеримую с функцией у пациентов, подверг-
шихся эндопротезированию плечевого сустава с
патологией неопухолевой природы. Однако из-за
малого количества и небольших сроков наблюде-
ний не представляется возможным получить ста-
тистически достоверные результаты, поэтому мы
считаем необходимым продолжать работу в этом
направление с целью накопления большего коли-
чества материала и клинических наблюдений. Основными ортопедическими критериями,
определяющими удачный исход оперативного
лечения пациентов с объемными резекциями
проксимального отдела плечевой кости, явля- р а в м а т о л о г и я и о р т о п е д и я р о с с и и
2014 – 4 (74) т р а в м а т о л о г и я и о р т о п е д и я р о с с и и 33 2014 – 4 (74) оригинальные статьи 3. Тепляков В.В., Карпенко В.Ю., Франк Г.А., Буланов А.А.,
Державин В.А., Шаталов А.М. Эндопротезирование при
опухолевом поражении длинных костей. Российский он-
кологический журнал: 2009; (5):11–14. ются: восстановление длины конечности, пра-
вильная установка и надежная фиксация со-
ответствующего эндопротеза, восстановление
точек естественного прикрепления мышц. Все
это позволяет сохранить конечность и достаточ-
ную для нормальной жизни функцию плечевого
сустава. Стоит отметить, что при использовании
ревизионных (длинных) ножек эндопротеза хи-
рург может столкнуться с определенными тех-
ническими трудностями во время их установки. Заключение g
9. Fuhrmann R.A., Roth A., Venbrocks R.A. Salvage of the upper
extremity in cases of tumorous destruction of the proximal
humerus. J Cancer Res Clin Oncol. 2000; 126:337–344. Применяемая нами методика эндопротези-
рования плечевого сустава с использованием
реверсивных систем показала в основном от-
личные и хорошие функциональные результа-
ты, вполне сопоставимые с результатами эндо-
протезирования, выполненного у пациентов с
поражениями плечевого сустава неопухолевого
генеза. В то же время однополюсное эндопроте-
зирование показало плохие результаты в обеих
исследуемых группах. Из этого следует, что на
сегодняшний день методом выбора, не ухудша-
ющим онкологическую составляющую лечения
этой категории пациентов, является эндопроте-
зирование плечевого сустава модульными ре-
версивными системами в сочетании с дополни-
тельными средствами фиксации мягких тканей. 10. Manfrini M., Vanel D., De Paolis M., Malaguti C., Innocenti
M., Ceruso M., Capanna R., Mercuri M. Imaging of
vascularized fibula autograft placed inside a massive allograft
in reconstruction of lower limb bone tumors. Am J Roentgenol. 2004;15(4):963–970. 11. Mayilvahanan N., Paraskumar M., Sivaseelam A., Natarajan
S. Custom mega-prosthetic replacement for proximal humeral
tumours. Int Orthop. 2006;30:158–162. p
12. Neer
C.S. Displaced
proximal
humeral
fractures. I. Classification and evaluation. J Bone Joint Surg Am. 1970;52:1077–1089. 13. Potter B.K., Adams S.C., Pitcher J.D. Jr, Malinin T.I., Temple
H.T. Proximal humerus reconstructions for tumors. Clin
Orthop. 2009;467:1035–1041. 14. Raiss P., Kinkel S., Sauter U., Bruckner T., Lehner B. Replacement of the proximal humerus with MUTARS tumor
endoprostheses. Eur J Surg Oncol. 2010;36:371–377. p
g
15. Rispoli D.M., Athwal G.S., Sperling J.W., Cofield R.H. The
anatomy of the deltoid insertion. J Shoulder Elbow Surg. 2009;18:386–390. 16. Rödl R.W., Gosheger G., Gebert C., Lindner N., Ozaki T.,
Winkelmann W. Reconstruction of the proximal humerus
after wide resection of tumours. J Bone Joint Surg Br. 2002;84:1004–1008. Обсуждение При погружении ножки в костный канал необ-
ходимо точно выставить ротацию компонента
и глубину посадки, восстановить дефект кости. Ошибка на любом из этих этапов может при-
вести к нестабильности сустава и повлиять на
функцию конечности. В то же время использо-
вание модульных систем в значительной степе-
ни упрощает восстановление длины конечности
и пространственное расположение компонентов
эндопротеза [11, 13, 14, 17]. у
4. Тепляков
В.В.,
Карпенко
В.Ю.,
Шаталов
А.М.,
Бухаров
В.А.,
Державин
В.А.,
Мысливцев
И.В. Эндопротезирование длинных трубчатых костей и су-
ставов при дефиците мягких тканей. Вестник москов-
ского онкологического общества. 2011 (4). Режим до-
ступа netoncology.ru/.uploads/press/1191/V-11-0495.file. doc (дата посещения 22.09.2014) 5. Aponte-Tinao L.A., Ayerza M.A., Muscolo D.L., Farfalli G.L. Allograft reconstruction for the treatment of musculoskeletal
tumors of the upper extremity. Sarcoma. 2013;2013:925413. 6. Cannon C.P., Paraliticci G.U., Lin P.P., Lewis V.O., Yasko
A.W. Functional
outcome
following
endoprosthetic
reconstruction of the proximal humerus. J Shoulder Elbow
Surg. 2009;18:705–710. 7. Enneking W.F., Dunham W., Gebhardt M.C., Malawer
M., Pritchard D.J. A system for the functional evaluation
of reconstructive procedures after surgical treatment
of tumors of the musculoskeletal system. Clin Orthop. 1993;(286):241–246. (
)
8. Enneking W.F. Modification of the system for functional
evaluation of surgical management of musculoskeletal tumors. In: Bristol–Myers/Zimmer Orthopaedic Symposium. Limb
salvage in musculoskeletal oncology. New York: Churchill
Livingstone; 1987. P. 626–639. Литература 1. Алиев М.Д., Соколовский В.А., Дзампаев А.З., Нисиченко
Д.В., Сергеев П.С., Хестанов Д.Б. Отдаленные результаты
применения мегапротезов в хирургии опухолей костей и
суставов. Вестник московского онкологического обще-
ства. 2011; (4). Режим доступа netoncology.ru/.uploads/
press/1191/V-11-0495.file.doc (дата посещения 22.09.2014) 1. Алиев М.Д., Соколовский В.А., Дзампаев А.З., Нисиченко
Д.В., Сергеев П.С., Хестанов Д.Б. Отдаленные результаты
применения мегапротезов в хирургии опухолей костей и
суставов. Вестник московского онкологического обще-
ства. 2011; (4). Режим доступа netoncology.ru/.uploads/
press/1191/V-11-0495.file.doc (дата посещения 22.09.2014) 17. Van de Sande M.A., Dijkstra P.D., Taminiau A.H. Proximal
humerus reconstruction after tumour resection: biological
versus
endoprosthetic
reconstruction. Int
Orthop. 2011;15(9):1375–1380. ( )
18. Yang Q., Li J., Yang Z., Li X., Li Z. Limb sparing surgery for
bone tumours of the shoulder girdle: the oncological and
functional results. Int. Orthop. 2010;15(6):869–875. p
(д
щ
)
2. Майков С.В. Пути повышения эффективности эндопротези-
рования плечевого сустава. Дис. канд. мед. наук. СПб.; 2012. p
(д
щ
)
2. Майков С.В. Пути повышения эффективности эндопротези-
рования плечевого сустава. Дис. канд. мед. наук. СПб.; 2012. Сведения об авторах: Микайлов Илкин Мугадасович – лаборант-исследователь научного отделения нейроортопедии с костной онкологией;
Григорьев Петр Владимирович – лаборант-исследователь научного отделения нейроортопедии с костной онкологией;
Пташников Дмитрий Александрович – д.м.н. профессор заведующий отделением № 18; Майков Сергей Валерьевич – младший научный сотрудник отделения спортивной травматологии и реабилитации СВЯЗЬ С АВТОРАМИ: СВЯЗЬ С АВТОРАМИ: СВЯЗЬ С АВТОРАМИ:
e-mail: mim17@mail.ru (Микайлов И.М.) СВЯЗЬ С АВТОРАМИ:
e-mail: mim17@mail.ru (Микайлов И.М.) Рукопись поступила 03.10.2014 т р а в м а т о л о г и я и о р т о п е д и я р о с с и и
Рукопись поступила 03.10.2014 т р а в м а т о л о г и я и о р т о п е д и я р о с с и и
2014 – 4 (74) 2014 – 4 (74) т р а в м а т о л о г и я и о р т о п е д и я р о с с и и 34 оригинальные статьи Vreden Russian Research Institute of Traumotology and Orthopedics, St. Petersburg, Russia Vreden Russian Research Institute of Traumotology and Orthopedics, St. Petersburg, Russia The purpose of the study was to evaluate the results of surgical reconstructions of the proximal humerus after transarticular
tumour resection, сcompare the functional results with the results of shoulder arthroplasty in patients with extensive damage of
the proximal humerus of non-neoplastic origin. Material and methods. Between 2001 and 2013 38 proximal humeral reconstructions were performed in our clinic: using
monopolar endoprostheses – 26 (68%) and modular systems with reversible head -12 (32%). The control group included 46
patients with extensive lesions of the proximal humerus of non-neoplastic origin, operated in our clinic in the period from 2006
to 2012. Term follow-up of patients ranged from 7 months to 9 years. Assessment of functional results was carried out in a period
from six months to one year. Results. The seven-year survival according the Kaplan – Meier method for patients with primary malignant tumors of the proximal
humerus (25 patients) was 77%. In the study group the average value of functional outcome MSTS score was 77.7%. Unipolar
prosthesis showed a bad result, both in the control (61,3% MSTS, 60,7 Neer) and in the main (67,7% MSTS, 61,1 Neer) study groups. C
l
i
C
tl
th
th d
f
h i
hi h d
’t i
i th
l
i
t
f t
t
t
ti
t
ith Results. The seven-year survival according the Kaplan – Meier method for patients with primary malignant tumors of the proximal
humerus (25 patients) was 77%. In the study group the average value of functional outcome MSTS score was 77.7%. Unipolar
prosthesis showed a bad result, both in the control (61,3% MSTS, 60,7 Neer) and in the main (67,7% MSTS, 61,1 Neer) study groups. Conclusion. Currently the method of choice which doesn’t impair the oncologic component of treatment patients with
shoulder neoplastic lesions is its replacement with modular reversible systems in combination with additional soft tissue fixation. Conclusion. Currently the method of choice which doesn’t impair the oncologic component
shoulder neoplastic lesions is its replacement with modular reversible systems in combination with ad Key words: tumors of the shoulder joint, shoulder prosthesis, modular endoprosthesis. 8. Enneking WF. Modification of the system for functional
evaluation of surgical management of musculoskeletal tumors. In: Bristol–Myers/Zimmer Orthopaedic Symposium. Limb
salvage in musculoskeletal oncology. New York: Churchill
Livingstone; 1987. P. 626–639. References Cannon CP, Paraliticci GU, Lin PP, Lewis VO, Yasko AW. Functional outcome following endoprosthetic reconstruction
of
the
proximal
humerus. J
Shoulder
Elbow
Surg. 2009;18:705–710. 6. Cannon CP, Paraliticci GU, Lin PP, Lewis VO, Yasko AW. Functional outcome following endoprosthetic reconstruction
of
the
proximal
humerus. J
Shoulder
Elbow
Surg. 2009;18:705–710. 17. Van de Sande MA, Dijkstra PD, Taminiau AH. Proximal
humerus reconstruction after tumour resection: biological
versus
endoprosthetic
reconstruction. Int
Orthop. 2011;15(9):1375–1380. 7. Enneking WF, Dunham W, Gebhardt MC, Malawer M,
Pritchard DJ. A system for the functional evaluation of
reconstructive procedures after surgical treatment of tumors of
the musculoskeletal system. Clin Orthop. 1993;(286):241–246. 18. Yang Q, Li J, Yang Z, Li X, Li Z. Limb sparing surgery for bone
tumours of the shoulder girdle: the oncological and functional
results. Int. Orthop. 2010;15(6):869–875. References 1. Aliyev MD, Sokolovskiy VA, Dzampayev AZ, Nisichenko
DV, Sergeyev PS, Khestanov DB. Otdalennyye rezul'taty
primeneniya megaprotezov v khirurgii opukholey kostey i
sustavov [Long-term results of megaprotezov in tumor surgery
of bones and joints.] Vestnik moskovskogo onkologicheskogo
obshchestva [Bulletin of Moscow Cancer Society]. 2011; (4). Available at: netoncology.ru/.uploads/press/1191/V-11-0495. file.doc (accessed 22.09.2014) g
9. Fuhrmann RA, Roth A, Venbrocks RA. Salvage of the upper
extremity in cases of tumorous destruction of the proximal
humerus. J Cancer Res Clin Oncol. 2000; 126:337–344. 10. Manfrini M, Vanel D, De Paolis M, Malaguti C, Innocenti M,
Ceruso M, Capanna R, Mercuri M. Imaging of vascularized
fibula autograft placed inside a massive allograft in
reconstruction of lower limb bone tumors. Am J Roentgenol. 2004;15(4):963–970. 2. Maykov
SV. Puti
povysheniya
effektivnosti
endoprotezirovaniya plechevogo sustava. Dis. kand. med. nauk [Ways to improve the efficiency of replacement of the
shoulder joint. Dr. med. nauk sci. diss.]. SPb.; 2012. 11. Mayilvahanan N, Paraskumar M, Sivaseelam A, Natarajan S. Custom mega-prosthetic replacement for proximal humeral
tumours. Int Orthop. 2006;30:158–162. shoulder joint. Dr. med. nauk sci. diss.]. SPb.; 2012. 3. Teplyakov VV, Karpenko VYu, Frank GA, Bulanov AA,
Derzhavin VA, Shatalov AM. Endoprotezirovaniye pri
opukholevom porazhenii dlinnykh kostey [Endoprosthesis
with neoplastic lesions of the long bones] Rossiyskiy
pediatricheskiy zhurnal [Russian Journal of Pediatrics]. 2009;
(5):11–14. p
12. Neer CS. Displaced proximal humeral fractures. I. Classification
and evaluation. J Bone Joint Surg Am. 1970;52:1077–1089. g
13. Potter BK, Adams SC, Pitcher JD Jr, Malinin TI, Temple HT. Proximal humerus reconstructions for tumors. Clin Orthop. 2009;467:1035–1041. 4. Teplyakov VV, Karpenko VYu, Shatalov AM, Bukharov VA,
Derzhavin VA, Myslivtsev IV. Endoprotezirovaniye dlinnykh
trubchatykh kostey i sustavov pri defitsite myagkikh tkaney
[Endoprosthesis of the long bones and joints with a deficit
of soft tissues] Vestnik moskovskogo onkologicheskogo
obshchestva [Internet]. 2011 (4). Available at: netoncology.ru/. uploads/press/1191/V-11-0495.file.doc (accessed 22.09.2014) 14. Raiss P, Kinkel S, Sauter U, Bruckner T, Lehner B. Replacement
of the proximal humerus with MUTARS tumor endoprostheses. Eur J Surg Oncol. 2010;36:371–377. 15. Rispoli DM, Athwal GS, Sperling JW, Cofield RH. The
anatomy of the deltoid insertion. J Shoulder Elbow Surg. 2009;18:386–390. 5. Aponte-Tinao LA, Ayerza MA, Muscolo DL, Farfalli GL. Allograft reconstruction for the treatment of musculoskeletal
tumors of the upper extremity. Sarcoma. 2013;2013:925413 16. Rödl RW, Gosheger G, Gebert C, Lindner N, Ozaki T,
Winkelmann W. Reconstruction of the proximal humerus
after wide resection of tumours. J Bone Joint Surg Br. 2002;84:1004–1008. 6. т р а в м а т о л о г и я и о р т о п е д и я р о с с и и
2014 – 4 (74) Author’s information: Mikailov Ilkin M. – research assistant of the scientific department of neuroorthopedics and bone tumors;
Grigoriev Petr V. – research assistant of the scientific department of neuroorthopedics and bone tumors;
h k
A
f
h h
d f d Mikailov Ilkin M. – research assistant of the scientific department of neuroorthopedics and bone tumors;
Grigoriev Petr V. – research assistant of the scientific department of neuroorthopedics and bone tumors;
Ptashnikov Dmitry A. – professor, the head of department N 18; Grigoriev Petr V. – research assistant of the scientific department of neuroorthopedics and bone tumors;
Ptashnikov Dmitry A. – professor, the head of department N 18; g
p
p
Ptashnikov Dmitry A. – professor, the head of department N 18; Ptashnikov Dmitry A. – professor, the head of department N 18;
k
h
f
d
h b l
d Maykov Sergei V. – researcher of sport trauma and rehabilitation department. Corresponding author:
e-mail: mim17@mail.ru (Mikailov IM) I.M. Mikailov, P.V. Grigoriev, D.A. Ptashnikov, S.V. Maykov I.M. Mikailov, P.V. Grigoriev, D.A. Ptashnikov, S.V. Maykov т р а в м а т о л о г и я и о р т о п е д и я р о с с и и Corresponding author: e-mail: mim17@mail.ru (Mikailov IM) 35 р а в м а т о л о г и я и о р т о п е д и я р о с с и и
2014 – 4 (74) 2014 – 4 (74)
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Paradigma indiciário: contribuições para a investigação da construção das identidades de futuros professores de línguas
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10.5216/sig.v27i2.35551 10.5216/sig.v27i2.35551 10.5216/sig.v27i2.35551 *
Professora Associada do Instituto de Letras da Universidade Federal da Bahia (UFBA).
E-mail: liviarad@yahoo.com. Resumo Este trabalho trata das contribuições do paradigma indiciário para o estudo das
identidades docentes e, particularmente, as dos futuros professores de línguas,
com ênfase para os do espanhol. Com essa finalidade, explicitaremos em que
consiste esse modelo e como pode contribuir para gerar maior inteligibilidade
a respeito de como futuros professores se envolvem nos processos de
construção identitária ao longo de seus percursos formativos. Sendo assim,
neste artigo indicaremos algumas contribuições desse paradigma para o exame
da problemática em foco, refletindo a respeito do seu potencial como construto
metodológico de cunho qualitativo e interpretativista. Este trabalho trata das contribuições do paradigma indiciário para o estudo das
identidades docentes e, particularmente, as dos futuros professores de línguas,
com ênfase para os do espanhol. Com essa finalidade, explicitaremos em que
consiste esse modelo e como pode contribuir para gerar maior inteligibilidade
a respeito de como futuros professores se envolvem nos processos de
construção identitária ao longo de seus percursos formativos. Sendo assim,
neste artigo indicaremos algumas contribuições desse paradigma para o exame
da problemática em foco, refletindo a respeito do seu potencial como construto
metodológico de cunho qualitativo e interpretativista. Palavras-chave: paradigma indiciário, formação de professores de espanhol,
identidades, Linguística Aplicada. Paradigma Indiciário: contribuições para a investigação da
construção das identidades de futuros professores de línguas Lívia Márcia Tiba Rádis BAPTISTA* Lívia Márcia Tiba Rádis BAPTISTA* 1 Sobre o paradigma indiciário: detalhes e singularidades O historiador italiano Carlo Ginzburg (2001, p. 143-179) examina
como, por volta do final do século XIX, emergiu, no campo das Ciências
Humanas, um modelo epistemológico conhecido como paradigma
indiciário, fundado na análise minuciosa do residual, do episódio, ou,
ainda, da singularidade. Apresenta, entre outras questões, os princípios
metodológicos desse modelo amplamente operante apesar de não ter sido
teorizado de forma explícita, o que contribuiria conforme Ginzburg (2001,
p. 143) para “sair dos incômodos da contraposição entre racionalismo e
irracionalismo”. Nesta seção, portanto, revisitaremos em que consiste o paradigma
indiciário e, com esse propósito, refletiremos sobre como ele pode
orientar a compreensão de determinados aspectos relacionados à
construção das identidades docentes, no âmbito da formação inicial de
futuros professores de línguas, em especial, a espanhola. Remontando à história desse paradigma, Ginzburg (2001, p.143-
144) menciona que entre 1874 e 1876 surgiu uma série de artigos
sobre pintura italiana na Zeitschrift für bildende Kunst assinada por
um estudioso russo, Ivan Lermolieff e traduzida para o alemão pelo
desconhecido Johannes Schwarze. Esses artigos propunham um
método novo para a atribuição da autoria dos quadros antigos em que
se recomendava examinar os pormenores considerados desprezíveis e,
consequentemente, não observados pelos imitadores no original, fato
que gerou polêmicas na época. Descobriu-se depois que o responsável
por essa proposta tinha sido o historiador de arte Giovanni Morelli;
razão pela qual passou a ser conhecido como método morelliano ou
método indiciário de Morelli. De acordo com Ginzburg (2001, p. 145-146), além de Morelli,
esse método foi empregado por Sherlock Holmes, personagem de
Arthur Conan Doyle e por Freud. Ginzburg (2001, p.150-151) esclarece
que o emprego do paradigma indiciário pelos três deu-se no final do
século XIX, no período de 1870 a 1880, fato que coincide com a
afirmação desse método no âmbito das Ciências Humanas. Ora, esse
método proporcionou aos que dele se valeram captar uma realidade
“mais profunda” e “de forma inatingível” através de uma análise
pormenorizada de pistas: os sintomas (no caso de Freud), os indícios (no
caso de Sherlock Holmes) e os signos pictóricos (no caso de Morelli). Contudo, as raízes desse paradigma se remontam aos primórdios
de nossa história, uma vez que como ressalta Ginzburg (2001, p.151) por milênios o homem foi caçador. 1 Sobre o paradigma indiciário: detalhes e singularidades Durante inúmeras perseguições,
ele aprendeu a reconstruir as formas e movimentos das presas invi-
síveis pelas pegadas na lama, ramos quebrados, bolotas de esterco,
tufos de pelos, plumas emaranhadas, odores estagnados. Aprendeu
a farejar, registrar, interpretar e classificar pistas infinitesimais como
fios de barba. Aprendeu a fazer operações mentais complexas com por milênios o homem foi caçador. Durante inúmeras perseguições,
ele aprendeu a reconstruir as formas e movimentos das presas invi-
síveis pelas pegadas na lama, ramos quebrados, bolotas de esterco,
tufos de pelos, plumas emaranhadas, odores estagnados. Aprendeu
a farejar, registrar, interpretar e classificar pistas infinitesimais como
fios de barba. Aprendeu a fazer operações mentais complexas com 566 Baptista, L. M. T. R. Paradigma Indiciário... rapidez fulminante, no interior de um denso bosque ou numa clarei-
ra cheia de ciladas. Ainda de acordo com Ginzburg (2001, p.152-155), “o caçador
teria sido o primeiro a ‘narrar uma história’ porque era o único capaz de
ler, nas pistas mudas (se não imperceptíveis) deixadas pela presa, uma
série coerente de eventos”. Assim, foi nessa sociedade de caçadores que
a narração e a leitura dos indícios se destacaram, mas foram igualmente
empregadas nas antigas civilizações mesopotâmica e grega, o que
sugere que esse saber indiciário não foi abandonado. Dando um salto temporal e situando-nos mais a frente, Ginzburg
(2001, p. 143-179) observa que o paradigma indiciário, ao emergir no
final do século XIX se defrontou com o galileano e, consequentemente,
o cartesiano. Quanto ao modelo proposto por Descartes, sabemos que
seguiu a tradição de Galileu e despontou nas sociedades modernas do
Renascimento no século XVI e passou a ser amplamente incorporado
à ciência. Consistiu em um conjunto institucionalmente articulado de
práticas e saberes, norteado por uma concepção de indivíduo livre
e racional, consciente de seus atos, sujeito de si e do conhecimento
bem como capaz de descobrir as leis da natureza. Segundo Japiassú e
Marcondes (2001, p. 194), essa concepção foi inaugurada por Francis
Bacon, Galileu e Descartes e amadureceu, por exemplo, com as
contribuições de Isaac Newton (1642-1727) no campo da Física, com
a formalização da Mecânica de Galileu e, ainda, com as investigações
em óptica e sobre a natureza da luz. 1 Sobre o paradigma indiciário: detalhes e singularidades Nietzsche questionou a noção de
sujeito racional de Descartes, embora com os filósofos iluministas
do século XVIII, a razão, a ciência e a lei natural tenham passado a
ser o alicerce de uma verdade universal que levaria ao progresso da
humanidade, libertando o homem da religião e do obscurantismo. Em
suma, estabeleceu-se o primado da razão e da vontade. Ginzburg (2001, p. 143) contrapõe o modelo galileano e, por
conseguinte, o cartesiano ao indiciário e salienta que, ao longo do tempo,
coexistiram, a despeito da ascensão do primeiro. Dada essa coexistência
tais paradigmas produziram seus efeitos e impactos epistemológicos
e, ao contrário do que se possa supor, o paradigma indiciário não é
propriamente uma perspectiva contemporânea nem tampouco foi
gestado no cenário atual. Portanto, interessa acentuar que esse modelo Signótica, Goiânia, v. 27 n. 2, p. 565-582, jul./dez. 2015 567 não é uma invenção ou uma moda a ser adotada sem nenhuma reflexão
a priori, mas faz parte de certa tradição interpretativista, ou de forma
mais ampla, de certa filosofia da ciência. Ora, em face desse panorama
é que podemos dizer que certos movimentos da ciência, em especial,
aqueles situados no campo epistemológico são reveladores das não
linearidades, incongruências, embates e conflitos acerca da definição
da natureza do conhecimento, da relação intricada entre os objetos e os
sujeitos. l Avançando em nossa reflexão, a seguir abordaremos a relação
entre paradigma, epistemologia, subjetividade e objetividade, o que nos
levará a apreciar como o paradigma indiciário pode contribuir para a
compreensão de questões concernentes à problemática das identidades
docentes, alvo de nossas considerações neste texto. Baptista, L. M. T. R. Paradigma Indiciário... 2 Paradigmas, epistemologias e subjetivismo/objetivismo:
algumas breves considerações Ao longo dessa seção, trataremos do que entendemos por
paradigma e, por extensão da relação entre epistemologias, subjetivismo
e objetivismo. Principiamos essa exposição com a definição platônica de
paradigma, com base em Japiassú e Marcondes (2001, p. 206), segundo
a qual esse está relacionado às formas ou ideias, ou seja, aos arquétipos,
modelos perfeitos ou “eternos imutáveis” dos objetos existentes no
mundo natural do qual seriam cópias e dos que, em certa medida,
participariam. Já o filósofo da ciência Thomas Samuel Kuhn (2003, p. 67-76), em seu livro A Estrutura das Revoluções Científicas, considera
o paradigma uma visão de mundo, a Wetanschaun, que envolve não
somente a teoria científica dominante como também determinados
princípios filosóficos, leis, procedimentos técnicos padronizados
empregados na resolução dos problemas e concepção metodológica. De
acordo com essa perspectiva, os modelos seguidos e dos quais surgem
as tradições da pesquisa científica são paradigmas, como, por exemplo,
a Mecânica de Galileu, a Mecânica Quântica. Conforme esse autor, um
paradigma é aquilo que os membros de uma determinada comunidade
científica partilham em termos de saber. Baptista, L. M. T. R. Paradigma Indiciário... 568 Contudo, ainda que se suponha certa homogeneidade ou
unidade constitutiva, o paradigma não deve ser visto como imutável
e estático, ou ainda, com uma natureza centrada e hermética. Desse
modo, se ocorre uma mudança de paradigma há mudanças de ordem
epistemológica, ou seja, transformações que afetam a própria teoria
do conhecimento em diferentes níveis, relacionados à forma da crítica,
à filosofia e à história das ciências. Quanto a esses níveis, conforme
Japiassú e Marcondes (2001, p. 84-85), o da forma da crítica do
conhecimento científico engloba o exame dos princípios, das hipóteses
e das conclusões das distintas ciências, considerando-se seu alcance e
o seu valor objetivo, enquanto os da filosofia e história das ciências
referem-se às modalidades como o Empirismo, o Racionalismo etc. Portanto, um exemplo de mudança de paradigma e, por extensão,
mudança epistemológica foi o da passagem da teoria astronômica
geocêntrica, de inspiração aristotélica-ptolomaica, para a heliocêntrica,
de inspiração copernicana e galileana e, além dessa, a da passagem do
modelo escolástico ao cartesianismo. Com estes modelos assumiu-se
a existência de uma verdade no mundo da natureza, distante da visão
teocêntrica. 2 Paradigmas, epistemologias e subjetivismo/objetivismo:
algumas breves considerações Devido a esse deslocamento, defende-se o pressuposto de
que se pode aceder à verdade, na medida em que essa é passível de ser
descoberta e transformada em leis capazes de expressar as regularidades
que dominam o movimento da natureza. Consoante com esse ponto de
vista, o método correto permitiria ao observador distinguir o verdadeiro
e o falso e, assim, o cientista descobriria algo pré-existente e exterior
a ele, ou seja, o objeto seria independente de sua subjetividade. Com
essa mudança de paradigma impôs-se o princípio de neutralidade
científica, que afirmou a isenção e imunidade do cientista em nome de
sua racionalidade objetiva. Mencionamos que no final do século XIX, o paradigma indiciário
veio à tona novamente, e no século XX observamos uma tendência à
redefinição da ideia de verdade/objetividade e falsidade/subjetividade. Dessa perspectiva, o objeto – quer fosse a sociedade ou quer fosse a
natureza – não existiria de modo objetivo a priori, mas seria construído
pelos sujeitos. Recolocou-se, assim, a problemática do ponto de vista,
da relação entre o sujeito e sua percepção e compreensão do real. 569 Signótica, Goiânia, v. 27 n. 2, p. 565-582, jul./dez. 2015 Esse questionamento, no seio dos mais diversos campos, a
respeito da natureza da verdade e da objetividade, esteve presente
na filosofia da ciência contraposta àquela que constitui o modelo
cartesiano. Neste sentido, problematizou-se a forma de representação
da relação estabelecida entre sujeito e objeto, entre indivíduo, natureza
e sociedade. Além disso, mostrou-se mais cautelosa quanto à afirmação
das verdades, ou, de modo mais radical, renunciou a propor qualquer
forma de verdade, fosse essa provisória ou não. Ainda quanto aos questionamentos a respeito da relação entre
sujeito e objeto, entre indivíduo, natureza e sociedade destacamos a
noção de regime de verdade proposta por Foucault (1985, p. 12),
conforme a qual cada sociedade tem seu regime de verdade, sua “política geral” de
verdade: isto é, os tipos de discurso que aceita e faz funcionar como
verdadeiros; os mecanismos e instâncias que permitem distinguir
entre sentenças verdadeiras e falsas, os meios pelos quais cada um
deles é sancionado; as técnicas e procedimentos valorizados na
aquisição da verdade; o status daqueles que estão encarregados de
dizer o que conta como verdadeiro. 2 Paradigmas, epistemologias e subjetivismo/objetivismo:
algumas breves considerações Ora, os regimes de verdade (FOUCAULT, 1985) instaurados na
e para a ciência evidenciam a tensa relação existente entre produção do
conhecimento e o poder na e para as sociedades humanas ao longo dos
séculos. Ainda segundo esse raciocínio, há tipos de discursos recebidos
e acolhidos que passam a funcionar como verdadeiros, embora
condicionados pelas mais diversas instâncias sociais, culturais e
históricas. E, além desses discursos, há mecanismos que diferenciam
os enunciados falsos dos verdadeiros, modos através dos quais uns e
outros enunciados são sancionados bem como procedimentos e formas
valorizados para a obtenção da verdade. Assim, a determinação da
noção de verdade está associada a processos históricos e linguísticos, a
estruturas de poder, segundo Foucault. Como observa Vaitsman (1995, p. 4), no interior dessa filosofia
da ciência sobre a qual estamos tratando, o conhecimento científico
e, particularmente o das Ciências Humanas, não se reduz a uma Baptista, L. M. T. R. Paradigma Indiciário... 570 “representação exata da realidade”, a uma espécie de “espelho da
natureza”. Ao contrário, é uma forma de representação, entre outras, da
realidade e as descrições científicas não são transparentes e objetivas,
são “construções da realidade” e “discursos sobre o mundo”; portanto,
são provisórias e reformuláveis, pois para essa autora (1995, p. 4), incerteza, contextualidade, contingência, singularidade, particulari-
dade, passaram a conviver ou mesmo a substituir, nesta visão de
ciência, a antiga concepção de conhecimento que pudesse abarcar
a totalidade, descobrir a verdade e estabelecer leis gerais sobre o
desenvolvimento histórico ou fenômenos da natureza. Diante do exposto, dada à natureza heterogênea, dinâmica,
negociável das identidades, assim como a dos processos identitários,
torna-se preciso orientar-se por uma abordagem que considere a
contingência do conhecimento, a complexa inter-relação entre a
subjetividade e os objetos, ou seja, as distintas vias de interpretação e
ressignificação do real/realidade. Quanto ao paradigma indiciário, se
filia a essa filosofia da ciência, enfatiza a idiossincrasia e a singularidade
dos dados, e, igualmente, propõe interpretações geradas a partir de uma
análise minuciosa das pistas para a compreensão do observado. i Por fim, gostaríamos de concluir anunciando que a seguir
trataremos de algumas das contribuições do paradigma indiciário
para a compreensão e análise das identidades no âmbito formativo. 2 Paradigmas, epistemologias e subjetivismo/objetivismo:
algumas breves considerações Com tal finalidade, discorreremos sobre alguns pontos em particular
relacionados com aspectos metodológicos, a saber, a natureza de dados
e o conceito de rigor e suas implicações para os estudos voltados à
problemática da constituição e construção das identidades, o que nos
permitirá avançar em nossa reflexão sobre a temática proposta. 3 Paradigma indiciário e estudo das identidades docentes:
algumas questões sobre os dados e o rigor Científico A opção pelo paradigma indiciário, assim como qualquer
outra, tem implicações que devem ser examinadas, em especial,
pelas repercussões na compreensão e na análise do objeto. Dessa
maneira, eleger esse modelo requer deslocar o foco da repetibilidade Signótica, Goiânia, v. 27 n. 2, p. 565-582, jul./dez. 2015 571 ou da replicabilidade para o da singularidade e da idiossincrassia
dos dados. Pressupõe, conforme Abaurre et al. (2001, p. 14-15),
definir critérios quanto à identificação dos dados a serem tomados
como representativos, ou seja, quanto à definição da singularidade
que revela e, igualmente, quanto ao conceito de rigor metodológico. Portanto, aqui nos ocuparemos fundamentalmente desses dois
aspectos, relacionando-os ao estudo das identidades docentes. Consistentes com o paradigma indiciário, consideramos
que os fenômenos sociais e, mais particularmente, os educativos,
conforme propõe Pérez Gómez (2007, p. 100) se definem por seu
“caráter radicalmente inacabado”, por sua “dimensão criativa,
autoformadora, aberta à mudança intencional” e, igualmente, por sua
dimensão semiótica, dada a “relação indeterminada” e “polissêmica”
instituída entre “o significante observável e o significado latente de
todo fenômeno social ou educativo”. Para a compreensão de como se
configura a dinâmica da construção identitária, em concreto no que
se refere à relação entre os sujeitos e suas identidades, acreditamos
que as contribuições do paradigma indiciário podem orientar a
geração e análise de dados. Assim sendo, a análise minuciosa de
certos indícios, reveladores da singularidade da ação dos sujeitos,
presentes nos diversos posicionamentos assumidos para ressignificar
e interpretar os saberes mobilizados, possibilita entrever a complexa
tessitura de significados que afetam as distintas maneiras de pensar,
sentir, expressar e atuar dos sujeitos nos percursos formativos. Essa
análise, por sua vez, precisa seguir o que seria o “rigor flexível”, como
veremos. Neste sentido, nos interessa focalizar a dinâmica dos processos
identitários, para cuja compreensão é relevante considerar as
sucessivas interações estabelecidas entre os sujeitos e os vários meios
sociais, culturais e profissionais nos quais se inserem nas distintas
ocasiões. Consideramos, assim, que essas interações promovem a
construção de identidades docentes diferenciadas, de acordo com as
distintas maneiras através das quais os sujeitos estabelecem relações
entre os saberes, experiências e contextos. Dessa forma, as identidades
são permanentemente renegociadas, reformuladas e reformuláveis,
visto que os sujeitos instauram processos de ressignificação e Baptista, L. M. T. R. Paradigma Indiciário... 572 interpretação, caracterizados, segundo Hoffnagel (2010, p. 64) como
uma “realização interacional e alcançada em eventos comuns, como
traços constitutivos dos encontros sociais”. 3 Paradigma indiciário e estudo das identidades docentes:
algumas questões sobre os dados e o rigor Científico Portanto, o processo identitário se produz ao longo das constantes
negociações dos sujeitos nos diversos espaços em que esse circula e
nos encontros em que participa. Destarte, é, simultaneamente, uma
construção dos sujeitos, situada histórica, social, cultural e politicamente,
na qual se imbricam diversos processos de interação e socialização,
diversas práticas discursivas e processos de autorreconhecimento e
heterorreconhecimento. No caso da formação inicial de professores
e, em especial, a de espanhol a qual nos dedicamos, esperamos
contribuir para a proposição de itinerários formativos críticos, nos
quais se questionem modelos profissionais que limitem a ação e a
prática docentes à dimensão técnica e/ou instrucionista e repercutam
na construção de suas identidades docentes. Por isso, acreditamos que
problematizar como os sujeitos negociam suas identidades e como
se percebem no processo de construção de suas identidades docentes
constitui uma alternativa mais condizente com uma formação reflexiva. Reiteramos, portanto, que esse olhar pressupõe admitir que a identidade
não é algo permanente e que sua construção implica os sujeitos, já que
como postula Pimenta (1997, p. 6) a identidade não é um dado imutável. Nem externo, que possa ser
adquirido. Mas, é um processo de construção do sujeito historica-
mente situado. A profissão de professor, como as demais, emerge
em dado contexto e momento históricos, como resposta a neces-
sidades que estão postas pelas sociedades, adquirindo estatuto de
legalidade. Diante das dimensões históricas e contextuais, no tocante aos
processos identitários docentes e à formação de professores, Silva
(2009, p. 48) refere-se aos impactos provocados pelas mudanças
sociais e educacionais vividas nos últimos vinte anos sobre a
identidade docente. Neste sentido, a crise de identidade docente se
traduz em diferentes manifestações que abrangem a insatisfação com
os problemas reais da prática e a identidade ideal, solicitações de
transferências, inibição com respeito ao trabalho realizado em sala Signótica, Goiânia, v. 27 n. 2, p. 565-582, jul./dez. 2015 573 de aula, desejo expresso de abandonar a docência, ausência laboral,
esgotamento, sentimentos como stress, ansiedade e depreciação do eu
e depressões. Além dessa crise, Silva (2009, p. 49) chama a atenção
para o fato de que [...] a formação prática da formação inicial deve permitir ao futuro
professor identificar-se a si próprio como professor e identificar-se
com os estilos de ensino que é capaz de utilizar, levando em con-
sideração os efeitos que os referidos estilos produzem nos alunos. 3 Paradigma indiciário e estudo das identidades docentes:
algumas questões sobre os dados e o rigor Científico [...] a formação prática da formação inicial deve permitir ao futuro
professor identificar-se a si próprio como professor e identificar-se
com os estilos de ensino que é capaz de utilizar, levando em con-
sideração os efeitos que os referidos estilos produzem nos alunos. Ora, em virtude do exposto é necessário estudar como, ao
longo desse processo formativo, se dá a construção identitária e,
particularmente, como os futuros professores de línguas percebem-se
e interpretam os diversos saberes docentes. Importa contribuir para
que os sujeitos reflitam sobre como certas percepções repercutem na
sua futura ação e prática e como se posicionam enquanto profissionais. li No entanto, não basta a reflexão com fim em si mesma, uma
vez que essa precisa alcançar a práxis pedagógica, considerando as
distintas possibilidades contextuais e de atuação e, igualmente, a
plausibilidade do ensino de línguas no contexto histórico e social em
que nos situamos. Contudo, essa opção não implica assumir uma visão
ingênua e idílica do papel da escola e dos professores, como observa
acertadamente Nóvoa (2014, p. 40-41) ao dizer que “os professores
não são certamente os ‘salvadores do mundo’, mas também não são
‘meros agentes’ de uma ordem superior que os ultrapassa”. Segundo
esse autor, somente por meio de uma reelaboração permanente de uma
identidade profissional “os professores poderão definir estratégias
de ação que não podem mudar tudo, mas que podem mudar alguma
coisa. E esta alguma coisa não é coisa pouca”. Condizente com nossas considerações anteriores é preciso rever
e problematizar concepções, tais como a do ensino como transmissão
cultural ou treinamento de habilidades (PÉREZ GÓMEZ, 2007,
p.67-97), contrastadas com a do ensino como mudanças conceituais,
afinado com uma perspectiva ecológica, que considera os contextos
de aprendizagem. Lembramos que o ensino centrado na transmissão
cultural, apoiado nas teorias comportamentalistas, destaca a primazia
dos ajustes ambientais, em especial, os materiais, conteúdos, Baptista, L. M. T. R. Paradigma Indiciário... 574 avaliação e demais recursos multissensoriais como fundamentais
para a aprendizagem. De acordo com essa concepção, se atingem os
objetivos ao empregarem-se corretamente as estratégias de ensino
e aprendizagem, daí o protagonismo do método. Já para o ensino
como treinamento de habilidades, importa vincular a formação de
capacidades aos conteúdos e ao contexto cultural, no qual habilidades
e tarefas tenham sentido. 3 Paradigma indiciário e estudo das identidades docentes:
algumas questões sobre os dados e o rigor Científico Ambas as visões valorizam os procedimentos
didáticos e podem ser relacionadas à primazia do método, uma vez que
propõem identificar e comparar métodos, abordagens e técnicas com
o intuito de determinar os que melhores, e, portanto, mais eficientes. Ainda que reconheçamos a dimensão técnica e procedimental,
é relevante a compreensão das diversas influências dos diferentes
contextos nos processos de aprendizagem, uma vez que como
propõem Williams e Burden (2008, p. 197) os enfoques sistêmicos ou
ecológicos enfatizam o em torno social do aluno, para que se explique
como e por que aprendem bem, como destacam o caráter dinâmico e
interativo das variáveis presentes e refutam a adoção de um ponto de
vista do tipo linear de causa e efeito. Em face de nossas observações anteriores, promover um
processo formativo crítico se fundamenta por uma relação dialógica,
por um constante movimento questionador e, no caso, um fio
condutor e desencadeador desse processo se relaciona às experiências
vividas pelos futuros docentes com respeito à conformação de suas
identidades. Neste sentido, vemos nos estágios supervisionados, em
suas diversas modalidades (participação, regência ou observação), um
dos primeiros e importantes espaços de socialização docente, ainda
que não o único e definitivo. Historiar as vivências coletivas nele
materializadas nos proporciona elementos para uma compreensão
de como são ressignificados os saberes docentes, relacionados
à(s) língua(s) e cultura(s), às relações entre sua autopercepção e a
realidade mais imediata, entre o ideal e o real bem como os constantes
reajustamentos e renegociações dos sujeitos com os sentidos que vão
estabelecendo para a sua ação. Dessa ótica, conforme Santos (2014,
p. 19), os estudos sobre a formação assumem novas perspectivas
e, sendo assim, importa entender como o professor constrói sua
identidade profissional. Ainda para essa autora (2014, p.19), na Signótica, Goiânia, v. 27 n. 2, p. 565-582, jul./dez. 2015 575 análise predominam os procedimentos interpretativos bem como se
enfatizam aspectos específicos da realidade e valorizam-se os aspectos
microssociais, em lugar das questões estruturais, além de interessar-se
pelo papel do agente-sujeito e pelas identidades sociais. Consideramos, por conseguinte, que os diversos contextos nos
quais os sujeitos participam, interagem e se relacionam são o eixo
dos processos de construção das identidades docentes. Os sujeitos se
implicam nessa construção, atribuindo sentidos ao ser professor e, no
caso, ao ser professor de línguas e da língua espanhola no e para o
contexto no qual estão inseridos ou se inserindo. 3 Paradigma indiciário e estudo das identidades docentes:
algumas questões sobre os dados e o rigor Científico As identidades, dada
à sua heterogeneidade, se embaralham, se confundem, se mesclam e
nessa dinâmica vão se delineando os processos identitários, com a
tentativa sempre fugaz de fixar determinados papéis e identidades. Assim, concordamos com Silva (2009, p. 54) que a identidade
docente é uma construção social, na qual convergem múltiplos
fatores que interagem entre si de forma contínua, como podem ser
suas histórias de vida, condições de trabalho, os imaginários sobre a
profissão e discursos que circulam nas diversas práticas sociais nas
quais os docentes se inserem e participam. Dessa forma, trazer à tona
questionamentos, confrontar visões, desnaturalizar sentidos e práticas,
implica ouvir a voz aos sujeitos, tendo em vista uma contextura mais
ampla que transcende, muitas vezes, modismos metodológicos,
vocações idealizadas ou pressupostos equivocados. No caso da formação inicial de professores de espanhol, os
relatórios produzidos nos estágios supervisionados contêm dados
reveladores sobre como esses sujeitos percebem-se e são percebidos:
se como estagiários, se como alunos, ou ainda, se como futuros
professores; sobre que significados esses sujeitos atribuem à ação
docente e à língua espanhola no contexto das práticas e das ações;
sobre quais saberes docentes e visões de ensino emergem nessas falas
e como são mobilizados e interpretados por eles. Esses indícios, daí
nossa aproximação do paradigma indiciário, nos revelam como os
sujeitos ressignificam a experiência vivenciada ao longo dos estágios
e, obviamente, ao longo do próprio percurso formativo. Dessa ótica,
os relatórios, registros de aulas, diários e narrativas elaboradas por
esses sujeitos são fontes de dados privilegiados ou cruciais, pois Baptista, L. M. T. R. Paradigma Indiciário... 576 geram um grande número de observações a respeito do observado e
nos permitem estabelecer interpretações e hipóteses sobre como se
processa a construção dos processos identitários em dados contextos
e sob dadas condições, evidenciando mais uma vez a não neutralidade
da relação entre conhecimento, saber e poder. Acrescentamos ainda que o rigor que orienta essas análises,
em consonância com o paradigma indiciário, diferencia-se daquele
exigido pelas metodologias experimentais, conforme observa A.A. Quartarolla (apud Abaurre et al., 2001, p.14-15). Neste sentido, o
olhar do pesquisador volta-se para a singularidade dos dados, para
o que eles nos dizem sobre essa relação entre sujeitos, identidades e
práxis pedagógica. Além disso, o modelo indiciário apoia-se na ideia
de que a realidade não é transparente e, igualmente, que a análise
detalhada de alguns sinais é central para a compreensão do observado. Signótica, Goiânia, v. 27 n. 2, p. 565-582, jul./dez. 2015 3 Paradigma indiciário e estudo das identidades docentes:
algumas questões sobre os dados e o rigor Científico Passaremos a seguir a tratar de duas questões relevantes
para aprofundarmos o exame acerca do paradigma indiciário e suas
contribuições para a compreensão dos processos identitários, no
âmbito da formação inicial de professores. A primeira delas se refere à
atuação do analista e à relação entre o sujeito e o objeto no paradigma
indiciário, já que de modo similar ao historiador e ao médico, o
nosso conhecimento acerca das identidades docentes em construção é
indireto, conjetural, indiciário e, portanto, jamais absoluto. A segunda
questão relaciona-se à forma de tratamento reservada aos dados e sua
análise. Como visto, no paradigma indiciário, prevalece o tratamento
qualitativo, a abordagem interpretativista e se defende a singularidade
do observado. Ora, essa opção metodológica implica lidar com
o contingente, com o idiossincrásico, com a provisoriedade e com
a contextualidade na proposição das interpretações e condução das
análises. Sendo assim, ao optar pelo paradigma indiciário valorizamos
os indícios que permitam traçar a história do sujeito ao vivenciar
as experiências docentes e os diversos conflitos e questionamentos
emergentes, materializados nos seus relatos, nas suas narrativas. Cabe-nos examinar como os sujeitos, de seu lugar de fala, percebem-
se por meio da análise de como se designam, como se categorizam
ou/e se denominam; por meio das representações que se revelam; do Signótica, Goiânia, v. 27 n. 2, p. 565-582, jul./dez. 2015 577 modo como enunciam, reforçando determinadas posições discursivas
ou apagando outras. Como analista, por conseguinte, é preciso
explorar as ocorrências que evidenciam um trabalho do sujeito com
a linguagem em dois sentidos: quando tomam posição e enunciam e,
ainda, quanto aos efeitos de sentido materializados/produzidos nessa
tomada de posição e suas implicações para e na construção de suas
identidades. Ainda no tocante à atuação do analista, optar pelo paradigma
indiciário para investigar a construção da identidade e, em especial,
os processos identitários pressupõe rever o conceito de rigor. Trata-
se de um “rigor flexível”, que orientará tanto a seleção dos dados
singulares quanto a sua interpretação. Lembramos, igualmente, que a
subjetividade será um elemento constitutivo do percurso investigativo
e, dessa maneira, inegável na condução e elaboração das propostas de
interpretações a respeito dessa história particular dos sujeitos. 4 Considerações finais Gostaríamos de finalizar dizendo que eleger o paradigma
indiciário pressupõe privilegiar a singularidade reveladora como dado
crucial e seguir procedimentos abdutivos, diferenciados dos indutivos
e dedutivos. Além disso, supõe assumir a existência, conforme postula
Foucault (1996, p. 26-27), da vinculação entre os sistemas de verdade
e as práticas sociais e políticas de onde são provenientes e aplicadas,
o que nos leva a falar do “caráter perspectivo” do conhecimento. De
acordo com essa ótica, não há uma essência do conhecimento, visto que
esse é “histórico e pontual”, contextual, contingente e provisório. Sendo assim, como propõe Foucault (1996, p. 25), o
conhecimento é (e será) “sempre uma certa relação estratégica em
que o homem se encontra situado”; será essa relação estratégica
responsável pela definição do efeito de conhecimento e, por isso,
esse será sempre “parcial, oblíquo, perspectivo”. Em outras palavras,
segundo esse autor (1996, p. 25) “o conhecimento esquematiza,
ignora as diferenças, assimila as coisas entre si, e isto sem nenhum
fundamento em verdade. Devido a isso, o conhecimento é sempre um
desconhecimento”. Baptista, L. M. T. R. Paradigma Indiciário... 578 Consoante com essa visão de conhecimento, concordamos com a
afirmação de Garcia et al. (2005, p. 54) de que, ao estudar as identidades
docentes, estamos diante de um saber que não é permanente e, assim,
torna-se necessário perder a ilusão de um conhecimento definitivo e de totalidade acer-
ca dessa questão, é uma precaução epistemológica importante se
considerarmos a heterogeneidade da categoria docente e a própria
instabilidade das identidades no mundo contemporâneo. perder a ilusão de um conhecimento definitivo e de totalidade acer-
ca dessa questão, é uma precaução epistemológica importante se
considerarmos a heterogeneidade da categoria docente e a própria
instabilidade das identidades no mundo contemporâneo. Em face de nossas considerações anteriores, o estudo das
identidades em construção, e, em especial, dos processos identitários
precisa considerar o complexo das relações entre sujeitos, práticas, ações
e contextos, consoante com uma visão perspectiva do conhecimento. Acreditamos, assim, que as identidades sociais são móveis, reformuláveis
e plásticas e que a construção identitária pressupõe a instauração de um
fluxo ininterrupto de (re)ssignificação e (re)interpretação, por parte dos
sujeitos, dos sentidos atribuídos à docência e à sua ação. Ainda conforme Garcia et al. (2005, p. Signótica, Goiânia, v. 27 n. 2, p. 565-582, jul./dez. 2015 Abstract This paper focuses on the contributions of the “indiciary paradigm” to the study
of teacher identity and specially the identity of prospective language teachers
with emphasis on the ones who teach Spanish. To this aim, we present the model
as well as its contributions to create more intelligibility about the way that
prospective teachers experience the processes of identity construction through
their formative paths. Thus, in this article, we indicate some possibilities of
this paradigm for the study of our central problem, reflecting on its potentiality
as a methodological construct for a qualitative, interpretive research. Keywords: indiciary paradigm, Spanish teacher education, identities, applied
linguistics. 4 Considerações finais 54) as investigações
recentes que problematizaram a relação entre a identidade e a
profissionalização docente parecem ter se fixado em “aspectos
unificadores e fundantes da identidade” como, por exemplo, a classe
social, a natureza do processo de trabalho, o gênero, a história de
vida, a formação profissional, etc”. Porém, diferentemente dessa via,
os autores sugerem que se busquem diferenças, descontinuidades e,
ainda, que se privilegiem as narrativas dos docentes sobre si mesmos
bem como seus contextos de trabalho. Ora, isso sugere ser preciso nos aproximarmos do que seria a
“dinâmica contraditória e fragmentada” dessa construção identitária e
neste sentido o paradigma indiciário e, em especial, o foco no residual,
nos indícios reveladores da singularidade e idiossincrasia, representa
um ganho em termos de possibilidades de análise. Nessa direção,
poderá trazer maior inteligibilidade sobre como os sujeitos se assumem,
se percebem e se reconhecem como docentes de língua espanhola
em seu processo inicial de formação. Consideramos, ainda, que essa
perspectiva de análise pode nos levar à problematização das diferentes
negociações que os sujeitos realizam quando refletem sobre esse Signótica, Goiânia, v. 27 n. 2, p. 565-582, jul./dez. 2015 579 processo, em consonância com uma (trans)formação crítico-reflexiva
situada histórica, espacial e temporalmente, mais próxima do cotidiano
das práticas. processo, em consonância com uma (trans)formação crítico-reflexiva
situada histórica, espacial e temporalmente, mais próxima do cotidiano
das práticas. Paradigma indiciario: sus aportes a la investigación de la
construcción de las identidades de futuros profesores de lenguas Resumen Resumen Este trabajo trata de los aportes del paradigma indiciario para el estudio de
las identidades docentes y, particularmente, las de los futuros profesores de
lenguas, con énfasis en los del español. Con esa finalidad, explicitaremos en qué
consiste ese modelo y cómo puede aportar para generar mayor inteligibilidad
a respecto de cómo futuros profesores se involucran en sus procesos de
construcción identitaria vividos a lo largo de sus itinerarios formativos. Así,
en este artículo indicamos algunas contribuciones de ese paradigma para el
examen de la problemática enfocada, reflexionando acerca de su potencial
como constructo metodológico de naturaleza cualitativa e interpretativista. Palabras clave: Paradigma Indiciario, Formación de profesores de español,
Identidades, Lingüística Aplicada. 580 Baptista, L. M. T. R. Paradigma Indiciário... Referências ABAURRE, Maria Bernadete Marques; FIAD, Raquel Salek; MAYRINK- ABAURRE, Maria Bernadete Marques; FIAD, Raquel Salek; MAYRINK-
SABINSON, Maria Laura Trindade. Cena I- Indícios. In: ______. Cenas de
aquisição da escrita. O sujeito e o trabalho com o texto. 2. ed. Campinas: ABAURRE, Maria Bernadete Marques; FIAD, Raquel Salek; MAYRINK-
SABINSON, Maria Laura Trindade. Cena I- Indícios. In: ______. Cenas de SABINSON, Maria Laura Trindade. Cena I- Indícios. In: ______. Cenas de
aquisição da escrita. O sujeito e o trabalho com o texto. 2. ed. Campinas:
Mercado das Letras, 2001. p. 13-36. FOUCAULT, Michel. Conferência I. In: ______. A verdade e as formas
jurídicas. Rio de Janeiro: Nau, 1996. p. 7-28. FOUCAULT, Michel. Microfísica do poder. Organização e Tradução Roberto
Machado. 5. ed. Rio de Janeiro: Edições Graal, 1985. GARCIA, Maria Manuela Alves; HYPOLITO, Álvaro Moreira; VIEIRA,
Jarbas Santos. As identidades docentes como fabricação da docência. In:
Educação e Pesquisa, São Paulo, v. 31, n. 1, jan./abr. ,2005. p. 45-56. GINZBURG, Carlo. Sinais: raízes de um paradigma indiciário. In: ______. Mitos, Emblemas, Sinais. Morfologia e história. 2. ed. São Paulo: Companhia
das Letras, 2001. p. 143-179. HOFFNAGEL, Judith Chambliss. Temas em Antropologia e Linguística. Recife: Bagaço, 2010. JAPIASSÚ, Hilton; MARCONDES, Danilo. Dicionário básico de filosofia. 3. ed. Rio de Janeiro: Zahar, 2001. KUHN, Thomas Samuel. “A prioridade dos paradigmas”. In: ______. A
Estrutura das Revoluções Científicas. 8. ed. Perspectiva, São Paulo, 2003, p. 67-76. NÓVOA, Antonio. Diz-me como ensinas, dir-te-ei quem és e vice-versa. In:
FAZENDA, Ivani (Org.). A pesquisa em educação e as transformações do
conhecimento. 12. ed. Campinas: Papirus, 2014. p. 29-41. PÉREZ GÓMEZ, Ángel Ignacio. Ensino para a compreensão. In: GIMENO
SACRISTÁN, José; PÉREZ GÓMEZ, Ángel Ignacio. Compreender e
transformar o ensino. 4. ed. Artmed. Porto Alegre: 2007. p. 67-97. PÉREZ GÓMEZ, Ángel Ignacio. Compreender o ensino na escola: modelos
metodológicos de investigação educativa. In: GIMENO SACRISTÁN, José;
PÉREZ GÓMEZ, Ángel Ignacio. Compreender e transformar o ensino. 4. ed. Porto Alegre: Artmed. 2007. p. 99-117. PIMENTA, Selma Garrido. Formação de professores - saberes da docência
e identidade do professor. In: Nuances, v. III, setembro de 1997, p. 5-14. 581 Signótica, Goiânia, v. 27 n. 2, p. 565-582, jul./dez. 2015 Disponível
em:
<http://revista.fct.unesp.br/index.php/Nuances/article/
view/50>. Acesso em: 24 abr. 2015. SANTOS, Lucíola Licinio de Castro Paixão. Formação do professor e
pedagogia crítica. In: FAZENDA, Ivani (Org.). A pesquisa em educação e as
transformações do conhecimento. 12. ed. Campinas: Papirus, 2014. Baptista, L. M. T. R. Paradigma Indiciário... Referências p. 17-28. SILVA, Maria de Lourdes Ramos. A complexidade Inerente aos Processos
Identitários Docentes. In: Notandum Libro 12 2009 CEMOrOC-Feusp /
IJI-Universidade do Porto, p. 45-58. Disponível em: <www.hottopos.com/
notand_lib_12/malu.pdf>. Acesso em: 24 abr. 2015. VAITSMAN, Jeni. Subjetividade e paradigma de conhecimento. In: Boletim
Técnico do Senac, v. 21, n. 2, maio/ago., 1995, p. 1-9. Disponível em: <http://
www.senac.br/bts/212/2102003009.pdf>. Acesso em: 28 abr. 2015. Williams, Marion & BURDEN, Robert L. El contexto de aprendizaje. In:
______. Psicología para profesores de idiomas. Enfoque del constuctivismo
social. Madrid: Edinumem, 2008. p. 195-209. Submetido em 13 de maio de 2015
Aceito em 12 de junho de 2015
Publicado em 21 de dezembro de 2015 582
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Psychoanalytic Reading of Decadence and Crisis in Ted Hughes' "Relic" and T.S. Eliot's "The Hollow Men"
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INTRODUCTION Literature often serves as a mirror reflecting the complexities of human experience, unraveling the intricate layers of emotion,
thought, and societal context. In twentieth century poetry, T.S. Eliot and Ted Hughes emerge as witnesses to the two consecutive
turbulent eras. Hughes’ “Relic” and Eliot’s “The Hollow Men” capture the essence of the two eras marked by profound uncertainty
and disquiet. Beyond the mere words on the page, these poems invite readers to delve into the depths of the human psyche, exploring
the interplay between decadence and crisis. The concept of decadence, often associated with moral and cultural decline, finds its resonance in these two poems. However, a psychoanalytic approach divulges deeper dimensions of the texts, showing the underlying psychological landscapes that
shaped their creations. Drawing from the theories of Sigmund Freud and Carl Gustav Jung, this research undertakes a journey to
illuminate the subconscious motivations, anxieties, and desires that find expression in “Relic” and “The Hollow Men”. Moreover, crises, whether individual or societal, act as a testing ground for both individuals and cultures. In the times of
Eliot and Hughes respectively, there were abundant upheavals including wars, economic changes, and shifts in cultural norms. “Relic” and “The Hollow Men” express the pervasive sense of disillusionment and fragmentation that characterized their times. By
using a psychoanalytic framework to explore the underlying psychological crisis, this study aims to unravel how individual minds
connect with the broader socio-cultural environment. This research undertakes a two-fold investigation: a psychoanalytic exploration of subconscious landscapes and an analysis
of how these landscapes intersect with the major crises of twentieth century. Through an examination of the two works of Hughes
and Eliot, this study strives to illuminate the intricate interplay between the human psyche, societal decadence, and existential crisis. While exploring their intricate poetry, this paper invites readers on a journey that goes beyond time and provides deep insights into
the human experience International Journal of Social Science And Human Research
ISSN (print): 2644-0679, ISSN (online): 2644-0695
Volume 06 Issue 09 September 2023
DOI: 10.47191/ijsshr/v6-i9-14, Impact factor- 6.686
Page No: 5446-5450 International Journal of Social Science And Human Research
ISSN (print): 2644-0679, ISSN (online): 2644-0695
Volume 06 Issue 09 September 2023
DOI: 10.47191/ijsshr/v6-i9-14, Impact factor- 6.686
Page No: 5446-5450 International Journal of Social Science And
ISSN (print): 2644-0679, ISSN (online): 2644-0695
Volume 06 Issue 09 September 2023
DOI: 10.47191/ijsshr/v6-i9-14, Impact factor- 6.686
Page No: 5446-5450 Psychoanalytic Reading of Decadence and Crisis in Ted Hughes’
“Relic” and T.S. Eliot’s “The Hollow Men” Md Mehedi Hasan artment of English Language and Literature, North Western University, Khulna-9000, Bangladesh ABSTRACT: This research employs psychoanalytic methods to explore themes of decadence and crisis in Ted Hughes’ “Relic”
and T.S. Eliot’s “The Hollow Men”. Drawing from Freudian and Jungian theories, it delves into the psychological aspects to reveal
expressions of the despair of modernity. The paper illustrates underlying psychological decay within the poems, examining
subconscious motives, suppressed desires, and conflicts. Through this exploration, intricate psychological states contributing to
perceptions of crisis come to light. Highlighting these literary pieces as reflections of contemporary turmoil, the poems expose the
erosion of both individual and collective identities. By using the lens of psychoanalysis, a deeper understanding of the interplay
between the human psyche and society emerges, offering insights into the profound implications of modern decadence and crisis. Ultimately, the application of psychoanalysis enriches comprehension of the connections between psychological intricacies and
societal disintegration, shedding light on the significance of the poets’ selected works. KEYWORDS: Crisis, decadence, fragmentation, hollow men, “Relic”, Psychoanalysis, Ted Hughes, T.S. Eliot, and twentieth
century TEXTUAL ANALYSIS AND INTERPRETATION Symbolism and Imagery: Freud’s view that symbols are the “fulfillments of wishes” (Freud, 1900) aligns with the notion that
symbols in literature often reflect subconscious desires and conflicts. By analyzing the symbols and imagery in “Relic” and “The
Hollow Men” with psychoanalysis, it is possible to discover deeper meanings beyond the obvious story. Character Analysis: Jung’s concept of individuation, a process of integrating conscious and unconscious elements, is pertinent to
character analysis. Jung proposed that “the conscious mind is thrown into the highest relief and revealed in all its peculiarity” during
the process of individuation (Jung, 1968). This idea resonates with analyzing the selected poems, revealing the narrator’s inner
struggles and psychological transformations. This approach shows how literature carries deep meanings, and creates links between personal psychology and the wider
social and cultural context. Psychoanalytic Reading of Decadence and Crisis in Ted Hughes’ “Relic” and T.S. Eliot’s “The Hollow Men”
DECADENCE AND CRISIS DECADENCE AND CRISIS Decadence: Both Freud’s “id” representing basic desires and Jung’s “shadow” symbolizing suppressed elements offer insights into
understanding decadence. According to Freud, “the id knows no judgments of value” (Freud, 1933), which implies that the portrayal
of decadence in the poems might be rooted in the uninhibited expression of suppressed urges. Jung’s idea that the shadow contains
both “positive and negative aspects” (Jung, 1968) relates to the complex interplay between societal norms and the darker, hidden
facets of the human psyche. Crisis: Freud’s theory of the unconscious mind as a reservoir of repressed traumas aligns with the crises depicted in the selected
poems. Freud’s concept of repression highlights the role of crisis in shaping the unconscious. Likewise, Jung’s study of archetypes
and the collective unconscious (Shelburne, 1976) offers a way to comprehend how characters in the poems struggle with existential
crises tied to universal themes. DISCUSSION Though Ted Hughes’ “Relic” and T.S. Eliot’s “The Hollow Men” differ in style and subject, they come together in depicting mental
decline, hidden wants, and twentieth century’s existential challenges. The study reveals common themes and profound psychological
insights found in the selected poems. LITERATURE REVIEW AND POINT OF DEPARTURE Lok Raj Sharma’s study of T.S. Eliot’s “The Hollow Men” reveals the poem’s profound exploration of men’s spiritual void and
inefficacy. In Sharma’s opinion, through vivid sections depicting communication barriers, existential uncertainties, and societal
detachment, the poem paints a bleak picture of modern human condition. Sharma’s analysis offers valuable insights into Eliot’s
portrayal of isolation, materialism, and spiritual hollowness in the poem: This poem is perhaps the most negative and pessimistic of all Eliot’s poems. It does not only reflect the paralyzed psyche of Post-
World War I , but it also reveals Eliot’s mental agony, the painful experience of collapsing marriage with Vivien Eliot, his
skepticism towards religious hope and love. (Sharma, 2021) Mariwan M Hasan’s analysis of T.S. Eliot’s “The Hollow Men” employs textual and reader-response approaches to reveal
its themes of religion, spirituality, and modernity. Hasan highlights Eliot’s depiction of hollow modern individuals and their quest
for meaning in a post-World War I world. The study provides insights into how Eliot’s poem serves as a reflection of societal
conditions and the search for faith and morality in a fragmented era: “His concerning about the outcomes of the modern time caused
him to be known as the spokesperson of the modern age. He has expressed the noticeable feelings and anxieties exposed to modern
man through the complexity of the modern period.” (Hasan, 2019) Dr. Maitali Khanna and Rajeshwari C. Patel analyze T.S. Eliot’s portrayal of moral dilemmas in “The Hollow Men” and
The Waste Land, revealing his insights into the societal breakdown during the wartime era. The research highlights Eliot’s keen
understanding of modern moral challenges, demonstrated in his poems: “Keeping in mind the disillusionment of the generation,
T.S. Eliot initially wrote “The Hollow Men”, to serve as an epilogue to The Waste Land.” (Khanna & Patel, 2022) While numerous research articles have extensively examined T. S. Eliot’s poem “The Hollow Men”, contemporary
scholarly investigations have yet to delve into a comparative analysis between Eliot’s “The Hollow Men” and Hughes’ “Relic” from
the vantage point of psychoanalysis. This void in existing literature serves as the research gap of this study. THEORETICAL FRAMEWORK Psychoanalytic literary criticism is a way of understanding literature analyzing the hidden thoughts and feelings in people’s minds. Sigmund Freud and Carl Jung came up with this idea. It helps readers see deeper meanings in stories by exploring what people
might not even realize they are thinking or feeling. Freud’s assertion “dreams are the royal road to the unconscious” (1900) highlights
the significance of exploring the hidden realms of the mind. Psychoanalysis is a process that aligns with the exploration of the
underlying psychological dimensions within the poems “Relic” by Ted Hughes and “The Hollow Men” by T.S. Eliot. IJSSHR, Volume 06 Issue 09 September 2023 www.ijsshr.in IJSSHR, Volume 06 Issue 09 September 2023 Page 5446 www.ijsshr.in Page 5446 Psychoanalytic Reading of Decadence and Crisis in T.S. Eliot’s “The Hollow Men” Psychoanalytic Reading of Decadence and Crisis in T.S. Eliot’s “The Hollow Men” T.S. Eliot’s poem “The Hollow Men” serves as a compelling canvas for a psychoanalytic exploration of decadence and
crisis. Using Sigmund Freud and Carl Jung's theories as guides, this perspective reveals deep symbolisms and psychological
struggles. It sheds light on how identity breaks down and the search for meaning in a world filled with emptiness. The two epigraphs, “Mistah Kurtz—he dead / A penny for the Old Guy,” (Eliot, 1925, p. 2383) echo the sense of loss and
the fleetingness of life. This fragmented opening reflects Freud’s notion of the unconscious mind as a repository of suppressed
desires and unresolved conflicts. The reference to Mistah Kurtz, an allusion to Conrad’s Heart of Darkness (1899), serves as a
reminder of the darkness within the human soul, resonating with Jung’s exploration of the collective unconscious. The line “We are the hollow men” (1925, p. 2383) reflects the fragmented and empty nature of the human psyche. The
term “hollow” can be seen as a metaphor for the suppression of authentic emotions and desires. Through a psychoanalytic lens, this
can be interpreted as the ego’s attempt to mask the unconscious id, resulting in a sense of emptiness and disconnection. The following
lines “We are the stuffed men / Leaning together / Headpiece filled with straw. Alas!” (1925, pp. 2383-2384) express a profound
disconnection from authentic emotions and genuine existence. This hollow state reflects the void-like nature of the unconscious,
mirroring Freud’s concept of the id—the reservoir of primal instincts that often remain unexamined and untamed. The imagery of “dried voices” (1925, p. 2384) that “whisper together / Are quiet and meaningless” (1925, p. 2384) evokes
the hollowness of communication, drawing parallels to the suppressed emotions and concealed desires within the human psyche. This notion aligns with Freud’s belief in the unconscious as a realm where repressed thoughts and emotions linger, shaping one’s
behavior without conscious awareness. Eliot’s portrayal of “Shape without form, shade without color” (1925, p. 2384) and “gesture without motion” (1925, p. 2384) captures the disintegration of self and identity, reflecting the fractured psyche. These descriptions mirror the fragmented
nature of modern identity, as individuals grapple with reconciling personal desires and societal expectations. This fragmentation
parallels Jung’s exploration of individuation—a journey towards integrating the conscious and unconscious aspects of the self. Psychoanalytic Reading of Decadence and Crisis in Ted Hughes’ “Relic” In Ted Hughes’ poem “Relic”, the depths of human experience and the shadows of the unconscious come to the forefront,
inviting a psychoanalytic exploration of decadence and crisis. Based on the theories of Sigmund Freud and Carl Jung, this
interpretation examines intricate layers of symbolism and meaning, focusing on the profound interplay between the human psyche
and the dissolution of societal structures. IJSSHR, Volume 06 Issue 09 September 2023 IJSSHR, Volume 06 Issue 09 September 2023 www.ijsshr.in Page 5447 www.ijsshr.in Psychoanalytic Reading of Decadence and Crisis in T.S. Eliot’s “The Hollow Men” The lines “This is the dead land / This is cactus land” (1925, p. 2385) paint a picture of desolation, underscoring the barren
psychological landscape that the hollow men inhabit. The supplication of “a dead man’s hand / Under the twinkle of a fading star”
(1925, p. 2385) evokes a sense of yearning for salvation amidst spiritual decay, echoing Freud’s concept of unfulfilled desires that
continue to influence human behavior. Eliot’s deliberate structural choices—fragmented sections, repetition of phrases—mirrors the repeating cycles of the
unconscious mind. The refrain “This is the way the world ends / Not with a bang but a whimper” (1925, p. 2386) encapsulates the
sense of disillusionment and crisis, suggesting that the end is not marked by a grand event but rather by a quiet fade into obscurity. Psychoanalytic Reading of Decadence and Crisis in Ted Hughes’ “Relic” and T.S. Eliot’s “The Hollow Men” The core of the poem holds a jawbone, a physical object that serves as a gateway to the mysterious realms of the subconscious. The
poem’s vivid imagery of “crabs, dogfish, broken by the breakers” (Hughes, 1960, p. 2588, line 2) creates a powerful scene reflecting
the wild sea and the chaotic unconscious. The lack of companionship in these depths shows a separation from awareness, echoing
Freud’s idea of the id, a place of basic instincts and desires. The sea’s unending devouring gains metaphorical meaning, reflecting the never-ending desires of the unconscious. The
action of being “gnawn bare” (p. 2588, line 8) reflects the ceaseless demands of the id, forever craving satisfaction. The
transformation of the jawbone into a “cenotaph” (p. 2588, line 16) evokes a poignant metaphor for the loss of identity and purpose,
reflecting the ego’s struggle to reconcile personal desires with societal expectations. Furthermore, the eternal presence of the sea resembles Freud’s idea of the timeless unconscious. As the sea brings ashore “spars of
purposes” (p. 2588, line 13), it holds unresolved psychological conflicts that were blocked, echoing Jung’s collective unconscious,
a reservoir of universal symbols and shared experiences. Within this complex web, Hughes’ “Relic” goes beyond just a poem—it becomes a reflection of intricate individual psychology and
societal collapse, echoing the chaos and disintegration of the modern world. Psychoanalytic Reading of Decadence and Crisis in Ted Hughes’ “Relic” and T.S. Eliot’s “The Hollow Men” on social media or in professional situations, can result in a fragmented self-perception. Many individuals find themselves wearing
different deliberate disguises online and offline, struggling to reconcile these various facets into a cohesive whole. Communication and Connection: The poem’s depiction of “dried voices” (1925, p. 2384) and “quiet and meaningless
whispers” (1925, p. 2384) reflects the challenges of communication in the digital era. While we are more connected than ever, truly
meaningful communication can be rare, often leading to superficial exchanges. The depiction of voices without depth illustrates the
reality of virtual interactions that lack emotional richness. Spiritual Vacuum: The references to “dead land” (1925, p. 2385) and “cactus land” (1925, p. 2385) evoke images of
spiritual desolation and a world devoid of higher values. In an age marked by consumerism, materialism, and a focus on immediate
gratification, many individuals may feel a void where deeper spiritual and ethical foundations should reside. The poem’s portrayal
of stone images and supplications to broken stone reflects the emptiness that can arise when material pursuits replace meaningful
connections and values. Fear of Irrelevance: The fear of being forgotten or becoming insignificant, as expressed in the refrain “Not with a bang
but a whimper,” (1925, p. 2386) echoes the anxieties of modern individuals. In a world saturated with information and fleeting
trends, the fear of being rendered irrelevant or forgotten is a genuine concern for many. The poem’s depiction of a quiet and
unremarkable end reflects the unease that arises from the potential obscurity that modern life can entail. Yearning for Meaning: The poem’s exploration of the “Shadow” and the yearning for something beyond the mundane
can be seen as a reflection of the human need for deeper meaning and purpose. In a world that often emphasizes material success
and external achievements, many individuals long for a sense of fulfillment that transcends superficial pursuits. The poem’s themes
resonate with the search for significance in an era of rapid change and uncertainty. “The Hollow Men” reflects modern society’s challenges with identity, communication, and meaning. It explores inner
struggles and emptiness, remaining relevant in a changing world searching for authenticity and purpose. A Comparative Study of Ted Hughes’ “Relic” and T.S. Eliot’s “The Hollow Men” from Psychoanalytic Perspective
“The Hollow Men” and “Relic” when analyzed through psychoanalytic perspective, reveal similar insights into the
complexities of the human mind. Relevance of “The Hollow Men” to Modern Society T.S. Eliot’s poem “The Hollow Men” resonates with modern society through its profound exploration of existential crisis,
disconnection, and the erosion of identity. This haunting portrayal of inner turmoil and societal decay finds uncanny parallels in the
complexities of contemporary existence, reflecting the challenges, anxieties, and uncertainties of the modern world. Existential Despair: The “hollow men” embody a sense of emptiness and existential despair that many individuals in
modern society can relate to. In a world marked by rapid changes, technological advancements, and shifting cultural norms,
individuals may grapple with a loss of meaning and struggle to find their place in the grand scheme of things. The poem’s portrayal
of individuals trapped in a state of spiritual desolation echoes the sense of disorientation and detachment experienced by many in
the face of a rapidly evolving world. Fragmentation of Identity: The fragmented nature of the hollow men symbolizes the fractured identities that can emerge
in the modern age. The widespread use of digital communication and constant exposure to various facets of one’s identity, whether IJSSHR, Volume 06 Issue 09 September 2023 Page 5448 Page 5448 www.ijsshr.in CONCLUSION Ted Hughes’ “Relic” and T.S. Eliot’s “The Hollow Men” converge in a psychoanalytic exploration. Themes of suppressed desires,
existential turmoil, and fragmented identity intertwine. Through Freudian and Jungian lenses, the selected poems resonate with
modern complexities, portraying the clash of conscious control and hidden impulses. The fading of self and the search for purpose
highlight twentieth century’s crisis, revealing the outcomes of ignoring hidden desires. These poems offer insights into our own
challenges, inviting us to explore the intricacies of the human mind, where hidden wishes and existential longings endure. Psychoanalytic Reading of Decadence and Crisis in Ted Hughes’ “Relic” and T.S. Eliot’s “The Hollow Men” Despite their differences, the poems intersect in portraying psychological decline, repressed wants,
and the existential challenges of the poets’ contemporary times respectively. Suppressed Desires and Fragmented Identity: In “Relic”, the sea’s consumption symbolizes insatiable desires, echoing
Freud’s concept of the id—the repository of repressed instincts. Similarly, “The Hollow Men” portrays individuals as “stuffed men”
(Eliot, 1925, p. 2384) with “dried voices” (1925, p. 2384), suggesting suppression of desires and fractured identity. This
fragmentation reflects the struggle to reconcile conscious identity with suppressed unconscious desires. Existential Emptiness and Crisis: Both poems explore existential emptiness. “Relic” presents the jawbone’s
transformation into a cenotaph, representing the loss of identity. In “The Hollow Men”, the titular figures embody spiritual
emptiness. These portrayals resonate with Freud’s concept of existential crises, wherein suppressed desires can lead to feelings of
meaninglessness and despair. Disconnection from Society: “Relic” alludes to the sea’s isolation, mirroring contemporary disconnection amidst desires. “The Hollow Men” speaks of individuals forgotten in death’s “twilight kingdom” (1925, p. 2385), reflecting detachment from
societal narratives. These portrayals align with Jung’s concept of individuation—the quest for self-integration amidst societal
disintegration. Search for Meaning and Salvation: In “Relic”, the jawbone’s transformation signifies the search for meaning amidst
decay, echoing Jung’s individuation journey. “The Hollow Men” portrays yearning for salvation, reflecting both Freud’s idea of
unfulfilled desires and Jung’s concept of transcendent aspirations. Fragmented Language and Repetition: Both poems use fragmented language and repetition to mirror the unconscious’s
fragmented nature. In “Relic”, phrase like “This is the sea’s achievement” (Hughes, 1960, p. 2588, line 10) demonstrates repetition’s
role in reflecting unconscious conflicts. In “The Hollow Men”, repeated lines emphasize how unfulfilled desires and a sense of
emptiness continue in an unending cycle. Modern Decay and Crisis: Both poems reflect modern decay and crisis. “Relic” portrays decay within the sea’s depths,
paralleling psychological decay. “The Hollow Men” evokes spiritual desolation and societal dissolution. Both poems offer poignant
reflections of contemporary anxieties and the psychological challenges posed by modernity. IJSSHR, Volume 06 Issue 09 September 2023 Page 5449 Page 5449 www.ijsshr.in REFERENCES 1) Eliot, T. S. (1925). The hollow men. In M. H. Abrams (Ed.), The Norton Anthology of English
Literature (7th ed.,
Vol. 2, pp. 2383-2386). W. W. Norton. 1) Eliot, T. S. (1925). The hollow men. In M. H. Abrams (Ed.), The Norton Anthology of English
Literature (7th ed.,
Vol. 2, pp. 2383-2386). W. W. Norton. d, S. (1900). The interpretation of dreams. York University. https://psychclassics.yorku.ca/Freud/Dreams/d 2) Freud, S. (1900). The interpretation of dreams. York University. https://psychclassics.yorku.ca/Freud/Dreams/dreams.pdf
3) Freud, S. (1915). The unconscious. General Psychological Theory: Papers on Metapsychology. pp. 116-150. 2) Freud, S. (1900). The interpretation of dreams. York University. https://psychclassics.yorku.ca/Freud/Dreams/dreams.pdf
3) Freud, S. (1915). The unconscious. General Psychological Theory: Papers on Metapsychology. pp. 116-150. C lli
B
k h
//
d /
i h/ df lib
/F
d U
i
df 3) Freud, S. (1915). The unconscious. General Psychological Theory: Papers on Metapsychology. pp. 116-150. Collier Books.https://www.sas.upenn.edu/~cavitch/pdf-library/Freud_Unconscious.pdf 3) Freud, S. (1915). The unconscious. General Psychological Theory: Papers on Metapsychol
Collier Books.https://www.sas.upenn.edu/~cavitch/pdf-library/Freud_Unconscious.pdf 4) Freud, S. (1933). New introductory lectures on psycho-analysis. Internet Archive. Pdf. 5) Hasan, M. (2019). A thematic investigation into T. S. Eliot’s “The hollow men:” with
approach. Critical Literary Studies, 1(2), pp.159-184. doi: 10.34785/J014.2019.680 6) Hughes, T. (1960). Relic. In M. H. Abrams (Ed.), The Norton Anthology of English Literature (7th e
2588). W. W. Norton. 7) Jung, C. G. (1968). Analytical psychology: its theory and practice. Academia. https://www.academia.edu/4977212/Analytical_Psychology_The_Theory_of_Car 7) Jung, C. G. (1968). Analytical psychology: its theory and practice. Academia. https://www.academia.edu/4977212/Analytical_Psychology_The_Theory_of_Carl_Jung 7) Jung, C. G. (1968). Analytical psychology: its theory and practice. Academia. https://www.academia.edu/4977212/Analytical_Psychology_The_Theory_of_Carl_Jung https://www.academia.edu/4977212/Analytical_Psychology_The_Theory_of_Carl_Jung 8) Khanna, M. & Patel, R. (2022). Moral predicament in T. S. Eliot’s “The hollow men” and “The waste land”. Research Journal of English, 7(1). pp. 7-15. https://www.researchgate.net/publication/357621278_Moral_Predicament_in_T_S_Eliot's_The_Hollow_Men_and_The_
Waste_Land https://www.researchgate.net/publication/357621278_Moral_Predicament_in_T_S_Eliot's_The_Hollow_Men_and_The_
Waste Land 9) Sharma, L. R. (2021). Men’s spiritual vacuity and inefficacy in Eliot’s poem “The hollow men”. Shanlax
I t
ti
l J
l f E
li h 10(1)
1 5 htt
//d i
/10 34293/
li h 10i1 4252 9) Sharma, L. R. (2021). Men’s spiritual vacuity and inefficacy in Eliot’s poem “The hollow men”. Shanlax
International Journal of English, 10(1), pp. 1-5. https://doi.org/10.34293/english.v10i1.4252 10) 10. Shelburne, W. A. (1976). C. G. Jung’s theory of the collective unconscious: a rational reconstruction. University of
Florida. https://ufdcimages.uflib.ufl.edu/UF/00/09/81/33/00001/cgjungstheoryofc00shel.pdf 10) 10. Shelburne, W. A. (1976). C. G. Jung’s theory of the collective unconscious: a rational reconstruction. University of
Florida. REFERENCES https://ufdcimages.uflib.ufl.edu/UF/00/09/81/33/00001/cgjungstheoryofc00shel.pdf SSHR, Volume 06 Issue 09 September 2023 www.ijsshr.in Page 5450
There is an Open Access article, distributed under the term of the Creative Commons Attribution
– Non Commercial 4.0 International (CC BY-NC 4.0)
(https://creativecommons.org/licenses/by-nc/4.0/), which permits remixing, adapting and
building upon the work for non-commercial use, provided the original work is properly cited. There is an Open Access article, distributed under the term of the Creative Commons Attribution
– Non Commercial 4.0 International (CC BY-NC 4.0) IJSSHR, Volume 06 Issue 09 September 2023 IJSSHR, Volume 06 Issue 09 September 2023 IJSSHR, Volume 06 Issue 09 September 2023 Page 5450 www.ijsshr.in Page 5450
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DAMPAK WABAH COVID-19 TERHADAP PENJUALAN JAHE MERAH DI SURAKARTA
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DAYA SAING Jurnal Ekonomi Manajemen Sumber Daya
Vol. 22, No. 2, Desember 2020 DAYA SAING Jurnal Ekonomi Manajemen Sumber Daya
Vol. 22, No. 2, Desember 2020 DAMPAK WABAH COVID-19 TERHADAP PENJUALAN
JAHE MERAH Hendi Yudhanto Adinugroho1, Liana Mangifera2, Rizcho Louistama Setyana Putra3,
Fida Nur Rahma4, Calista Amelia Salsabila5, Diah Ayu Martiningsih6
Manajemen Fakultas Ekonomi dan Bisnis Universitas Muhammadiyah Surakarta
Email: Hendi.yudhanto@gmail.com, Abstrak Penelitian ini bertujuan menganalisis dampak wabah Covid-19 terhadap penjualan jahe merah
di Kawasan kota Surakarta. Variabel yang digunakan adalah Pemahaman & Pengetahuan
tentang Covid-19 dan Persepsi & Sikap Masyarakat untuk mengantisipasi Covid-19. Populasi
penelitian ini adalah penjual jahe merah yang pernah melakukan transaksi jual beli jahe merah
di saat situasi Covid-19 yang berlokasi di kawasan Kota Surakarta dan Sekitarnya . Pemilihan sampel menggunakan metode purposive sampling. Jumlah sampel yang diambil
dalam penelitian ini sebanyak 120 orang responden yang tersebar di Surakarta, Sukoharjo,
Karanganyar, Boyolali, Sragen, Klaten dan Wonogiri. Penelitian ini menggunakan data primer
yang diambil menggunakan wawancara terstruktur dengan bantuan kuesioner. Analisis data
yang digunakan adalah analisis regresi berganda. Simpulan yang dihasilkan adalah adalah
Persepsi & Sikap Masyarakat untuk mengantisipasi Covid-19 berpengaruh positif dan
signifikan terhadap tingkat penjualan jahe merah, sedangkan Pemahaman dan Pengetahuan
tentang Covid-19 berpengaruh positif tetapi tidak signifikan terhadap tingkat penjualan jahe
merah. Persepsi & Sikap Masyarakat untuk mengantisipasi Covid-19 paling berpengaruh
dalam penjualan jahe merah di kawasan Kota Surakarta. Kata Kunci: Covid-19, penjualan, jahe merah Abstract This study aims to analyze the impact of the Covid-19 outbreak on red ginger sales in the
Surakarta city area. The variables used are Understanding & Knowledge about Covid-19 and
Perceptions & Attitudes of the Community to anticipate Covid-19. The population of this study
were red ginger sellers who had bought and sold red ginger during the Covid-19 situation,
located in the city of Surakarta and its surroundings. The sample selection used purposive
sampling method. The number of samples taken in this study were 120 respondents spread
across Surakarta, Sukoharjo, Karanganyar, Boyolali, Sragen, Klaten and Wonogiri. This study
uses primary data which is taken using a structured interview with the help of a questionnaire. The data analysis used is multiple regression analysis. The resulting conclusion is that the
perception & attitude of the community to anticipate Covid-19 has a positive and significant
effect on the level of red ginger sales, while the understanding and knowledge of Covid-19 has a
positive but not significant effect on the level of red ginger sales Public perceptions & attitudes
to anticipate Covid-19 are the most influential in selling red ginger in the Surakarta City area. Keywords: Covid-19, sale, Red Ginger Pendahuluan (Buana, 2020). Penyakit pernapasan virus
akut yang dinamai penyakit Corona virus
2019 (COVID-19) berawal mula mewabah
di Wuhan Cina disebabkan oleh Coronavirus Maraknya wabah COVID-19 di berbagai
negara
meyebabkan
banyak
masyarakat
berupaya meningkatkan daya tahan tubuhnya. 93 Dampak Wabah Covid-19... (93-100) novel, merupakan Sindrom Pernafasan Akut
Parah Coronavirus 2 (SARS-CoV-2). Menurut
penelitian yang dilakukan oleh Li, dkk (2020
) Coronavirus (CoVs) sejauh ini merupakan
kelompok terbesar dari RNA indra-positif
yang diketahui virus memiliki berbagai inang
alami. Dalam beberapa dekade terakhir, baru
Coronavirus yang berkembang telah menjadi
ancaman global bagi kesehatan masyarakat. Organisasi
Kesehatan
Dunia
(WHO)
telah mendaftarkan epidemi coronavirus
pneumonia baru sebagai keadaan darurat
kesehatan masyarakat yang menjadi perhatian
internasional.(Darmawan et al., 2020) paparan virus tersebut dengan penerapan
protokol kesehatan yaitu pencegahan dengan
melalukan social distancing, belajar dirumah
yang dilakukan oleh instansi pendidikan,
physical distance, pemakaian masker hingga
lockdown di beberapa wilayah di dunia dan
menjaga kebersihan dengan rajin cuci tangan
dengan sabun, serta meningkatkan imunitas
dalam tubuh. Hal tersebut mempengaruhi
perilaku
dan
sikap
masyarakat
dalam
mengantisipasi penularan virus covid 19
melalui konsumsi minuman herbal jahe. (Redi Aryanta, 2019) dan masyarakat percaya
akan manfaat jahe yang dipercaya mampu
untuk mengobati sesak nafas serta Di dukung
dengan aneka kuliner yang terdapat di daerah
Surakarta, masyarakat cenderung lebih mudah
mendapatkan minuman-minuman herbal jahe
tersebut seperti di warung hik pinggir jalan
yang menyediakan berbagai minuman khas
tradisional berbahan rempah seperti wedang
jahe, susu jahe, the jahe, wedang uwuh yang
berkhasiat
untuk
meningkatkan
stamina
sekaligus menghangatkan badan (Mangifera,
Isa and Wajdi, 2018). Perilaku masyarakat ini
merupakan tindakan- tindakan individu yang
secara langsung terlibat dalam mendapatkan
produk, megkonsumsi dan menghabiskan
produk yang diinginkan.(Medika, 2020). Di Indonesia sendiri Pemerintah telah
mengeluarkan status darurat bencana terhitung
mulai tanggal 29 Februari 2020. Menurut
data situs covid 19, per 1 november 2020
data jumlah korban di Indonesia yang positif
terinfeksi covid 19 adalah 415.402, sembuh
345.566 dan meninggal 14.044 orang. Hal ini
berdampak terhadap seluruh sistem kehidupan
dan aktivitas sosial di seluruh sektor terutama
bagi masyarakat di wilayah Surakarta dan
sekitarnya. Kebijkana yang dilakukan oleh
pemerintah untuk dapat menyelesaikan
kasus luar biasa ini, antara lain dengan
mensosialisasikan gerakan Social Distancing. Konsep ini menjelaskan bahwa untuk dapat
mengurangi bahkan memutus mata rantai
infeksi Covid-19 seseorang harus menjaga
jarak aman dengan manusia lainnya minimal
2 meter, dan tidak melakukan kontak langsung
dengan orang lain, menghindari pertemuan
massal. Pendahuluan Pemerintah
juga
meliburkan
aktivitas sekolah dan perkuliahan diganti
dengan pembelajaran secara online dan
memberlakukan bekerja didalam rumah,
namun kondisi ini justru malahan dimanfaatkan
oleh banyak masyarakat untuk berlibur dan
belum bisa mengatasi permasalahan bencana
covid 19.(Yanti et al., 2020). Pilihan atau keputusan pembelian jahe
dipengaruhi oleh empat faktor psikologis
utama yaitu motivasi, persepsi, pembelajaran,
serta kepercayaan dan sikap.(Isa, Mardalis
and Mangifera, 2018) Pengertian Persepsi
Konsumen disini bahwa Persepsi adalah
suatu
proses
memilih,
mengatur
dan
menginterpretasikan informasi mengenai
suatu produk barang atau jasa oleh konsumen. (Mauliansyah, 2015). Persepsi tidak hanya
terjadi dalam bentuk rangsangan fisik tapi
juga dipengaruhi oleh kondisi pemasaran
yang ada. Hal ini selaras dengan yang
disampaikan beberapa ahli. Menurut Pride
& Ferrel dalam Fadila (2013:45), Persepsi
adalah proses pemilihan, pengorganisasian
dan penginterprestasian masukan informasi,
sensasi yang diterima melalui penglihatan,
perasaan,
pendengaran,
penciuman
dan Hingga saat ini belum ditemukan obat
antivirus covid-19, status kekebalan tubuh
sendiri menjadi salah satu faktor paling
penting yang mempengaruhi perkembangan
penyakit dan prognosis. Himbauan kepada
masyarakat untuk melindungi diri dari 94 DAYA SAING Jurnal Ekonomi Manajemen Sumber Daya
Vol. 22, No. 2, Desember 2020 sentuhan, untuk menghasilkan makna. Persepsi tersebut muncul karena daya tahan
tubuh merupakan kunci yang sangat penting
dalam situasi pandemi global COVID-19. Oleh karena itu banyak masyarakat memilih
meningkatkan
imun
daya
tahan
tubuh
melalui alternatif lain dengan membuat dan
mengkonsumsi jahe merah sebagai upaya
untuk meningkatkan daya tahan tubuh dalam
upaya melawan COVID19. mengantisipasi Covid-19 , variable dependent
adalah tingkat penjualan Jahe di kawasan kota
Surakarta dan sekitarnya. Analisis data yang
digunakan adalah analisis regresi berganda,
yang terdiri dari koefisien diterminan, uji
simultan (uji F), dan uji hipotesis (uji T) dan
content analysis. Hasil Penelitian dan Pembahasan Penelitian ini dilakukan didaerah Surakarta
yang terdiri dari 6 kabupaten yaitu Kota
Surakarta, Kabupaten Sukoharjo, Wonogiri,
Klaten, Karanganyar, Sragen, dan Boyolali. Dimana hamper semua wilayah tersebut
menjadi zona merah persebaran COVID-19
yang sampai saat ini belum ditemukan anti
virusnya atau vaksinnya. Sehingga masyarakat
dan sekitarnya mencoba mensiasati hal tersebut
dengan mengkonsumsi obat-obatan herbal
seperti jahe merah yang di olah menjadi
minuman/ wedang. Dengan permintaan jahe
merah yang tinggi di duga adanya dampak
COVID-19 terhadap tingkat penjualan jahe. Dengan adanya persepsi masyarakat
terhadap wabah Covid 19 tersebut di duga
memberi dampak terhadap pola perilaku
masyarakat dalam mengkonsumsi minuman
herbal khususnya jahe merah. Dengan adanya
COVID-19 menyebabkan minat masyarakat
akan jahe merah tinggi sehingga diduga
mengakibatkan tingkat penjualan jahe merah
di wilayah solo raya akan mengalami kenaikan. Hal ini merupakan suatu permasalahan yang
menarik untuk di teliti. Untuk mendukung hal
tersebut, maka diadakan penelitian ini dengan
tujuan untuk mengetahui dampak COVID-19
terhadap tingkat penjualan jahe merah di kota
Surakarta dan sekitarnya. COVID-19 membawa dampak yang
begitu kompleks di semua sector industry
dan kehidupan masyarakat Surakarta dan
sekitarnya, salah satu sector yang terdampak
sangat parah adalah perekonomian, banyak
tenaga kerja yang terkena PHK bahkan kondisi
UMKM sangat memprihatinkan sehingga
banyak UMKM yang gulung tikar. Hampir
Mayoritas masyarakat terkena dampak akan
situasi dan Kondisi tersebut tanpa terkecuali
merah di wilayah kota Surakarta dan sekitarnya.i Metode Penelitian Penelitian
ini
menggunakan
metode
diskriptif
kuantitatif. Populasi
penelitian
ini adalah Penjual Jahe merah yang berada
di wilayah Surakarta, Sukoharjo, Klaten,
Wonogiri, Sragen Karanganyar, Boyolali. Pemilihan sampel menggunakan metode
purposive sampling. Kriteria tersebut adalah
Penjual jahe Merah yang berdomisili di Kota
Surakarta dan Sekitarnya. Jumlah sampel yang
diambil dalam penelitian ini sebanyak 120
pedagang jahe merah yang tersebar di Surakarta
dann kabupaten Sukoharjo, Wonogiri, Sragen,
Klaten, Karanganyar dan Boyolali. Penelitian
ini menggunakan data primer yang diambil
menggunakan wawancara terstruktur dengan
bantuan kuesioner. Variabel independent dalam
penelitian ini adalah persepsi responden tentang
Pemahaman dan Pengetahuan tentang Covid-19
dan Persepsi dan Sikap Masyarakat untuk Profil responden dalam penelitian ini
adalah Sebanyak 120 pedagang yang menjadi
responden, yang terdiri dari 15,8 % Laki-laki
dan 84,2 % perempuan. Sedangkan untuk aspek
Pendidikan sebanyak 1,67% tidak bersekolah,
21,7% Tamatan SD, 37,5% tamatan SMP. 37,5% tamatan SMA, 0,83% tamatan D3 dan
0,83% tamatan S1. Terkait penjualan terdiri dari
sistem penjualan Offline dan untuk Distribusi
lokasi penjualan berasal dari Surakarta,
Boyolali, Wonogiri, Karanganyar, Sukoharjo,
Sragen dan Klaten. 95 Dampak Wabah Covid-19... (93-100) Tabel 1. Distribusi Lokasi Penjualan Tabel 1. Distribusi Lokasi Penjualan Rata-rata harga jahe dari 120 pedagang
yang menjadi responden diperoleh informasi
harga/Kg pada saat Covid-19 yang dihimpun
dari berbagai daerah di Surakarta dan
sekitarnya yang hasilnya yaitu, karanganyar
berkisar Rp. 50.000, Sragen berkisar Rp. 49.100, Surakarta Rp.46.700, Klaten berkisar
Rp. 46.500, wonogiri berkisar Rp. 46.250 dan
Sukoharjo berkisar Rp. 46.100. Sehingga dapat
disimpulkan bahwa disaat Covid-19 wilayah
karanganyar menjadi daerah dengan harga
komoditas jahe merah paling tinggi yaitu
sebesar Rp. 50.000/Kg dan Sukoharjo menjadi
daerah dengan harga jahe merah yang paling
rendah yaitu sebesar Rp. 46.100/Kg. Boyolali
berkisar Rp.47.200. Tabel 1. Distribusi Lokasi Penjualan Tabel 1. Distribusi Lokasi Penjualan Dari Sebanyak 120 pedagang yang
menjadi
responden
diperoleh
informasi
mengenai rata-rata harga jahe merah/Kg pada
saat sebelum Covid-19 yang dihimpun dari
berbagai daerah di Surakarta dan sekitarnya
yang hasilnya yaitu, Wonogiri berkisar
Rp.33.000, Surakarta berkisar Rp31.000,
karanganyar berkisar Rp.30.000, Boyolali
berkisar Rp.28.800, Klaten berkisar Rp. 28.300, Sukoharjo berkisar Rp. 28.100 dan
Sragen berkisar 26.100. Sehingga dapat
disimpulkan bahwa harga jahe merah/kg disaat
sebelum Covid-19 di level harga paling tinggi
berada di Kab. Wonogiri sebesar Rp.33.000
dan terendah di Kab. Sragen yaitu sebesar Rp. 26.100 Tabel 3. Distribusi harga Jahe merah saat covid-19 Tabel 3. Distribusi harga Jahe merah saat covid-19 Tabel 3. Distribusi harga Jahe merah saat covid-19 Tabel 2. Distribusi harga jahe merah sebelum
covid-19. Berdasarkan data diantara sebelum dan
disaat Covid-19 yang telah dipaparkan diatas
maka diperoleh prosentase kenaikan yang
terjadi di daerah Surakarta dan Sekitarnya
sebagai berikut yaitu, Wonogiri mengalami
kenaikan harga sebesar 71.3%, Surakarta
mengalami kenaikan harga sebesar 65,5%,
Boyolali sebesar 61,7%, Sukoharjo sebesar
60,8%, Klaten sebesar 60,7%, Karanganyar
sebesar 60% dan Sragen sebesar 53,2%. Sehingga dapat disimpulkan bahwa wilayah
Wonogiri menjadi daerah dengan tingkat
kenaikan harga komoditas jahe merah paling
tinggi yaitu sebesar 71,3% dan Sragen menjadi Berdasarkan data diantara sebelum dan
disaat Covid-19 yang telah dipaparkan diatas
maka diperoleh prosentase kenaikan yang
terjadi di daerah Surakarta dan Sekitarnya
sebagai berikut yaitu, Wonogiri mengalami
kenaikan harga sebesar 71.3%, Surakarta
mengalami kenaikan harga sebesar 65,5%,
Boyolali sebesar 61,7%, Sukoharjo sebesar
60,8%, Klaten sebesar 60,7%, Karanganyar
b
60% d
S
b
53 2% Tabel 2. Distribusi harga jahe merah sebelum
covid-19. 96 DAYA SAING Jurnal Ekonomi Manajemen Sumber Daya
Vol. 22, No. 2, Desember 2020 DAYA SAING Jurnal Ekonomi Manajemen Sumber Daya
Vol. 22, No. 2, Desember 2020 daerah dengan kenaikan harga komoditas
jahe merah yang paling rendah yaitu sebesar
53,2%. Tingkat penjualan jahe merah seperti
dalam table 4 diatas, diperoleh informasi
bahwa tingkat penjualan Jahe Merah tertinggi
berada di daerah Sukorharjo Dimana Sukoharjo
menjadi daerah yang mengalami peningkatan
penjualan terbesar yaitu dengan prosentase
sebesar 81,75 % lalu di susul Klaten dengan
Prosentase sebesar 79,20%, Wonogiri dengan
Prosentase sebesar 69,16%, Boyolali dengan
Prosentase sebesar 64,89%, Sragen dengan
Prosentase sebesar 60,37%, Karanganyar
dengan Prosentase sebesar 59,89% dan
Surakarta dengan Prosentase sebesar 58,24%. Sehingga dapat di simpulkan bahwa wilayah
wonogiri menjadi daerah dengan tingkat
peningkatan penjualan tertinggi dan sedangkan
Surakarta menjadi daerah dengan tingkat
peningkatan paling rendah . Tabel 4. Tingkat Penjualan jahe merah Tabel 4. Tingkat Penjualan jahe merah Tabel 4. Tingkat Penjualan jahe merah Hasil analisis regresi linear berganda
dapat di lihat pada tabel 5 di bawah ini. Tabel 5. Hasil Analisis Regresi Linier Berganda
Variabel Independen
Koefisien
t-ratio
Sig. Pemahaman dan pengetahuan tentang Covid-19 (X1)
0,107
1.720
.088
Persepsi dan sikap masyarakat untuk mengantisipasi Covid-19 (X2)
0,203
3.506
.001
Konstanta = 8.304
R2 = 0.140
F = 9.486 (Sig. 0,000)
Sumber: Hasil Analisa Data (2020). Tabel 5. Hasil Analisis Regresi Linier Berganda Tabel 5. Hasil Analisis Regresi Linier Berganda Sumber: Hasil Analisa Data (2020). Tabel 3. Distribusi harga Jahe merah saat covid-19 Variabel Pemahaman
& Pengetahuan tentang Covid-19 (X1)
berpengaruh positif tetapi tidak signifikan
pada alpha 5% terhadap penjualan jahe
merah di kawasan kota Surakarta dan Hasil
uji T untuk variabel Persepsi & Sikap
Masyarakat untuk mengantisipasi Covid-19
menunjukkan nilai T seebsar 3.506 dengan
nilai sig 0, 01. Artinya variabel Persepsi &
Sikap Masyarakat untuk mengantisipasi
Covid-19 (X2) berpengaruh positif dan
signifikan pada alpha 5% terhadap penjualan
jahe merah di kawasan kota Surakarta. Dari hasil analisis Variable Pemahaman
& Pengetahuan tentang Covid-19 di atas
berpengaruh positif dan tetapi tidak signifikan
terhadap penjualan jahe merah di kota
Surakarta dan sekitarnya. Hal ini di karenakan
tingkat
pemahaman
responden
belum
sepenuhnya paham dan mengetahui tentang
Covid-19. Sedangkan Persepsi & Sikap Masyarakat
untuk mengantisipasi Covid-19 berpengaruh
positif dan signifikan terhadap penjualan jahe
merah di kota Surakarta dan sekitarnya. Hal
ini dikarenakan adanya dorongan persepsi
masyarakat dalam melakukan keputusan
pemilihan komoditas jahe merah tersebut. Menurut Kotler dan Keller (2016:153),
keputusan pembelian seseorang dipengaruhi
oleh faktor psikologi utama, antara lain. persespsi serta keyakinan dan pendirian. Pendapat di atas menunjukkan adanya
hubungan antara persepsi konsumen dengan
keputusan untuk membeli suatu produk. Sehingga jika tingkat pembelian masyarakat
akan Jahe Merah meningkat maka akan
berkorelasi dengan tingkat penjualan dari
pedagang Jahe Merah tersebut. Pengujian asumsi klasik digunakan untuk
memastikan bahwa data yang digunakan
dalam kondisi yang baik. Uji ini meliputi
uji
normalitas,
heterokedastisitas
dan
multikolinieritas. Hasil uji normalitas untuk
mengetahui apakah data dari masing- masing
variabel memiliki sebaran yang normal
ataukah tidak. Uji kolmogorof- smirnov
digunakan menguji normalitas data. (Ghazali,
2015). Hasil analisis menunjukkan bahwa
semua nilai asymp. Sig dia ats 5% sehingga
disimpulkan data dalam penelitian ini memiliki
sebaran data yang normal. Uji Heteroskedastisitas digunakan untuk
mendeteksi ada tidaknya heteroskedastisitas
dilakukan dengan uji glejser, yaitu dengan
melakukan
regresi
variabel
independen
terhadap absolut residual variabel dependen
dalam regresi utama (Ghazali, 2015). Hasil
analisis menunjukkan bahwa besarnya nilai
signifikan untuk hasil uji heterokedastisitas
terhadap semua variabel memiliki nilai lebih
besar dari 0,05 sehingga disimpulkan bahwa
semua variabel penelitian bebas dari masalah
heterokedastisitas. Tabel 3. Distribusi harga Jahe merah saat covid-19 Berdasarkan tabel 5 dapat dibuat persaman
regresi linear berganda sebagai berikut: (X2) sebesar 0,203 . Setiap kenaikan 1 satuan,
maka tingkat penjualan jahe merah di kawasan
kota Surakarta meningkat sebesar 0,203. 2 Y = 8.304 + 0,107 (X1) + 0,203 (X2) (0,88)
(0,01) Nilai R2 sebesar 0.104 . Variasi tingkat
penjualan jahe merah di kawasan kota Surakarta
dijelaskan oleh variabel Pemahaman &
Pengetahuan tentang Covid-19 dan Persepsi &
Sikap Masyarakat untuk Hasil analisis regresi linear berganda ini
di atas menunjukkan bahwa Koefisien regresi
variabel Pemahaman dan Pengetahuan
tentang
Covid-19
(X1)
sebesar
0,107. Setiap kenaikan 1 satuan pemahaman dan
pengetahuan masyarakat terhadap covid19,
maka tingkat penjualan jahe merah di
kawasan kota Surakarta meningkat sebesar
0,107. Koefisien regresi Persepsi dan Sikap
Masyarakat untuk mengantisipasi Covid-19 mengantisipasi Covid-19 sebesar 10,4
%, sedangkan sisanya 89,6 % dijelaskan oleh
variabel lainnya diluar model yang diteliti. Hasil uji F menunjukkan nilai 9, 486
dengan signifikansi 0,000. Model penelitian
ini adalah tepat (fit) dan Pemahaman &
Pengetahuan tentang Covid-19 serta Persepsi
& Sikap Masyarakat untuk mengantisipasi 97 Dampak Wabah Covid-19... (93-100) Covid-19
secara
simultan
mempunyai
pengaruh signifikan terhadap tingkat penjualan
Jahe merah di kawasan kota Surakarta. Hasil uji T untuk variabel dan
Pemahaman & Pengetahuan tentang
Covid-19 menunjukkan nilai T sebesar 1.720
dengan nilai sig 0.88. Variabel Pemahaman
& Pengetahuan tentang Covid-19 (X1)
berpengaruh positif tetapi tidak signifikan
pada alpha 5% terhadap penjualan jahe
merah di kawasan kota Surakarta dan Hasil
uji T untuk variabel Persepsi & Sikap
Masyarakat untuk mengantisipasi Covid-19
menunjukkan nilai T seebsar 3.506 dengan
nilai sig 0, 01. Artinya variabel Persepsi &
Sikap Masyarakat untuk mengantisipasi
Covid-19 (X2) berpengaruh positif dan
signifikan pada alpha 5% terhadap penjualan
jahe merah di kawasan kota Surakarta. dengan melihat nilai VIF dari masing-
masing variabel. Jika nilai VIF lebih rendah
dari 10, maka disimpulkan bahwa tidak ada
multikolinieritas yang serius antara variabel
independen dalam model (Ghazali, 2015). Hasil
uji
multikolinearitas
menujukkan
bahwa nilai VIF pada uji multikolinearitas
variabel penelitian memiliki nilai kurang
dari 10 sehingga dapat disimpulkan bahwa
semua variabel penelitian tidak terjadi
multikolinearitas. Hasil uji T untuk variabel dan
Pemahaman & Pengetahuan tentang
Covid-19 menunjukkan nilai T sebesar 1.720
dengan nilai sig 0.88. Penutup Simpulan dalam penelitian ini adalah
wabah Covid 19 memberi dampak terhadap
tingkat penjualan jahe merah di wilayah
Surakarta dan sekitarnya. Persepsi & Sikap
Masyarakat untuk mengantisipasi Covid-19
berpengaruh positif dan signifikan terhadap
tingkat penjualan jahe merah dikota Surakarta
dan sekitarnya. Selain adanya pengaruh
akan Persepsi & Sikap Masyarakat untuk
mengantisipasi Covid-19 ada hal yang Uji
Multikolinieritass
adalah
suatu
keadaan di mana salah satu atau lebih
variabel independen dapat dinyatakan sebagai
kombinasi linier dari variabel independen
lainnya. Uji
multikolinieritas
dilakukan 98 DAYA SAING Jurnal Ekonomi Manajemen Sumber Daya
Vol. 22, No. 2, Desember 2020 & Sikap Masyarakat untuk mengantisipasi
Covid-19 adalah salah satu Variable yang
mempengaruhi tingkat penjualan jahe merah
di Kota Surakarta dan Sekitarnya di saat
situasi Pandemi Covid-19. Implikasi terhadap
manajerialnya
adalah
pentingnya
dalam
mengelola Persepsi & Sikap Masyarakat
untuk mengantisipasi Covid-19 yang di
implementasikan ke dalam bentuk penjualan
jahe merah di tengah situasi Covid - 19 . membuat permintaan dan harga jahe merah
mengalami kenaikkan yang cukup signifikan
yaitu karena dipengaruhi oleh cuaca yang
berkorelasi melalui kondisi media tanam dan
minat daya beli masyarakat disaat terdampak
Covid-19 serta juga dipengaruhi oleh musim
tanam dan panen. & Sikap Masyarakat untuk mengantisipasi
Covid-19 adalah salah satu Variable yang
mempengaruhi tingkat penjualan jahe merah
di Kota Surakarta dan Sekitarnya di saat
situasi Pandemi Covid-19. Implikasi terhadap
manajerialnya
adalah
pentingnya
dalam
mengelola Persepsi & Sikap Masyarakat
untuk mengantisipasi Covid-19 yang di
implementasikan ke dalam bentuk penjualan
jahe merah di tengah situasi Covid - 19 . Implikasi teoritis penelitian ini adalah
membuka
penelitian
terbaru
terkait
penjualan jahe merah, dimana Persepsi Daftar Pustaka Aryanta, I. W. R. (2019). Manfaat Jahe Untuk Kesehatan. Widya Kesehatan, 1(2), 39-43 Buana, D. R. (2020) ‘Analisis Perilaku Masyarakat Indonesia dalam Menghadapi Pandemi
Virus Corona (Covid-19) dan Kiat Menjaga Kesejahteraan Jiwa’, SALAM: Jurnal Sosial
dan Budaya Syar-i, 7(3). doi: 10.15408/sjsbs.v7i3.15082. Darmawan, D. et al. (2020) ‘Sikap Keberagamaan Masyarakat Muslim Menghadapi Covid-19’,
Religious: Jurnal Studi Agama-Agama dan Lintas Budaya, 7249, pp. 116–124. Isa, M., Mardalis, A. and Mangifera, L. (2018) ‘Analisis Keputusan Konsumen Dalam
Melakukan Pembelian Makanan dan Minuman di Warung Hik’, Jurnal Manajemen
Dayasaing, 20(1), pp. 44–51. doi: 10.23917/dayasaing.v20i1.6590. Mangifera, L., Isa, M. and Wajdi, M. F. (2018) ‘Faktor-Faktor yang Mempengaruhi Konsumen
Dalam Pemilihan Kuliner di Kawasan Wisata Alam Kemuning’, Jurnal Manajemen
Dayasaing, 20(1). doi: 10.23917/dayasaing.v20i1.6415. Mauliansyah, R. (2015) ‘Analisis Pengaruh Motivasi, Persepsi Kualitas dan Sikap Konsumen
terhadap Keputusan Pembelian Sepeda Motor Honda di Kota Langsa’, Jurnal Manajemen
dan Keuangan Unsam, 4(1), p. 196986. Medika, J. M. (2020) ‘Hubungan Pengetahuan dan Sikap Masyarakat Terhadap Covid-19:
A literature Review’, Jurnal Menara Medika https://jurnal.umsb.ac.id/index.php/
menaramedika/index JMM 2020 p-ISSN 2622-657X, e-ISSN 2723-6862, 3(1), pp. 46–53. Redi Aryanta, I. W. (2019) ‘Manfaat Jahe Untuk Kesehatan’, Widya Kesehatan, 1(2), pp. 39–
43. doi: 10.32795/widyakesehatan.v1i2.463. Yanti, N. P. E. D. et al. (2020) ‘Gambaran Pengetahuan Masyarakat tentang Covid-19 dan
Perilaku Masyarakat di Masa Pandemi Covid-19’, Jurnal Keperawatan Jiwa, Vol. 8
No.(3), pp. 485–490. Pradana, A. A., & Casman, C. (2020). Pengaruh Kebijakan Social Distancing pada Wabah
COVID-19 terhadap Kelompok Rentan di Indonesia. Jurnal Kebijakan Kesehatan
Indonesia: JKKI, 9(2), 61-67. Kriswibowo, A., & Utomo, S. A. P. (2020). Ekonomi Politik Indonesia di Tengah Pandemi
Covid- 19 Penerbit Cakradewa Ilmu. Ramadhani, V. A. (2018). Pengaruh Persepsi, Motivasi Dan Gaya Hidup Konsumen Usia
Muda Terhadap Keputusan Pembelian Makanan Cepat Saji Di Mcdonald’s Sudirman
Pekanbaru (Studi Kasus Pada Mahasiswa UIN Suska Riau Fakultas Sains dan Teknologi
Jurusan Teknik Industri S1) (Doctoral dissertation, Universitas Islam Negeri Sultan Syarif 99 Dampak Wabah Covid-19... (93-100)
Kasim Riau Dampak Wabah Covid-19... (93-100)
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Journal of Business & Entrepreneurship, 2(2), 83-92. 100
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The Influence of Technology on Accounting Practices in India: A Survey of Accounting Software Utilization by Micro Enterprises in Delhi
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European journal of theoretical and applied sciences
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Abstract: significant in urban areas like Delhi, where micro
enterprises form a vital component of the local
economy. The advent of various accounting
software solutions has enabled these businesses
to manage their financial transactions more Abstract: Suggested Citation
Gupta, R. (2023). The Influence of
Technology on Accounting
Practices in India: A Survey of
Accounting Software Utilization by
Micro Enterprises in Delhi. European Journal of Theoretical and
Applied Sciences, 1(6), 1041-1053. DOI: 10.59324/ejtas.2023.1(6).101 Suggested Citation
Gupta, R. (2023). The Influence of
Technology on Accounting
Practices in India: A Survey of
Accounting Software Utilization by
Micro Enterprises in Delhi. European Journal of Theoretical and
Applied Sciences, 1(6), 1041-1053. DOI: 10.59324/ejtas.2023.1(6).101 This research paper investigates the impact of technology adoption
on accounting practices within micro enterprises in Delhi, India. The
primary objectives of this study were to identify the factors
influencing the selection of accounting software, examine the
relationship
between
technology
adoption
and
perceived
improvements in financial efficiency, and uncover the specific
challenges faced by micro enterprises in integrating technology into
their accounting processes. To achieve these objectives, a
comprehensive survey was conducted among a diverse sample of
micro enterprises in Delhi. The survey assessed the criteria used by
these businesses to choose accounting software, their level of confidence in data security, and their
satisfaction with the software's functionality. It also explored the challenges encountered during software
adoption and identified the features that micro enterprises deemed essential for software improvement. The key findings of this research indicate that micro enterprises in Delhi prioritize cost-effectiveness and
functionality when selecting accounting software. While technology adoption positively impacts
confidence in data security and satisfaction levels, it does not consistently lead to perceived improvements
in financial efficiency. Challenges in software adoption are closely related to the nature of the business,
emphasizing the need for tailored approaches. Desired software improvements include enhanced tax
compliance features, cloud-based access, improved reporting tools, and scalability. The implications of
this study extend beyond Delhi's micro enterprises, offering insights for policymakers, business owners,
and the accounting profession. Policymakers can use these findings to design initiatives that support
digital transformation in small businesses. Business owners can make informed decisions when selecting
accounting software, and accountants can adapt their skills to the evolving landscape of technology
integration in finance. gg
Gupta, R. (2023). The Influence of
Technology on Accounting
Practices in India: A Survey of
Accounting Software Utilization by
Micro Enterprises in Delhi. European Journal of Theoretical and
Applied Sciences, 1(6), 1041-1053. DOI: 10.59324/ejtas.2023.1(6).101 Keywords: technology adoption, accounting practices, micro enterprises, accounting software, data security, financial
efficiency, digital transformation. The Influence of Technology on Accounting Practices in India:
A Survey of Accounting Software Utilization
by Micro Enterprises in Delhi Renu Gupta
Department of Commerce, Jesus and Mary College, University of Delhi, India Introduction The integration of technology into accounting
practices, particularly in the context of micro
enterprises in India, represents a profound
transformation in the financial management
landscape. This shift has been especially This work is licensed under a Creative Commons Attribution 4.0 International License. The license permits unrestricted
use, distribution, and reproduction in any medium, on the condition that users give exact credit to the original author(s)
and the source, provide a link to the Creative Commons license, and indicate if they made any changes. This work is licensed under a Creative Commons Attribution 4.0 International License. The license permits unrestricted
use, distribution, and reproduction in any medium, on the condition that users give exact credit to the original author(s)
and the source, provide a link to the Creative Commons license, and indicate if they made any changes. efficiently and effectively, marking a significant
departure from traditional manual methods. adapting
to
new
technologies
and
methodologies (Kamau and Ilamoya, 2023). This
research
will
contribute
to
a
better
understanding of these changes in the context of
Indian micro enterprises, highlighting the
nuances of technology adoption in a sector that
significantly differs from large corporations. The evolution from manual book keeping to
sophisticated accounting software is a pivotal
development in the accounting domain. It
signifies not just a change in the tools used for
accounting but also represents a fundamental
shift in the skills and mindset required within the
accounting
profession. This
technological
revolution, often encapsulated in the term
"accounting information systems" (AIS), has had
a far-reaching impact on how accounting data is
collected, processed, and reported (Gnana
Prakash, 2023). Moreover, the relevance of this research extends
beyond the academic realm; it has practical
implications for business owners, policymakers,
software
developers,
and
accounting
professionals. By examining the utilization of
accounting software in micro enterprises, this
study will provide valuable insights into the
effectiveness of these tools in improving
business operations and compliance with
financial regulations. Furthermore, it can guide
policymakers in developing strategies to support
the digital transformation of small businesses, a
crucial step in enhancing the overall economic
growth of the country. In India, and particularly in its capital, New
Delhi, the adoption of technology in accounting
poses both opportunities and challenges for
micro enterprises. While these small businesses
typically operate with limited resources, making
the adoption of technology a critical and
strategic decision, the potential benefits of such
adoption are significant. Introduction They
investigated the extent to which these systems
have been adopted and the consequent impact
on the financial management practices of
MSMEs (Cahyani & Amna, 2022). examining the challenges, benefits, and overall
impact on their business operations. The
findings of this study are expected to contribute
significantly to the literature on technology in
accounting and provide practical insights for
various stakeholders in the accounting and
business sectors. By delving into the specific
context of Delhi's micro enterprises, this study
will shed light on a critical aspect of India's
economic fabric, offering a comprehensive
understanding
of
the
interplay
between
technology and accounting in the small business
sector. Wicaksono, Kartikasary, and Salma (2020)
delved
into
cloud
accounting
software
implementation and security systems. Their
research provided insights into the adoption of
cloud-based accounting solutions by MSMEs
and the security considerations associated with
such implementations (Wicaksono et al., 2020). The work by Vysochan et al. (2021) focused on
the selection of accounting software for small
and medium enterprises using the Fuzzy Topsis
Method. This study presented a novel approach
to choosing appropriate accounting software,
considering various factors critical for small and
medium-sized businesses (Vysochan et al.,
2021). Wicaksono, Kartikasary, and Salma (2020)
delved
into
cloud
accounting
software
implementation and security systems. Their
research provided insights into the adoption of
cloud-based accounting solutions by MSMEs
and the security considerations associated with
such implementations (Wicaksono et al., 2020). The work by Vysochan et al. (2021) focused on
the selection of accounting software for small
and medium enterprises using the Fuzzy Topsis
Method. This study presented a novel approach
to choosing appropriate accounting software,
considering various factors critical for small and
medium-sized businesses (Vysochan et al.,
2021). Introduction Improved efficiency,
accuracy,
and
compliance
with
financial
regulations are among the foremost advantages. The use of popular accounting software like
Tally
and
QuickBooks,
which
offer
functionalities catering to the specific needs of
small-scale businesses, has gained prominence in
this segment (Utami & Yulianto, 2019). The study of technology's influence on
accounting in micro enterprises is not just about
the adoption of new tools; it's about
understanding the broader context in which
these businesses operate. Factors such as the
ease of use of the software, its cost-effectiveness,
and the training required for effective utilization
are critical aspects that influence the decision-
making process of small business owners. Additionally, the study will explore the impact of
such technological adoption on the financial
performance and growth prospects of these
enterprises. The significance of studying the impact of
technology on accounting practices in micro
enterprises in Delhi lies in understanding how
widespread these tools are and the consequent
effects on accounting practices. As Fauzi et al. (2022)
point
out,
the
advancement
of
information
technology
has
a
profound
influence on the development of accounting
practices (Fauzi et al., 2022). This study aims to
bridge the gap in literature by focusing
specifically on micro enterprises in a developing
economy, offering insights into how these
businesses are navigating the technological
landscape in accounting. This research is also significant in the context of
the global shift towards digitalization. As
economies around the world increasingly
embrace digital technologies, understanding
how this trend impacts the grassroot level of the
business ecosystem in developing countries like
India becomes crucial. The insights gained from
this study could be instrumental in shaping
policies and strategies not only for India but also
for other developing nations facing similar
challenges and opportunities in the digital era. The evolution of accounting practices under the
influence of technology has broader implications
for the accounting profession itself. As noted by
Kamau and Ilamoya (2023), the accounting
profession is undergoing significant changes, In summary, this research aims to explore the
extent of technology adoption in accounting
practices among micro enterprises in Delhi, www.ejtas.com
EJTAS
2023 | Volume 1 | Number 6
1042 1042 2023 | Volume 1 | Number 6 2023 | Volume 1 | Number 6 www.ejtas.com Anindya, 2021). In their 2022 study, Cahyani and
Amna analyzed the application of accounting
information systems in micro, small, and
medium
enterprises
(MSMEs). Literature Review Literature Review Literature Review of Scholarly Works Literature Review of Scholarly Works Literature Review of Scholarly Works The intersection of technology and accounting
practices, particularly in the context of micro
enterprises, has been the subject of extensive
research over the past few years. This literature
review examines following scholarly works that
provide insights
into
the
utilization
of
accounting software in micro enterprises,
highlighting the evolution and impact of this
trend. Apriyanti and Yuvitasari (2021) researched the
role of digital utilization in accounting to
enhance MSMEs' performance during the
COVID-19
pandemic. Their
case
study
highlighted the increased reliance on digital
accounting tools during the pandemic and its
positive impact on the performance of MSMEs
in Semarang, Indonesia (Apriyanti & Yuvitasari,
2021). In a 2019 study, Suhayati and Riandani
explored an accounting application tailored for
small and medium enterprises. Their research
contributed to understanding how tailor-made
accounting applications can meet the specific
needs of SMEs, facilitating more efficient
financial management practices (Suhayati &
Riandani, 2019). Ezenwoke (2017) examined the
organizational determinants and implementation
of e-accounting systems in micro and small
enterprises in Nigeria. This study provided a
comprehensive analysis of factors influencing
the adoption of electronic accounting systems in
small businesses in a developing country context
(Ezenwoke, 2017). Sumarna (2023) examined the usage of
computerized accounting systems in micro-small
enterprises. The study emphasized the increasing
trend of digitalization in accounting practices
among smaller businesses, highlighting the
benefits and challenges faced during the
transition
from
traditional
methods
to
computerized systems (Sumarna, 2023). In
Shbeilat and Al-Hajaia (2022), the focus was on
electronic accounting applications in micro-
enterprises. The paper discussed the challenges
and the impact of these applications on financial
inclusion, underscoring the role of technology in
enhancing
accessibility
and
efficiency
in
accounting for micro-enterprises (Shbeilat & Al-
Hajaia, 2022). Rahman and Anindya (2021) explored how the
utilization of accounting software could increase
the performance of MSMEs. Literature Review of Scholarly Works Their findings
suggested that the adoption of such software
directly correlates with improved business
efficiency and financial performance (Rahman & These scholarly works collectively highlight the
evolving landscape of accounting practices in the www.ejtas.com
EJTAS
2023 | Volume 1 | Number 6
1043 1043 2023 | Volume 1 | Number 6 2023 | Volume 1 | Number 6 www.ejtas.com Sampling
Technique
Stratified Random Sampling
Data Collection
Time
March – May 2023
Response Rate
60% [out of 90 firms response
was received from 54 firms
Data Collection
Tool
Structured Questionnaire (See
Appendix: Questionnaire)
Pilot Study
Conducted on a group of 10
firms to pre-test the
questionnaire for clarity, context of micro enterprises. The transition to
digital and computerized systems has brought
about significant changes in how these
businesses manage their financial transactions. Despite the challenges, the overall impact has
been positive, with improvements in efficiency,
financial performance, and inclusion. Identification of Literature Gap The existing literature extensively explores the
adoption and impact of accounting software in
micro enterprises, with a global perspective that
often includes developing countries. However, a
noticeable gap exists in the specific study of the
influence of technology on accounting practices
within the context of micro enterprises in Delhi,
India. While previous studies have addressed the
general utilization of accounting software in
small and medium-sized enterprises, there is a
lack of focused research on the unique
challenges and opportunities faced by micro
enterprises in a densely populated and
economically diverse city like New Delhi. Variables for the Study To effectively analyse the influence of
technology on accounting practices in micro
enterprises in Delhi, it is essential to categorize
the variables into Independent, Dependent, and
Controlled Variables, based on the questionnaire
provided in the appendix. Table 2. Variables for the Study
Variable Type
Variables
Independent
Variables
• Type of Micro Enterprise
•
Nature of Business (Goods,
Services, Both)
•
Accounting Software in Use
•
Duration of Current
Accounting Software Usage,
Dependent
Variables
•
Satisfaction Level with
Current Software
•
Factors Contributing to
Satisfaction
•
Confidence in Financial
Data Security
•
Reasons for
Dissatisfaction
•
Frequency of
Accounting Software Updates
•
Challenges Faced in
Accounting Software Adoption
•
Perceived Improvement
in Financial Efficiency
•
Justification of Cost for
Software Benefits Table 2. Variables for the Study Addressing this gap is significant for several
reasons. New Delhi, as a major economic hub in
India, presents a unique environment where
traditional business practices and modern
technological
advancements
converge. Understanding how micro enterprises in this
region adopt and utilize accounting software can
provide valuable insights into the effectiveness
of these technologies in varied economic
landscapes. Additionally, this research can guide
policymakers and software developers in
creating more tailored accounting solutions and
support mechanisms for micro enterprises in
similar urban settings. Research Methodology Table 1. Sampling & Data Collection Source
Element
Description
Sample Size
90 firms
Source of Data
Online Surveys and In-Person
Questionnaires
Geographical Area
Delhi, India www.ejtas.com
EJTAS
2023 | Volume 1 | Number 6
1044 1044 2023 | Volume 1 | Number 6 2023 | Volume 1 | Number 6 Controlled
Variables
•
Gender
•
Age
•
Education Level
•
Years in Business
•
Annual Turnover
•
Factors Considered in
Software Selection
•
Desired Features for
Software Improvement Controlled
Variables
•
Gender
•
Age
•
Education Level
•
Years in Business
•
Annual Turnover
•
Factors Considered in
Software Selection
•
Desired Features for
Software Improvement Table 3. Results of Pilot Testing of the
Questionnaire/Instrument or Reliability
Analysis
Variables
Cronbach's
Alpha Values
No. of
Items
Type of Micro
Enterprise
0.73
4
Nature of Business
0.78
3
Accounting Software in
Use
0.82
5
Duration of Accounting
Software Usage
0.75
4
Satisfaction Level with
Current Software
0.81
5
Factors Contributing to
Satisfaction
0.84
8
Confidence in Financial
Data Security
0.77
5
Reasons for
Dissatisfaction
0.79
7
Frequency of
Accounting Software
Updates
0.72
4
Challenges in
Accounting Software
Adoption
0.80
6
Perceived Improvement
in Financial Efficiency
0.85
5
Justification of Cost for
Software Benefits
0.74
5 The data collected through the survey was
analysed using the following inferential statistical
tests: 1. Frequency Count & Percentage:
Applied to controlled variables to understand
the demographic and business profile of the
respondents. 2. Correlation Matrix: This tool was used
to assess the relationship between all dependent
and independent variables. It helped in
understanding how variables such as the type of
software used, duration of its use, and nature of
the business relate to satisfaction levels, security
confidence,
and
perceived
efficiency
improvements. 2. Correlation Matrix: This tool was used
to assess the relationship between all dependent
and independent variables. It helped in
understanding how variables such as the type of
software used, duration of its use, and nature of
the business relate to satisfaction levels, security
confidence,
and
perceived
efficiency
improvements. 3. Regression Analysis: This analysis was
conducted to evaluate the predictive power of
independent variables on all the dependent
variables. The beta coefficient indicated the
strength and direction of these relationships,
while the p-value helped in determining the
statistical significance. This analysis is crucial in
understanding which factors most strongly
predict satisfaction with accounting software,
challenges
in
adoption,
and
perceived
improvements in financial processes. Research Methodology To test the reliability, a pilot test was conducted
on a subset of responses to the questionnaires. It was found that all the Cronbach's alpha values
were above the threshold of 0.70, indicating a
high level of internal consistency and reliability
in the measures used in the survey. This result
validates the reliability of the questionnaire for
the larger study. Table 4. Socio-demographic Characteristics
of the Respondents (n=54)
Demographic
Information
Frequency
Count
Percentage
(%)
Gender
Male
39
72.22
Female
15
27.78
Age
18-24
10
18.52
25-34
16
29.63 Table 4. Socio-demographic Characteristics
of the Respondents (n=54) Results and Analysis This section presents the findings from the
research, offering a comprehensive analysis of
the data collected. It includes various tables and
figures that illustrate the key points of the
research. www.ejtas.com
EJTAS
2023 | Volume 1 | Number 6
1045 1045 2023 | Volume 1 | Number 6 www.ejtas.com www.ejtas.com EJTAS 35-44
12
22.22
45-54
8
14.81
55 and above
8
14.81
Education
High School or below
5
9.26
Bachelor's Degree
18
33.33
Master's Degree
20
37.04
Doctorate or
Professional Degree
11
20.37
Type of Enterprise
Sole Proprietorship
20
37.04
Partnership
12
22.22
Private Limited
Company
16
29.63
Others
6
11.11
Business Nature
Goods
15
27.78
Services
25
46.30
Both Goods and
Services
14
25.93
Years in Business
Less than 1 year
6
11.11
1-5 years
24
44.44
6-10 years
16
29.63
More than 10 years
8
14.81
Annual Turnover (Rs.)
Less than 1 crore
12
22.22
1-2 crore
18
33.33
2-4 crore
16
29.63
4-5 crore
8
14.81 35-44
12
22.22
45-54
8
14.81
55 and above
8
14.81
Education
High School or below
5
9.26
Bachelor's Degree
18
33.33
Master's Degree
20
37.04
Doctorate or
Professional Degree
11
20.37
Type of Enterprise
Sole Proprietorship
20
37.04
Partnership
12
22.22
Private Limited
Company
16
29.63
Others
6
11.11
Business Nature
Goods
15
27.78
Services
25
46.30
Both Goods and
Services
14
25.93
Years in Business
Less than 1 year
6
11.11
1-5 years
24
44.44
6-10 years
16
29.63
More than 10 years
8
14.81
Annual Turnover (Rs.)
Less than 1 crore
12
22.22
1-2 crore
18
33.33
2-4 crore
16
29.63
4-5 crore
8
14.81 The socio-demographic characteristics of the
respondents (n=54) provide a diverse overview
of micro enterprises in Delhi. The majority of
respondents are male (72.22%), highlighting a
gender disparity in micro enterprise ownership. Age-wise, most respondents are within the 25-34
(29.63%) and 35-44 (22.22%) age brackets,
indicating a relatively young entrepreneurial
base. In terms of education, a significant
proportion holds a Master's Degree (37.04%),
followed by those with a Bachelor's Degree
(33.33%). This suggests a relatively high level of
educational attainment among the micro
enterprise owners. The most common type of
micro enterprise is a sole proprietorship
(37.04%), followed by private limited companies
(29.63%) and partnerships (22.22%). This
distribution indicates a variety of business
structures in the micro enterprise sector. Regarding the nature of business, a larger
percentage deals in services (46.30%) compared
to goods (27.78%) or both (25.93%). This aligns
with the growing trend of service-oriented
businesses in urban areas like Delhi. Most
businesses have been operating for 1-5 years
(44.44%), suggesting a substantial number of
relatively new enterprises. this dataset, warmer colors (such as red and
orange) denote stronger positive correlations,
while cooler colors (like blue) represent weaker
or negative correlations. The descriptive statistics for the key variables in
the study are presented in Table 4.3. The mean
values range from 2.15 (Type of Micro
Enterprise) to 4.61 (Satisfaction Level with
Current Software), indicating varied levels of
these characteristics among the respondents. The standard deviation values, which measure
the amount of variation or dispersion in the data,
range from 0.54 to 1.31, suggesting a moderate
spread in the responses. Notably, all the
skewness and kurtosis values lie within the range
of -1 to +1, confirming the normality of the data. This is crucial for the validity of further statistical
analyses such as correlation and regression. A
skewness or kurtosis value within this range
indicates that the data distribution does not
significantly deviate from a normal distribution,
which is a key assumption in many statistical
tests. Note: Correlation is significant at the 0.05 level
(2-tailed). This table represents the diagonal
correlation matrix which shows the relationship
between
all
dependent
and
independent
variables. The correlation values range from -1
(perfect negative correlation) to +1 (perfect
positive correlation), with 0 indicating no
correlation. Figure 1 presents the correlation matrix, which
illustrates the relationships between various
independent and dependent variables of the
study. The correlations range between -1 and 1,
where values close to 1 or -1 indicate a strong
positive or negative relationship, respectively,
and values around 0 suggest little to no
correlation. For instance, a notable positive
correlation between 'Duration of Accounting
Software Usage' and 'Accounting Software in
Use' (0.89) suggests that longer use is associated
with specific types of software. Conversely, a
strong negative correlation between 'Nature of
Business' and 'Satisfaction Level with Current
Software' (-0.62) indicates that the nature of the
business significantly impacts satisfaction levels,
possibly due to varying software needs. Overall, these descriptive statistics provide a
foundational understanding of the data's
distribution and central tendencies, setting the
stage for more in-depth inferential analyses. Figure 1. Correlation Matrix Heat Map The
matrix
also
highlights
interesting
relationships like the strong negative correlation
between 'Confidence in Financial Data Security'
and 'Perceived Improvement in Financial
Efficiency' (-0.92), suggesting that higher
confidence in data security might not always
correlate with perceived improvements in
efficiency. This work is licensed under a Creative Commons Attribution 4.0 International License. The license permits unrestricted
use, distribution, and reproduction in any medium, on the condition that users give exact credit to the original author(s)
and the source, provide a link to the Creative Commons license, and indicate if they made any changes. www.ejtas.com In terms of annual
turnover, the most common range is 1-2 crore
INR (33.33%), followed by 2-4 crore INR
(29.63%), indicating a moderate level of business
scale among the respondents. Table 5. Descriptive Statistics for Key Variables Table 5. Descriptive Statistics for Key Variables
Key Variables
Mean
Standard Deviation
Skewness
Kurtosis
Type of Micro Enterprise
2.15
0.54
-0.79
0.17
Nature of Business
4.30
0.94
-0.72
0.89
Accounting Software in Use
3.96
0.60
-0.38
0.32
Duration of Current Accounting Software Usage
4.15
1.31
0.74
-0.29
Satisfaction Level with Current Software
4.61
1.30
0.81
0.11
Factors Contributing to Satisfaction
2.49
0.64
0.59
0.53
Confidence in Financial Data Security
4.51
0.87
0.34
-0.05
Reasons for Dissatisfaction
3.53
1.16
0.91
0.67
Frequency of Accounting Software Updates
2.78
0.90
-0.75
-0.31
Challenges Faced in Accounting Software
Adoption
3.15
1.02
-0.93
-0.49
Perceived Improvement in Financial Efficiency
2.18
0.96
-0.63
-0.91
Justification of Cost for Software Benefits
3.93
0.58
-0.81
-0.97 www.ejtas.com
EJTAS
2023 | Volume 1 | Number 6
1046 1046 2023 | Volume 1 | Number 6 Additionally, the 'Challenges in
Software Adoption' show a notable positive
correlation with the 'Nature of Business' (0.75),
indicating that the type of business significantly
influences the challenges faced in software
adoption. Figure 1. Correlation Matrix Heat Map This figure presents a visual representation of
the correlation matrix in the form of a heat map. The color intensity and the values in each cell
indicate the strength and direction of the
correlations between the variables studied. In These correlations provide valuable insights into
how different aspects of business operations and
characteristics interact with the adoption and
satisfaction levels of accounting software, guiding further analysis and discussion in the
study. a strong and statistically significant relationship
with satisfaction levels. Frequency of Software
Updates shows a very high positive beta
coefficient (1.567), suggesting a significant
influence on satisfaction, although the p-value
(0.078) is slightly higher than the conventional
significance level. The p-values, especially those
below 0.05, indicate statistically significant
relationships that can be confidently interpreted
within the context of this study. Table 6. Regression Analysis
Independent
Variables
Beta
Coefficients
p-
Values
Type of Micro
Enterprise
0.195
0.053
Nature of Business
0.861
0.057
Accounting Software in
Use
0.411
0.093
Duration of Software
Usage
0.180
0.007
Factors Contributing to
Satisfaction
-0.305
0.009
Confidence in Data
Security
0.584
0.002
Reasons for
Dissatisfaction
-0.250
0.083
Frequency of Software
Updates
1.567
0.078
Challenges in Software
Adoption
1.855
0.087
Perceived Financial
Efficiency
-0.466
0.098
Justification of Cost for
Benefits
1.167
0.080 Table 6. Regression Analysis Figure 2. Beta Coefficients from Regression
Analysis Figure 2. Beta Coefficients from Regression
Analysis This figure illustrates the beta coefficients from
the regression analysis, visually representing the
strength and direction of the relationships
between the independent variables and the
dependent variable 'Satisfaction Level with
Current Software'. Each bar in the plot
corresponds to an independent variable, with the
length and direction indicating the size and
direction of its beta coefficient. Positive
coefficients are depicted to the right, suggesting
a positive relationship with satisfaction levels,
while negative coefficients extend to the left,
indicating an inverse relationship The regression analysis in Table 4.5 evaluates the
predictive power of various independent
variables on the dependent variable 'Satisfaction
Level with Current Software'. The beta
coefficients indicate the strength and direction
of these relationships, while the p-values provide
insight into their statistical significance. Nature
of Business and Challenges in Software
Adoption have strong positive beta coefficients
(0.861 and 1.855, respectively), suggesting they
are significant predictors of satisfaction levels
with accounting software. Factors Contributing
to
Satisfaction
and
Perceived
Financial
Efficiency show negative beta coefficients (-
0.305 and -0.466), indicating an inverse
relationship with the satisfaction level. This
suggests that these factors may adversely affect
satisfaction. Confidence in Data Security has a
significant positive impact (beta coefficient of
0.584) with a very low p-value (0.002), indicating 1048 EJTAS 2023 | Volume 1 | Number 6 2023 | Volume 1 | Number 6 www.ejtas.com EJTAS Frequency Count & Percentage: C
Variables
Table 7. Factors Considered in
Selection
Factors Considered
Frequency
Count
Per
(%)
Cost
48
88. User-friendliness
42
77. Customer support
29
53. Features
and
functionalities
45
83.3
Reputation
of
the
software
31
57.4
Other (Specify)
7
12.9
In Table 7, it can be observed that cost
and features and functionalities (83.33%
most critical factors considered when
accounting software for micro enter
Delhi. User-friendliness (77.78%)
reputation of the software (57.41%)
significant roles in the decision-making
Additionally, a small percentage of res
(12.96%) specified other factors that in
their software selection. Table 8. Desired Features for
Improvement
Desired Features for
Improvement
Frequency
Count
Per
Advanced analytics
and reporting tools
32
Better integration with
other business
systems
37
Enhanced mobile
accessibility and app
functionality
21
More customizable
invoicing and billing
options
34
Improved data
security and privacy
features
29
AI for financial
forecasting and
insights
25
Easier tax compliance
and updates for
changing regulations
38 More extensive
inventory
management features
27
50.00
Automated bank
reconciliation
31
57.41
Simplified payroll
processing
33
61.11
Better scalability for
growing businesses
36
66.67
User interface and
usability
enhancements
29
53.70
Cloud-based access
and data backup
40
74.07
24/7 customer
support and more
extensive training
28
51.85
Other (Specify)
2
3.70 Frequency Count & Percentage: Controlled
Variables Table 7. Factors Considered in Software
Selection Table 7. Factors Considered in Software
Selection
Factors Considered
Frequency
Count
Percentage
(%)
Cost
48
88.89
User-friendliness
42
77.78
Customer support
29
53.70
Features
and
functionalities
45
83.33
Reputation
of
the
software
31
57.41
Other (Specify)
7
12.96 In Table 7, it can be observed that cost (88.89%)
and features and functionalities (83.33%) are the
most critical factors considered when selecting
accounting software for micro enterprises in
Delhi. Discussion The findings of this study provide valuable
insights into the influence of technology on
accounting practices in micro enterprises in
Delhi, India. In this section, we will discuss and
interpret the results obtained from the data
analysis, examining their implications and
significance in filling the literature gap. User-friendliness (77.78%) and the
reputation of the software (57.41%) also play
significant roles in the decision-making process. Additionally, a small percentage of respondents
(12.96%) specified other factors that influenced
their software selection. In Table 7, it can be observed that cost (88.89%)
and features and functionalities (83.33%) are the
most critical factors considered when selecting
accounting software for micro enterprises in
Delhi. User-friendliness (77.78%) and the
reputation of the software (57.41%) also play
significant roles in the decision-making process. Additionally, a small percentage of respondents
(12.96%) specified other factors that influenced
their software selection. Table 8 illustrates the desired features for
software improvement as indicated by micro
enterprise owners. Easier tax compliance and
updates for changing regulations (70.37%) and
cloud-based access and data backup (74.07%)
are among the top priorities for improvement. Enhanced
mobile
accessibility
and
app
functionality (38.89%) and AI for financial
forecasting and insights (46.30%) are also areas
where
businesses
seek
enhancements. Furthermore, respondents provided specific
suggestions
for
additional
improvements,
indicating a range of software needs in this
sector. Table 8. Desired Features for Software
Improvement
Desired Features for
Improvement
Frequency
Count
Percentage
(%)
Advanced analytics
and reporting tools
32
59.26
Better integration with
other business
systems
37
68.52
Enhanced mobile
accessibility and app
functionality
21
38.89
More customizable
invoicing and billing
options
34
62.96
Improved data
security and privacy
features
29
53.70
AI for financial
forecasting and
insights
25
46.30
Easier tax compliance
and updates for
changing regulations
38
70.37 Desired Software Improvements selection. This suggests that businesses in this
segment recognize the value of technology in
streamlining
their
financial
management
processes. The prominence of user-friendliness
(77.78%) and features and functionalities
(83.33%) further underscores the importance of
software that is accessible and tailored to the
specific needs of small businesses. The data also shed light on the features and
improvements that micro enterprises in Delhi
desire in their accounting software. Easier tax
compliance
and
updates
for
changing
regulations (70.37%) emerge as a top priority,
reflecting the complex and ever-evolving tax
landscape in India. Cloud-based access and data
backup (74.07%) also feature prominently,
indicating a growing demand for remote
accessibility and data security. The strong positive correlation between
'Duration of Software Usage' and 'Accounting
Software in Use' (0.89) indicates that businesses
tend to stick with a chosen software once
adopted, emphasizing the stability and reliability
of these tools. This stability may be attributed to
the training and investment required to
transition to a new system, which can be a barrier
for micro enterprises with limited resources. Advanced analytics and reporting tools (59.26%)
and better integration with other business
systems (68.52%) demonstrate a desire for
software that provides comprehensive insights
and streamlines various aspects of business
operations. These findings align with the
broader trend of businesses seeking not only
efficient bookkeeping but also value-added
features that can enhance decision-making. Adoption and Utilization of Accounting
Software Adoption and Utilization of Accounting
Software The data reveal that the adoption of accounting
software is prevalent among micro enterprises in
Delhi, with a significant proportion (88.89%)
considering cost as a crucial factor in software www.ejtas.com
EJTAS
2023 | Volume 1 | Number 6
1049 1049 EJTAS 2023 | Volume 1 | Number 6 2023 | Volume 1 | Number 6 EJTAS Desired Software Improvements Factors Influencing Satisfaction Levels The regression analysis highlights several factors
that significantly influence satisfaction levels
with
accounting
software
among
micro
enterprises in Delhi. Notably, the 'Nature of
Business' and 'Challenges in Software Adoption'
have strong positive beta coefficients (0.861 and
1.855, respectively), suggesting that the type of
business and the difficulties faced during
software adoption are significant predictors of
satisfaction. Filling the Literature Gap Practical Implications The practical implications of this research are
multifaceted. For micro enterprises in Delhi, the
study's findings can serve as a guide for selecting
and optimizing accounting software. Business
owners can consider factors such as the nature
of their business, the challenges they might face
during adoption, and the level of data security
provided by the software when making informed
decisions. The main findings of the study suggest that the
choice of accounting software is significantly
influenced by the nature of the business and the
challenges faced in software adoption. Micro
enterprises in Delhi prioritize cost-effectiveness
and functionality when selecting accounting
software. User-friendliness,
features,
and
reputation of the software also play important
roles in the decision-making process. These
findings align with the global trend of businesses
seeking cost-efficient solutions that cater to their
specific needs, especially in the case of small-
scale enterprises. Software developers and providers can leverage
the insights on desired software improvements
to tailor their offerings to the specific needs of
micro enterprises in Delhi. Enhancements
related to tax compliance, data security, and
cloud-based access can be a key selling point in
this market. Policymakers can use these findings to design
initiatives and policies that support the digital
transformation of micro enterprises. Addressing
the challenges in software adoption, such as
providing training and support, can help
businesses fully harness the benefits of
technology. Moreover, the study highlights that while
technology adoption in accounting positively
impacts factors like confidence in data security
and satisfaction levels, it does not always
translate into perceived improvements in
financial efficiency. The strong correlation
between confidence in data security and the
adoption of accounting software underscores
the importance of trust in technology, especially
in a digital age where data integrity is paramount. Additionally, the study reveals that challenges in
software adoption are closely related to the
nature of the business, indicating that tailored
approaches are required to address the unique
needs of micro enterprises. Therefore, this research paper has explored the
influence of technology on accounting practices
in micro enterprises in Delhi, India. The findings
highlight the prevalence of accounting software
adoption, the factors influencing satisfaction
levels, and the desired software improvements in
this segment. By addressing the literature gap
and providing practical insights, this study
contributes to a better understanding of
technology adoption in micro enterprises and its
implications for businesses, software developers,
and policymakers. Practical Implications As technology continues to
shape the landscape of accounting practices,
research in this area remains essential to guide
businesses towards more efficient and effective
financial management. Furthermore, the desired features for software
improvement identified in the study reflect the
evolving demands of micro enterprises in Delhi. The focus on tax compliance, cloud-based
access, and enhanced reporting tools highlights
the need for software providers to continuously
adapt their offerings to meet the changing
regulatory and operational requirements of these
businesses. The importance placed on scalability
also suggests that many micro enterprises have
growth aspirations, and their software solutions
should accommodate this potential expansion. Filling the Literature Gap This study addresses the literature gap by
focusing specifically on micro enterprises in
Delhi, India, within the context of technology
adoption in accounting practices. While previous
research has explored technology adoption in
small and medium-sized enterprises globally, the
unique economic and cultural factors in Delhi
necessitate a closer examination. The strong negative beta coefficient for 'Factors
Contributing to Satisfaction' (-0.305) indicates
that businesses with more factors contributing to
satisfaction tend to report lower overall
satisfaction levels. This paradoxical finding may
be attributed to businesses with higher
expectations and a greater understanding of
what constitutes satisfactory software. The study findings confirm that micro
enterprises in Delhi are actively adopting
accounting software, driven by factors such as
cost, user-friendliness, and innovative features. However, the influence of the 'Nature of
Business' and the 'Challenges in Software
Adoption' are significant in shaping their
satisfaction levels. These insights provide a
nuanced understanding of the dynamics of
technology adoption in a densely populated and
economically diverse city like Delhi. Interestingly, 'Confidence in Data Security'
emerges as a significant predictor of satisfaction,
with a positive beta coefficient of 0.584 and a
very low p-value (0.002). This finding highlights
the importance of data security in shaping the
overall satisfaction of micro enterprises with
their chosen accounting software. Given the
increasing concerns about data breaches and
privacy, businesses that have greater confidence
in the security of their financial data are more
likely to be satisfied with their software. The findings also underscore the critical role of
data security in shaping satisfaction levels,
highlighting the need for software developers to
prioritize security features. Additionally, the
desire for improvements in tax compliance and
cloud-based access reflects the unique regulatory
and infrastructure challenges faced by businesses www.ejtas.com
EJTAS
2023 | Volume 1 | Number 6
1050 1050 2023 | Volume 1 | Number 6 2023 | Volume 1 | Number 6 www.ejtas.com in Delhi, further emphasizing the importance of
tailored accounting solutions. practices in micro enterprises in Delhi, India, by
conducting
a
comprehensive
survey
of
accounting software utilization. The findings
from the study shed light on several key aspects
of technology adoption in this specific context
and contribute to filling the existing literature
gap. Conclusion In conclusion, this research paper has explored
the influence of technology on accounting www.ejtas.com
EJTAS
2023 | Volume 1 | Number 6
1051 1051 2023 | Volume 1 | Number 6 2023 | Volume 1 | Number 6 www.ejtas.com EJTAS COVID-19 Pandemic: Case Study in Semarang,
Central Java, Indonesia. In Complex, Intelligent and
Software
Intensive
Systems. https://doi.org/10.1007/978-3-030-79725-
6_49 COVID-19 Pandemic: Case Study in Semarang,
Central Java, Indonesia. In Complex, Intelligent and
Software
Intensive
Systems. https://doi.org/10.1007/978-3-030-79725-
6_49 The broader implications of this research extend
beyond the specific context of Delhi's micro
enterprises. The study offers insights that can
inform policy decisions and strategies not only
in India but also in other developing economies
facing similar challenges and opportunities in the
digital era. Policymakers can use these findings
to design initiatives that support the digital
transformation of small businesses, thereby
contributing to overall economic growth. Cahyani, J., & Amna, L. S. (2022). Analysis of the
Application of Accounting Information Systems
In Micro, Small and Medium Enterprises
(MSMEs). JASa (Jurnal Akuntansi, Audit dan
Sistem
Informasi
Akuntansi),
6(1). https://doi.org/10.36555/jasa.v6i1.1846 For business owners, the research underscores
the importance of making informed decisions
when
selecting
accounting
software. Understanding the factors that influence
satisfaction and efficiency can help micro
enterprises make choices that align with their
specific needs and objectives. This knowledge
empowers them to harness the full potential of
technology for improved financial management. Ezenwoke,
O. (2017). Organisational
determinants
and
e-accounting
system
implementation in micro and small enterprises
(mses)
in
south-west
Nigeria. http://eprints.covenantuniversity.edu.ng/9509 Fauzi, E., Sinatrya, M. V., Ramdhani, N. D.,
Ramadhan, R., & Safari, Z. M. R. (2022). Pengaruh
kemajuan
teknologi
informasi
terhadap perkembangan akuntansi. Jurnal riset
pendidikan
ekonomi. https://doi org/10 21067/jrpe v7i2 6877 In the broader context of the accounting
profession, this research underscores the
transformative
impact
of
technology
on
traditional practices. It emphasizes the need for
accountants and financial professionals to adapt
their skills and mindset to effectively utilize
accounting software. The evolving role of
accountants, from data entry to data analysis and
decision support, highlights the importance of
continuous learning and upskilling in the digital
age. https://doi.org/10.21067/jrpe.v7i2.6877 Gnana Prakash, A. P. (2023). Digitalization of
Accounting Applications: From Clay Tablets to
Smart
Accounting
Applications. https://doi.org/10.58830/ozgur.pub105.c887 Kamau, C., & Ilamoya, S. L. (2023). Accounting
Profession: African Perspective Review of steps
into
the
future. SU+
digital
repository. Conclusion https://doi.org/10.48039/mjtum.v2i1.43 In conclusion, the research on the influence of
technology on accounting practices in micro
enterprises in Delhi provides valuable insights
into a rapidly evolving field. The findings
contribute to a deeper understanding of the
complex interplay between technology adoption,
business characteristics, and financial outcomes. As the global trend towards digitalization
continues, the lessons learned from this study
are invaluable for guiding the future of
technology integration in the world of finance,
particularly in the realm of micro enterprises. Rahman, A., & Anindya, J. P. (2021). Increasing
MSMEs
Performance
by
Utilization
of
Accounting
Software. https://journal.kapin.org/index.php/conferenc
e/article/view/19 Shbeilat, M. K., & Al-Hajaia, M. E. (2022). Electronic accounting applications in micro-
enterprises: Challenges and impact on financial
inclusion. Journal of Governance and Regulation,
11(3). https://doi.org/10.22495/jgrv11i3art14 Suhayati, E., & Riandani, I. (2019). Accounting
Application for Small Medium Enterprises. https://doi.org/10.1088/1757-
899X/662/3/032056 References References
Apriyanti, H. W., & Yuvitasari, E. (2021). The
Role of Digital Utilization in Accounting to
Enhance
MSMEs’
Performance
During References
Apriyanti, H. W., & Yuvitasari, E. (2021). The
Role of Digital Utilization in Accounting to
Enhance
MSMEs’
Performance
During 1052 EJTAS 2023 | Volume 1 | Number 6 2023 | Volume 1 | Number 6 https://doi.org/10.1109/ICIMTECH50083.20
20.9211271 www.ejtas.com EJTAS Sumarna, A. D. (2023). The analysis of
computerized accounting systems usage in the
micro-small enterprises. Akuntabilitas, 17(1). https://doi.org/10.29259/ja.v17i1.17489 Using the Fuzzy Topsis Method. TEM Journal,
10(3),
1348-1356. Using the Fuzzy Topsis Method. TEM Journal,
10(3),
1348-1356. https://doi.org/10.18421/TEM103-43 Wicaksono, A., Kartikasary, M., & Salma, N. (2020). Analyze Cloud Accounting Software
Implementation and Security System for
Accounting in MSMEs and Cloud Accounting
Software Developer. In International Conference on
Information
Management. Utami, N., & Yulianto, H. D. (2019). Significant
Influence of Information Technology on the
Use
of
Modern
Accounting
Software. https://doi.org/10.1088/1757-
899X/662/2/022003 https://doi.org/10.1109/ICIMTECH50083.20
20.9211271 Vysochan, O., Vysochan, O., Yasinska, A., &
Hyk, V. (2021). Selection of Accounting
Software for Small and Medium Enterprises www.ejtas.com
EJTAS
2023 | Volume 1 | Number 6
1053 1053 2023 | Volume 1 | Number 6 2023 | Volume 1 | Number 6 www.ejtas.com
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English
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ESG Index Construction and China's ESG System
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BCP business & management
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ESG Index Construction and China's ESG System
Lanyi Duan* Lanyi Duan*
School of Economics, University College Dublin, Dublin, Ireland
*Corresponding author: lanyi.duan@ucdconnect.com Abstract. The Environmental, Social, and Governance (ESG) system has gained popularity recently
as concerns over issues like corporate responsibility, labor conditions, and climate change become
more pressing. At the same time, investors from around the world have become increasingly
interested in the ESG Thematic Index because it offers greater risk resilience and returns than
traditional trading strategies. Building a Chinese financial ESG system is crucial to advancing the
development of ESG in China since Chinese finance needs to create and enhance its own ESG
system but there is still a lack of systematic study in this area. This article first defines the meaning
of ESG-themed indices, then selects representative ESG-themed indices from the global market,
analyzes the construction processes and methodologies of the indices, and finally makes pertinent
recommendations to promote the growth of ESG-themed indices in China using inductive,
comparative, and case study methods. eywords: ESG; ESG Thematic Index; Green Finance; Sustainable Development. Keywords: ESG; ESG Thematic Index; Green Finance; Sustainable Development. BCP Business & Management
Volume 38 (2023) BCP Business & Management
Volume 38 (2023) EMFRM 2022 1. Introduction The ESG Bond
Index chooses sample bonds based on two factors, the investment of funds raised and the bond issuer, 175 BCP Business & Management
Volume 38 (2023) EMFRM 2022 either to ascertain whether the funds raised are invested in ESG-related projects based on clear criteria
or to ascertain whether the bond issuer has a good ESG performance; in both cases, the index is based
on the bond price of the sample bonds. either to ascertain whether the funds raised are invested in ESG-related projects based on clear criteria
or to ascertain whether the bond issuer has a good ESG performance; in both cases, the index is based
on the bond price of the sample bonds. Few institutions and organizations throughout the world have ESG indicators that are properly
defined, and the system of ESG indicators is still highly unfinished and fragmented. Table 1 illustrates
the main international ESG-related indicators. Table 1. Indicators related to ESG from major international institutions and organisations
Subject
Relevant indicators involving ESG
Global Reporting Initiative
Pay attention to society and the environment. Materials, energy, and the
environment are covered by 12 categories and 34 items. Materials, energy,
wastewater and waste, air emissions, and other topics are covered by 12
categories and 34 items under the environmental heading; similarly, labor
practices, human rights, social effect, and product responsibility are covered
by 30 categories and 48 things under the social heading [5,6]. Committee on Non-Profit
Sustainability
The entirety of the Sustainability Code includes 79 sectors in 10 categories,
including those associated with governance and the environment [7]. International Integrated
Reporting Committee
The link between financial performance and society and the environment
[8]. European Parliament and
European Commission
Environmental issues, social and employee issues, human rights, anti-
corruption and bribery issues, and Board and Management Diversity [9,10]. World Bank
To concentrate on society and the environment. Starting with a goal of
sustainable development and ending with the funding of investment
initiatives Environmental and social norms, as well as their annexes, are
discussed [11]. International Finance
Corporation
The sustainability framework focuses on the environment and society. Asian Development Bank
Information on the environment, society, and governance. Agriculture and
food safety, climate change, gender and development, social development
and poverty, and water are some of the issues that need to be addressed
[11,12]. ble 1. Indicators related to ESG from major international institutions and organisations
Subject
Relevant indicators involving ESG 1. Introduction The ESG Thematic Index integrates the three dimensions of environmental, social, and corporate
governance as a crucial tool for ethical investors to participate in the ESG investment market,
reflecting the ability of the position sample in environmental protection, social responsibility, and
their own sustainable development [1]. It is also a crucial financial tool for directing social capital
flow to sustainable development projects to support sustainable economic development. The use of
social capital as a financial resource to fund sustainable development initiatives is crucial for
promoting sustainable economic growth. ESG measures sustainability and ethical impact in three key areas, including gender inequality,
labor, product responsibility, corruption, bribery, social development, and poverty, as well as
shareholder rights, institutional investors, stakeholders, disclosure and transparency, board
responsibility, and management diversity [1,3]. These areas are all covered by ESG. The E (Environmental) in ESG stands for a company's proactive attitude to the environment, which
includes adherence to current regulatory frameworks and consideration of potential effects in the
future. The combination of the governance environment, governance structure, governance
mechanism, and governance behavior results in G (Governance) [2]. The system's base is its
governance environment, its carrier is its governance structure, and its operational mechanism for the
interaction of its components—a process of continual improvement—is its governance behavior or
process. p
A number of ESG-themed indices have been produced recently by fusing ESG themes with index
products, as more and more financial institutions have started to concentrate on the ESG investing
sector and continue to strengthen their efforts in the invention and development of ESG financial
products. These index products may be roughly divided into two groups based on whether they have
the qualities necessary to engage in market trading, namely ESG stock indices and ESG bond indices. The screening and weighted aggregation of listed firms by various index companies and ESG rating
organizations based on their performance in the three dimensions of E, S, and G are referred to as
ESG stock indexes among them [4]. They reflect the overall changes in the share prices of businesses
with sustainable development capabilities by giving more weight and exposure to stocks with better
ESG performance, thereby encouraging businesses to actively fulfill their obligations in
environmental protection and fostering social development through capital flows. 2.1 Construction method The majority of the aforementioned metrics are challenging to properly quantify and were not
created with ESG in mind. As a result, the assessment agency first gathers data and information from
businesses and other outside organizations. It then researches the current industry best practices for
each assessment element, evaluates the distance between businesses and the best practices in each
area, and assigns a score. The composite score is then determined after each score has been given a
weight. Since various organizations have different ideas on what the ESG framework should include
and the weighting of each component, there is no standardized ESG assessment technique. The
MSCIESG series (Global/US/Emerging Markets), the FTSE4Good series, and the Bloomberg ESG
Index are currently significant international ESG rating indexes. y
g
g
The MSCI ESG family of indices has criteria for each of the three ESG dimensions, which are
scored and added to form an overall ESG score, with AAA denoting the best sustainable performance
and C denoting the worst sustainable performance [9]. This family of indices provides investment
decision support tools for over 5,000 clients in pension funds and hedge funds [6]. In order to assist
investors understand the environmental, social, and corporate governance (ESG) risks and
opportunities in their portfolios and to assess issuers' ability to manage long-term financial-related
ESG issues, MSCI developed the ESG Rating System [10]. 176 BCP Business & Management
Volume 38 (2023) BCP Business & Management
Volume 38 (2023) EMFRM 2022 To identify important risks and opportunities in each sector and to build a framework of indicators,
MSCI employs quantitative models. The indicator system is made up of 35 ESG Key Issues (ESG
Key Issues), 10 themes (Themes), 3 primary categories (Pillars), and hundreds of indicators. The
MSCI ESG Key Issues are assigned to each sector based on the extent of ESG's impact on that sector,
and to each GICS sub-sector based on the extent and duration of the impact of environmental and
social risks on that sector [3]. Governance issues are always crucial and will be taken into
consideration when calculating all company scores. As a result, the final ESG rating score assigned
to a firm is not an absolute number but rather a measure of how well the company performed in
comparison to its competitors. 2.1 Construction method p
p
The FTSE4Good Index focuses on five key categories, including labor standards in the supply
chain, support for human rights, and environmental sustainability. bribery issues and tackling climate
change mitigation. ESG concerns are taken into account for both positive and negative screening, in
addition to theme factors (such as minimal carbon emissions). In comparison to the parent index, the
index's members will exclude from the index any firms involved in questionable business practices
or those that do not adhere to the UN Global Compact Principles. To ensure that the relative weighting
of constituents at the nation and sector level is equal to that in the parent index, components are also
weighted in accordance with FTSE's internal ESG score and thematic risk ratings (such as carbon
emissions). By rating 20,000 of the most regularly traded public firms based on these disclosures in four
categories—environmental, social, governance, and ESG composite scores—the Bloomberg ESG
Index pushes businesses to publish more ESG data. The eligible constituents of the Large/MidCap
ESG Index, which covers global and regional markets, respectively, are used to create the S&P Global
Large/MidCap ESG Index and the Emerging Large/MidCap ESG Index. The eligible constituents are
chosen by evaluating the business practices of the companies and/or their scores in accordance with
the UN Global Compact Principles, as well as by excluding the companies in each sector with the
lowest quartile of S&P's internal metrics. A target of 75% of the outstanding market capitalization of
eligible constituents in each sector is then selected from the remaining candidates, weighted by market
capitalization and chosen in descending order of internal ESG score. 2.2 ESG Index Construction Process Currently, the creation of an international ESG stock index typically involves five steps. A
benchmark index is chosen in the first stage, and the benchmark index's sample stocks are utilized as
the starting reference. The second stage involves "scanning" the sample stocks for negativity based
on the sample businesses' industry, the prevalence of contentious problems like human rights
concerns, and other factors. The final ESG index sample is chosen in the third phase, which involves
evaluating the ESG scores by combining the pertinent components, ranking them, and rescreening
them in accordance with the various aims of the index design. The weighting of sample stocks is done
in the fourth phase, and the weighting guidelines are often established under circumstances like the
goal of the index design, geographic distribution, and compatibility with the benchmark index. To
accomplish the proper index design goal, three weighting strategies are typically used: weighting by
market capitalization, equal weighting, and skewing towards certain metrics. The regular upkeep,
revision, and updating of the index constitute the fifth step. 2.3 Case Study - MSCI ESG Leaders Index The MSCI ESG Leaders Index is a leading ESG index that was introduced in 2020 with the goal
of drawing market attention to top-tier, leading ESG businesses and fostering ESG investment. Its
benchmark index is the "five-step" MSCI Global Investable Market Indexes (GIMI), which accounts
for 50% of the index's market capitalization. "Extremely controversial" items like biochemical and incendiary weapons, "seriously
controversial" items like nuclear missiles and related accessories, and "more controversial" items like
nuclear missiles and related accessories are all categorized in the negative screening process 177 BCP Business & Management
Volume 38 (2023) EMFRM 2022 according to the level of controversy in the business sector. All businesses engaged in the four
business levels mentioned above are not included in the MSCI ESG Leaders Index, also known as
the ESG Leaders Index. Businesses. according to the level of controversy in the business sector. All businesses engaged in the four
business levels mentioned above are not included in the MSCI ESG Leaders Index, also known as
the ESG Leaders Index. Businesses. The index demands that businesses have an ESG rating of BB or above (there are seven categories,
from AAA to CCC), as well as a controversy index score of 3 or higher (on a scale of 10, with a score
of zero being the most controversial). The four main themes for the environment are climate change,
natural resources, pollution and waste, and opportunities for development related to the environment
[13,14]. The four main themes for society are human resources, product responsibility, stakeholder
conflict, and opportunities for development related to social responsibility. The four main themes for
governance are corporate governance and corporate behavior. Additionally, the index considers ESG
trend scores, current index inclusion, and sector-adjusted ESG scores. The final index, which is
maintained and modified on a quarterly basis, is created by liquid market capitalization weighting the
index. 3.1 Differences in domestic and international ESG systems First off, China's ESG system is still in its infancy compared to other nations, and the climate for
information sharing and the quality of the data base are both lacking. There have been restrictions in
the independent study on firms' fulfillment of social responsibility or green development in recent
years, and China's ESG system still trails far behind that of other nations. The quantity and caliber of
corporate information disclosure are weak, and the disclosure structure for ESG-related information
is still unclear. Second, compared to other nations, China's base for data collecting is weak. Thirdly,
the data disclosed in China's social responsibility reports and other reports primarily focuses on
performance enhancement and hardly ever includes unfavorable data. Second, whereas ESG systems are mostly governed by the government in China, they are founded
on spontaneous movements in other nations. The public's spontaneous movement and the promotion
of non-profit organizations marked the beginning of the ESG system in the international sphere. Subsequently, internationally significant organizations, like the United Nations, started to develop
ESG-related principles and frameworks and to encourage the adoption of ESG disclosure standards
by national exchanges [13]. The early ESG system in China was created by the government issuing
pertinent papers, which was followed by a top-to-bottom promotion of ESG awareness among
businesses and the general public. The government still plays a significant role in directing the
development of an ESG system in China, despite the fact that numerous non-governmental
organizations in China also publish their own ESG ratings and investment recommendations [15,16]. 3.2 Suggestions for China's ESG system 3.2.1 Strengthening information disclosure capabilities 3.2.3 Improving ESG database construction Based on international experience, an effective ESG system has to be well backed by data. More
comprehensive fundamental data will result in more credible evaluation outcomes, especially in the
evaluation of ESG performance. The results are more trustworthy the more data there is available. Some organizations offer their processed data directly to the public, but the data base and data
processing remain mainly undisclosed. Processing ESG data is a complicated process that needs a lot
of time, effort, and resources, and it is regularly updated and maintained. The database is routinely
maintained and updated. Therefore, it is advised that the pertinent departments support the company
or institution in creating an ESG database as part of an overall ESG strategy. Therefore, it is advised
that the pertinent departments help businesses or institutions create their ESG database as part of an
overall ESG strategy. 3.2.2 Playing a government-led role In contrast to the growth of public movements in other nations, the government and pertinent
agencies should still direct the ESG system in China. In order to improve the corresponding
supporting policies and ultimately reflect the effect at the enterprise level, this necessitates ongoing
communication and cooperation between the industry itself, industry associations, and regulatory
authorities (including industry and commerce, taxation, environmental protection, finance, etc.)
[16,17]. Examples include preferential rules for bidding and tax exemptions, as well as access to
financial resources, for businesses (particularly listed businesses) with strong ESG performance
evaluations. Companies should be cautioned or penalized if they fail to provide the necessary
disclosures, provide false information, and perform badly during performance reviews. Information disclosure, performance evaluation, and investment advice are the three components
of the ESG system. These three components are interconnected and naturally interwoven. In addition
to aggressively promoting and improving the entire institutional framework of ESG in China,
government departments and pertinent institutions should increase their general awareness of ESG
ideas and systems. In particular, they should encourage industry associations and regulatory bodies
to establish ESG disclosure and evaluation systems, encourage rating agencies to actively carry out
ESG performance ratings of businesses and make them public in a timely manner, support businesses
in implementing ESG principles in their operations, and increase investor awareness of ESG
principles and responsible investment. 3.2.1 Strengthening information disclosure capabilities Globally speaking, ESG-related disclosures are becoming more and more common in many
nations and areas. More than 30 nations have passed legislation requiring corporate ESG disclosure,
including important stock markets in Europe, the United States, Japan, and Singapore, as well as
South Africa, Brazil, Canada, India, Malaysia, Sri Lanka, Thailand, and Turkey. By 2030, the United
Nations mandates that all significant businesses produce sustainability reports. China can get more insight from the aforementioned experience with information disclosure in
developed capital markets and create a set of ESG information disclosure policies that are appropriate
for its own national circumstances. Additionally, it is recommended that the appropriate authorities
increase the standards for financial ESG reports, harmonize information disclosure guidelines,
standardize third-party certification of the reports, and gradually establish the principles, procedures,
and individuals in charge of third-party audits to ensure the reliability of the published reports. Furthermore, financial ESG disclosure has to be standardized. Information on the effects of financial 178 BCP Business & Management
Volume 38 (2023) EMFRM 2022 institutions' own operations, investment and financing activities, as well as the effects of ESG on their
possibilities and dangers, should also be included in financial ESG disclosure. institutions' own operations, investment and financing activities, as well as the effects of ESG on their
possibilities and dangers, should also be included in financial ESG disclosure. References [1] Friede, G., M. Lewis, A. Bassen, T. Busch, ESG and Financial Performance: Aggregated Evidence from
More than 2000 Empirical Studies, Journal of Sustainable Finance and Investment, 2015. [2] Atan, R., M. Alam, J. Said, and M. Zamri, The Impacts of Environmental, Social, and Governance Factors
on Firm Performance: Panel Study of Malaysian Companies, Management of Environmental Quality: An
International Journal, 2018, 29(2), 182-194. [3] Aupperle, K., A. Carroll, and J. Hatfield, An Empirical Examination of the Relationship between
Corporate Social Responsibility and Profitability, Academy of Management Journal,1985, 28(2), 446-
463. [4] Blank H., S. Sgambati, and Z. Truelson, Best Practices in ESG Investing. The Joumal of Investing, 2016,
25 (2), 103-112. [5] Brooks, C. and I. Oikonomou, The Effects of Environmental, Social and Governance Disclosures and
Performance on Firm Value: A Review of the Literature in Accounting and Finance, The British
Accounting Review, 2018, 50(1), 1-142. [6] Clark, G., F. Andreas, and V. Michael, From the Stockholder to the Stakeholder: How Sustainability Can
Drive Financial Outperformance, Arabesque Asset Management and Oxford University, 2015. [7] Coleman, L., K. Maheswaran, and S. Pinder, Narratives in Managers Corporate Finance Decisions,
Accounting & Finance, 2010, 50(3), 605-633. [8] Cucari N., S. Falco, and B. Orlando, Diversity of Board of Directors and Environmental Social
Governance: Evidence from Italian Listed Companies, Corporate Social Responsibility and
Environmental Management, 2018, 25(2), 250-266. [9] Fang Zhiyong, Green Credit Innovation Practices of Commercial Banks and Related Policy
Recommendations, Financial Supervision and Regulation Research, Vol. 6, 2016, 57-72. [10] Huang Zhijie, Implementation and Innovation of Corporate Governance and Capital Market Legal System,
Tsinghua University Press, 2013, 231-280. [11] Lu Zhengwei and Fang Qi, Financial Regulation and Green Financial Development: An Overview of
Practice and Research, Financial Regulation Research, Vol. 11, 2018, 1-13. [12] Luo Sheng and Yin Aichao, A study on governance risks of financial institutions based on the systemic
theory of corporate governance, Insurance Research, Vol. 12, No. 57, 2008. [13] Zhang Lin and Lian Yongke, Green Credit, Bank Heterogeneity and Bank Financial Performance,
Financial Supervision Research, Vol. 2, 2019, No. 43-61. [14] Cheung Kong Securities. Emerging investment concepts in China: The rise of ESG [R/OL]. Cheung Kong
Securities Research Report, 2018-06-21. https:/V/www.sohu.com/a/237046611_675170. [15] Jin Heisi. The experience of global ESG investment development and implications for China IV. Modern
Management Science, 2018(9). [16] He Zhiquan. Environmental, social and governance (ESG) investment and disclosure in China. BCP Business & Management
Volume 38 (2023) BCP Business & Management
Volume 38 (2023) BCP Business & Management
Volume 38 (2023) EMFRM 2022 4. Conclusion Environmental, social and governance (ESG) measures the three core elements of sustainability
and ethical impact in a company or corporate investment. ESG is also gaining attention in China,
where more and more companies are disclosing their social responsibility reports, but its development
is still in its infancy. The ESG Thematic Index, which integrates the three dimensions of
environmental, social and corporate governance, is an important tool for ethical investors to
participate in the ESG investment market, reflecting the ability of a sample of positions in
environmental protection, social responsibility and their own sustainable development. It is also an
important financial tool for directing social capital flows to sustainable development projects to
support sustainable economic development. The use of social capital as a financial resource to finance
sustainable development initiatives is essential to promote sustainable economic growth. Important
international ESG rating indices currently include the MSCIESG series, the FTSE4Good series and
the Bloomberg ESG Index. There is still a gap between the current Chinese ESG system and the
international one. China needs to strengthen its information disclosure capacity, play a leading role
in government, improve the ESG database construction and gradually improve the ESG system. 179 References China
Capital Finance and Accounting, 2015(34). [17] Chong Zhengwei, Tang Weiqi. ESG disclosure by listed companies. Assessment and application [R]. Industrial Research, 2017-04-04. 180
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“Leisure” spaces within work environments in the “Performance society”
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“Leisure” spaces within work environments
in the “Performance society”
Espaços de “lazer” em ambientes de trabalho
na “Sociedade de desempenho” Simone Jubert [I]
Lúcia Leitão [II] Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024
http://dx.doi.org/10.1590/2236-9996.2024-5909.e Introduction Richard Sennett, in Building and dwelling: Ethics
for the city (2018), recounts his experience of
visiting the Googleplex, in New York, remarking
that the Googleplex is "in the city but not of it"
(Sennett, 2018, p. 170). It is a space “meant to
be self-contained” (ibid., p. 171), in such a way
that staff do not need to leave the workplace
in order to engage in a wide range of activities,
such as attending a medical consultation or
even sleeping to relax after the extended
working day. In addition to this brief introduction, the
text is divided into two parts. In the first, the
concept of leisure is defined, and we present
a brief historical overview of the incorporation
of leisure spaces into the workplace. The
second part then examines how the idea
of surveillance in the “disciplinary society”
developed by Michel Foucault can be seen to
shape productivity, indicating the existence of
an orchestrated division of the representations
of power, in such a way that the surveillance
and control of individuals is exercised and
represented by a “social cell”. We then present
Deleuze’s notion of the “control society”
(1995), in which surveillance becomes virtual,
and then move on to Han’s concept of the
“performance society” (2015 and 2017), in
which self-exploitation is associated with a
sense of freedom. This discussion is essential
for understanding the novel contours that
surveillance has assumed in the contemporary
world and the way in which workers play an
active role in a phenomenon that appropriates
their non-work time by providing spaces for
leisure in the workplace. In addition to all the arguments in favor
of the provision of such spaces for leisure,
socializing and even sleeping,1 such facilities
have resulted in the Google complex becoming
the inspiration for the emergence of so-called
"creative class"2 offices around the world. In
Brazil, the offices of major companies, such as
Walmart (Figure 1), Unilever, Vivo (Figures 2
and 3), Serasa, OLX (Figure 4) and Locaweb, and
those of smaller companies, such as OutPromo,
SolveSystem, Arizona and the like, are also
incorporating spaces designed for leisure and
relaxation into the working environment. Abstracti This article proposes a reflection on the increasing
trend of integrating leisure spaces into work
environments. It begins with the hypothesis that
such spaces constitute a form of appropriation, by
employers, of their employees' non-working time. The conceptual references primarily stem from the
notions of Disciplinary Society (Foucault), involving
the idea of docile bodies shaped by surveillance,
and Performance Society (Han), demonstrating
how the experience within these spaces that
exploit employees' free time can easily be mistaken
for a sense of freedom. The article concludes
by highlighting the worker’s active participation
in their own exploitation and pointing out the
need to discuss the psychosocial implications
arising from this rapidly expanding practice within
contemporary capitalism. Este artigo propõe uma reflexão sobre a crescente
inserção de espaços de lazer em ambientes de tra
balho. Parte-se da hipótese de que esses espaços
constituem uma forma de apropriação, por parte
do empregador, do tempo de não trabalho de seus
empregados. As referências conceituais vêm, prin
cipalmente, das noções de “Sociedade disciplinar”
(Foucault), com a ideia de corpos dóceis conforma
dos pela vigilância, e de “Sociedade de desempe
nho” (Han), mostrando como a vivência nesses es
paços de exploração do tempo livre dos empregados
pode ser facilmente confundida com uma experiên
cia de liberdade. Conclui-se assinalando a participa
ção ativa do trabalhador na sua própria exploração
e apontando a necessidade de discutir as implica
ções psicossociais advindas dessa prática em franca
expansão no capitalismo contemporâneo. Palavras-chave: arquitetura e urbanismo; lazer;
trabalho; vigilância; desempenho. Keywords: architecture and urbanism; leisure;
work; surveillance; performance. Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024
http://dx.doi.org/10.1590/2236-9996.2024-5909.e Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024
http://dx.doi.org/10.1590/2236-9996.2024-5909.e Artigo publicado em Open Acess
Creative Commons Atribution Artigo publicado em Open Acess
Creative Commons Atribution Artigo publicado em Open Acess
Creative Commons Atribution Simone Jubert, Lúcia Leitão aim of the present study is to reflect on these
questions and to propose the hypothesis that
such environments constitute a subtle form of
appropriation, by employers, of the non-work
time of their employees. This hypothesis is
explored here using Foucault’s concept of the
“disciplinary society” (1995), and the notion of
the “performance society” developed by Han
(2015 and 2017). Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 Introduction What, however, are the implications,
of the inclusion of leisure spaces in the
workplace?3 And why, generally speaking, do
the production, proliferation and adoption of
such spaces tend not make workers feel that
their non-work time is being appropriated
by employers but are instead perceived as
motivational factors and benefits?4 The main 192 “Leisure” spaces within work environments... Source: Office Snapshots.5
Source: Maurício Grego/ Exame.6
Source: Galeria da Arquitetura.8
Source: Maurício Grego/Exame.7
Figura 1
Figura 2
Figura 3
Figura 4 Source: Maurício Grego/ Exame.6
Figura 2
Figura 4 Source: Office Snapshots.5
Figura 1
Figura 3 Figura 2 Source: Office Snapshots.5 Source: Maurício Grego/ Exame.6 Figura 3 Figura 4 Source: Galeria da Arquitetura.8
Source: Maurício Grego/Exame.7 Source: Galeria da Arquitetura.8 Source: Maurício Grego/Exame.7 Source: Maurício Grego/Exame.7 Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 Leisure of the community determined the cycles of
production. In other words, there was a direct
relation between nature and community, not
only influencing what communities produced
and when, but also how they produced it. It was, therefore, with the emergence of
industrial societies that a different way of
ordering time and a new relation to it came
into being. As a result, Corbin, in L’Avènement des Loisirs [The Advent
of Free Time] (1995), remarks that, up to the
19th century, work time was discontinuous,
in such a way that work was intertwined with
other everyday activities, and often liberally
interspersed with pauses and periods of free
time. The seasons of the year and the life 193 193 Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 Simone Jubert, Lúcia Leitão work to which men, women, the elderly and
children were subjected ended up motivating
the workers’ struggle for the eight-hour
working day, with eight hours of leisure and
eight hours of rest. The abuses perpetrated
by employers necessitated the creation of
guaranteed free time for all workers. Leisure,
as we now know it, is therefore a right that has
been won. [...] the modern problematization and
ideology of free time appeared between
the late 18th and early 19th centuries,
alongside the phenomenon of mass
urbanization, which was closely related
to industrial mechanization. Likewise,
the dramatic transformation effected
by the separation of housing from the
workplace – with the family ceasing to
function as the physical center of the
economy – played a significant role at
this time. [...] It is thus fair to say that
free time properly speaking, as opposed
to work time, bears specific traces,
characteristic of the civilization born of
the industrial revolution. The evolution
of free time has therefore reflected the
ensuing social struggles concerning labor
legislation, within capitalist society or
with a view to surpassing it. (Gaspar,
2003, pp. 104-105) [...] the modern problematization and
ideology of free time appeared between
the late 18th and early 19th centuries,
alongside the phenomenon of mass
urbanization, which was closely related
to industrial mechanization. Likewise,
the dramatic transformation effected
by the separation of housing from the
workplace – with the family ceasing to
function as the physical center of the
economy – played a significant role at
this time. Figure 5 – Confederation Generale du Travail (C.G.T)
poster advocating the eight-hour working day
Source: Doumenq Félix, 1919. Figure 5 – Confederation Generale du Travail (C.G.T)
poster advocating the eight-hour working day Figure 5 – Confederation Generale du Travail (C.G.T)
poster advocating the eight-hour working day gure 5 – Confederation Generale du Travail (C.G
poster advocating the eight-hour working day
Source: Doumenq Félix, 1919. Source: Doumenq Félix, 1919. The exploitation of the working classes
culminated in a series of stand-offs and
demands for improvements in working and
living conditions and generated a series of
responses on the part of society. Workers’
demands were subsequently examined in
accordance with the prevailing system of
rationality of the time, in order to provide
legitimacy for any possible changes. This clearly
shows how the special place that science
had come to occupy, along with changes in
architecture and urban planning, the value
accorded to data and statistics, and other
factors, all had a part to play in the rational
system that was under development at the
time, strengthening its position and causing After capital had taken centuries to
extend the working day to its normal
maximum limit, and then beyond this
to the limit of the natural day of 12
hours, there followed, with the birth of
largescale industry in the last third of
the eighteenth century, an avalanche of
violent and unmeasured encroachments. Every boundary set by morality and
nature, age and sex, day and night, was
broken down. Even the ideas of day and
night, which in the old statutes were of
peasant simplicity, became so confused
that an English judge, as late as 1860,
needed the penetration of an interpreter
of the Talmud to explain ‘judicially’ what
was day and what was night. Capital was
celebrating its orgies. (Marx, 2017, pp. 439-440) After capital had taken centuries to
extend the working day to its normal
maximum limit, and then beyond this
to the limit of the natural day of 12
hours, there followed, with the birth of
largescale industry in the last third of
the eighteenth century, an avalanche of
violent and unmeasured encroachments. Every boundary set by morality and
nature, age and sex, day and night, was
broken down. Even the ideas of day and
night, which in the old statutes were of
peasant simplicity, became so confused
that an English judge, as late as 1860,
needed the penetration of an interpreter
of the Talmud to explain ‘judicially’ what
was day and what was night. Leisure [...] It is thus fair to say that
free time properly speaking, as opposed
to work time, bears specific traces,
characteristic of the civilization born of
the industrial revolution. The evolution
of free time has therefore reflected the
ensuing social struggles concerning labor
legislation, within capitalist society or
with a view to surpassing it. (Gaspar,
2003, pp. 104-105) Dumazedier thus regards work as
being in fact essential for the very existence
of leisure as the result of a historical
relationship between work time and non-
work time. The present article adopts this
reasoning, defining leisure as non-work time
for the worker (that is nevertheless based
in principle on the existence of work), and
understanding it as an historical right to free
time that has been won. It was thus in the context of the Industrial
Revolution that the concept of leisure first
began to emerge. The French sociologist
Dumazedier, who is one of the foremost
authorities and pioneers in the field of the
sociology of leisure, has remarked that "leisure
is not idleness, it does not supplant work;
it presupposes it. It represents a periodic
liberation from work at the end of the day,
week, year, or working life" (1979, p. 28). The present article also accepts one
important criticism of Dumazedier’s position,
concerning the adoption of a functionalist
approach to leisure, seeing it as something
that serves as compensation for work. We are
interested principally here, however, in the way
leisure is understood as something achieved,
the fruit of a long process of workers’ struggles
for better living and working conditions,
without which employers would readily have
allowed work to occupy all their workers’
waking hours. Dumazedier’s view of the nature of
leisure derives from an understanding of the
fact that, while work time and non-work time
were, as Corbin and Gaspar both note, once
intertwined, with the Industrial Revolution,
work time came to hold sway over other kinds
of time, in such a way that the exhausting day’s The way in which work came to dominate
every minute of every day is clearly described
in a passage from Karl Marx’s Capital, first
published in September 1867: 194 194 Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 “Leisure” spaces within work environments... Figure 5 – Confederation Generale du Travail (C.G.T)
poster advocating the eight-hour working day
Source: Doumenq Félix, 1919. Capital was
celebrating its orgies. (Marx, 2017, pp. 439-440) 195 Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 Simone Jubert, Lúcia Leitão it to gain acceptance throughout society. This
would therefore be the prism through which
the idea of time free of work would come to be
examined and finally accepted over time. would generate self-intoxication. This also led
them to criticize excessive sporting activity
(ibid.). Thus, according to Corbin, it was now
understood to be wrong to see the body as a
machine, since the body is subject to the laws
of fatigue (ibid., p. 337). The struggle for a day divided into three
periods of eight hours – eight for work, eight
for rest, and eight for leisure – was one of the
demands that was especially carefully analyzed
in the light of scientific studies of the time. Scholars pored over figures relating to the gains
and losses expected to result from this model
of the working day, in an attempt to justify the
stern resistance of a significant sector of the
economic elite to any reduction to the length of
the working day. This period also saw the appearance
of studies of the capacity to resist fatigue
and the factors influencing this. The bad
habits studied included lack of sleep, a
poor diet, and being overweight, and it
was understood, while excessive physical
exercise caused fatigue, “training – which
was the subject of numerous experiments
– tended to increase the resistance of the
muscles and of the brain” (ibid., p. 338). New connections between work, rest and
fatigue were therefore identified, with
thresholds established for each, taking into
consideration that fact that each kind of
activity has the capacity to become harmful
in certain contexts. “As a whole, this research
activity provided a scientific basis for the
need for a periodic restoration of strength”
(ibid.) and consequently justified examination
of specific studies of industrial fatigue. This
led to the emergence of what Corbin calls
(ibid.), “a period of rational management of
human effort” (p. 339), based on studies of
fatigue. The roots of this intensive scientific
activity are various. Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 Figure 5 – Confederation Generale du Travail (C.G.T)
poster advocating the eight-hour working day
Source: Doumenq Félix, 1919. Research was
spurred by a desire to use science
to justify the demand for and the
introduction of the ‘three eights’ – which
even socialist theorists had proved
incapable of bringing about [...] There
was a widespread general impression
that surmenage9 [overwork] was one of
the many scourges that blighted the late
19th century. In that age of a veritable
“medical coup d’état,” facilitated by
the success of Pasteur’s theories,
physiologists and psychologists found, in
the study of fatigue, a way of bolstering
the authority of their message and
extending their influence. (Corbin et al.,
2001, p. 336) Corbin also notes that the First World
War sparked numerous studies of industrial
fatigue, principally among the Allies. He
goes on to remark that it would be fair to
describe the post-World War I period as “the
golden age of this new branch of knowledge,
especially on the North American side of the
Atlantic” (ibid., p. 341). It should be borne Corbin notes that, in the late 19th century
and early 20th century, a series of studies of
fatigue demonstrated that “it is a chemical
process that affects the body as a whole and not
just the organ that appears to affected” (ibid.). The researchers of the time believed that any
prolonged period of intensive muscular effort 196 196 Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 “Leisure” spaces within work environments... in mind, however, that all of this activity and
interest surrounding fatigue aimed to provide
improved understanding of the mechanisms
of the human body in relation to its limits
as a way of augmenting the productivity of
workers. Fatigue researchers thus became
involved in a wide range of fields, including
studies of sensory fatigue, such as that
produced by noise, studies of the need for
sleep (generating greater appreciation for this,
since tiredness can affect sleeping and reduce
productivity on the following day), and studies
devoted to the psychological and physiological
effects of work. Ensuring that there is time for meals and
breaks at work led to the creation within factories
of leisure spaces such as cafeterias, refectories,
canteens, and restrooms. This preceded the
creation of such spaces in the offices of white-
collar workers. However, according to a study
conducted by Resende (2018), it was in the latter
that leisure spaces would eventually develop at a
much rapider pace. Figure 5 – Confederation Generale du Travail (C.G.T)
poster advocating the eight-hour working day
Source: Doumenq Félix, 1919. Resende notes that leisure spaces were
already being incorporated into workplaces
in the late 19th century, in office blocks
and company campuses. These included
skyscrapers, such as the Pullman Building in
Chicago, designed in 1883 by Solon S. Beman,
for the Pullman Palace Car Company, and Frank
Lloyd Wright’s 1904 Larkin Building, in New
York, along with company campuses, such
those of AT&T (built in 1941, with additional
buildings constructed in 1974), in Murray
Hills, New Jersey, and Connecticut General
Life Insurance Company (1954), in Bloomfield,
Connecticut, designed by S.O.M. under the
influence of Mies Van der Rohe. The scientific debate and discussion
explicitly referred to the process of legitimation
of practices, narratives, and consensuses. It is
therefore fair to say that between 1870 and
1914, the issue of fatigue featured widely in
scientific studies, paving the way for acceptance
within society of the right to time free of work
and setting in motion processes that would
result in the creation of periods of time that we
now call leisure time (ibid., p. 345), such as the
weekends, paid holidays, and other benefits. These spaces contained a leisure
infrastructure designed for workers and
their families, with facilities such as libraries,
restaurants, bowling alleys, and, in some
cases, housing. Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 Leisure at work Awareness that the body is not a machine and
that lack of rest may harm productivity and
hence reduce profits led to the development
of a series of scientific studies within the
workplace, principally in the fields of
psychology, ergonomics, and sociology, that
indicated the benefits of leisure time for
productivity and staff motivation. According to Resende, some offices
moved from the city to out-of-town campuses
and business parks because cities to some
extent no longer catered for the new spatial
needs of businesses. Nor, in the post-World
War II period, were they able to cope with the
increased road traffic and overpopulation in
city centers, with all the disadvantages these 197 197 Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 Simone Jubert, Lúcia Leitão bring, leading companies to move to out-
of-town green areas and suburbs, as a way
of finding peace and quiet and encouraging
greater interaction among employees of the
same company. Examination of the history of workplaces
and the leisure spaces contained within them
reveals that, although such spaces were
intended to enhance the well-being and the
social integration of workers and to provide
breaks for rest, the true purpose was to create
a healthy workforce even more willing to work. Alongside this movement to the
periphery, there was also rapid
development of information and
communications technologies,
establishing new paradigms in society
and the world of work, some of which
had already been included in the
functioning of such buildings. This was,
as Peter Drucker (2002) called it, for the
first time, in 1959, the beginning of the
Knowledge Society, which would have
organizational and spatial implications
for workplaces, in particular office
spaces. (Ibid., p. 73) a healthy workforce even more willing to work. In the 1980s, however, new smaller
business campuses surrounding universities
began to appear in the United States. At this
time, most office work now involved working
in “cubicles”, since these constituted the least
expensive, most accessible, most flexible,
and most easily reproduceable option. Most
workplaces thus still depended heavily on
organizational culture for the provision of
leisure. The idea of the campus, however,
combined with the experience of young
researchers and workers—many of them still
at college or recently graduated—provided
this class of workers with workplaces that
were replete with the leisure and recreational
facilities typical of a university, including sofas,
games rooms, gymnasia and so forth. Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 Leisure at work Social activities
similar to those at universities, such as
picnics, barbecues, and afternoon teas,
were also introduced. History shows that the production
of leisure spaces within workplaces has
shifted from a more formal, hierarchical,
corporative mentality to one that is more
informal, horizontal and focused on the needs
of individuals. Gradual appropriation of the
world of leisure by the world of work has
occurred in tandem with technological and
economic transformations, and this has led
to the emergence of new kinds of workplaces
that incorporate leisure spaces, indicating
the emergence of a new stage of capitalism. Apart from increased productivity, employee
motivation is a common argument advanced
by employers to justify the adoption of such
measures. In a study conducted by Resende
(2018), employees testified that the existence
of leisure spaces in the workplace History shows that the production
of leisure spaces within workplaces has
shifted from a more formal, hierarchical,
corporative mentality to one that is more
informal, horizontal and focused on the needs
of individuals. Gradual appropriation of the
world of leisure by the world of work has
occurred in tandem with technological and
economic transformations, and this has led
to the emergence of new kinds of workplaces
that incorporate leisure spaces, indicating
the emergence of a new stage of capitalism. Apart from increased productivity employee It is interesting to note that, little by
little, the spaces dedicated to leisure within
workspaces came to be absorbed more by
way of entropy, in so far as they broke down
the boundaries between the two categories
of space. It thus became increasingly common
for workspaces to contain not only areas set
aside for leisure but also leisure activities
installed within workspaces, with computers
and workstations interspersed with pool tables,
foosball tables, pinball machines, and other
leisure facilities. Apart from increased productivity, employee
motivation is a common argument advanced
by employers to justify the adoption of such
measures. In a study conducted by Resende
(2018), employees testified that the existence
of leisure spaces in the workplace The tone of informality that came to
imbue workspaces, flexible working hours,
and the possibility of remote working from a
‘home office,’ with "leisure as much a cause
as a consequence of longer hours being spent
at the office" (ibid.) reveals a clear tendency
for the worlds of work and leisure to overlap. Leisure at work Other
smaller companies and laboratories were
connected to this ecosystem and there was a
climate of constant interchange. Workers in the
emerging society of knowledge tended, at that
time, to hail from a privileged background, and
the much-vaunted idea that a multi-million-
dollar business could start out in a garage is
largely a myth (Avendaño, 2014). Drucker’s belief that "the real and
controlling resource and the absolutely decisive
'factor of production' is now neither capital, nor
land, nor labor…[but]… knowledge" (Drucker,
1993, p. 15) reveals this new paradigm of work,
as reflected in the production of spaces based
on new production processes. New working practices were also
established. In the 1960s, there was an
increasing emphasis on “organizational
culture,” which systematized rewards and
shaped the spirit, the environment, and values
of an organization (Resende, 2018, p. 77). This
“organizational culture” aimed to encourage
employees to develop as sense of belonging
to and feeling at home in the company as if
it were a family. In addition to the provision
of physical leisure infrastructure, systematic
efforts were undertaken to create an equally
conducive emotional environment. When Internet companies began to
emerge, open-plan offices became more
popular. These were open indoor areas, with no
fixed predefined workstations, that encouraged
a constant exchange of ideas. According to
Resende (2018, p. 87): 198 Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 “Leisure” spaces within work environments... and non-work time intertwined in accordance
with the cycle of the seasons. Now, the
intertwining of work and leisure is governed
by the need for speed and efficiency. Work
and leisure are merged together for the
purpose, ultimately, of ensuring that work
can be accomplished and goals achieved. The
success that has been experienced and the
profits made by companies in Silicon Valley
from the 1990s onwards has inspired other
employers to do their best to replicate the
same spatial formula. Companies once again started to value
the possibility of chance meetings of
different people to exchange ideas. This spontaneity was associated
with diversion, as a social factor, and
companies thus began to break down
the boundaries between work and
leisure in the workplace. The spaces
provided by such firms were far superior
to those of other companies or even
of universities, and it was not unusual
to find foosball tables, basketball,
volleyball, tennis and racquetball courts,
football pitches, swimming pools and
recreation centers. helped to consolidate interpersonal
relations [...], to enhance physical and
mental well-being, to develop cognitive
and creative abilities, and to increase
productivity, [...] in addition to improving
the quality of life both at work and
outside of the workplace. (ibid., p. 167) helped to consolidate interpersonal
relations [...], to enhance physical and
mental well-being, to develop cognitive
and creative abilities, and to increase
productivity, [...] in addition to improving
the quality of life both at work and
outside of the workplace. (ibid., p. 167) Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 Productivity through
surveillance The image of the panopticon proposed
by Jeremy Bentham perfectly sums up the idea
of surveillance in a disciplinary society. This led
Foucault to conclude that "visibility is a trap". Its architectural composition involves, One key to a fuller understanding of why
workers submit voluntarily to the encroachment
of work time on their non-work time in the
contemporary world and react positively to the
phenomenon of leisure spaces located within
the workplace may be found in the work of
Michel Foucault. In particular in Vigiar e punir
(2018), Foucault identified the methods used
to subjugate, coerce and punish the human
body throughout history, describing the system
of power relations that prevailed each period,
the ramifications of the discourse used, and the
development of mechanisms to achieve their
objectives. For Foucault, a study of the control
of human bodies in western countries reveals
clearly discriminable periods during which certain
discourses of power hold sway. These discourses
are, in turn, reflected in the institutions, politics
and forms of policing established, and also in
architecture and urban planning at the periphery, an annular building;
at the centre, a tower [...]. All that is
needed, then, is to place a supervisor in a
central tower and to shut up in each cell
a madman, a patient, a condemned man,
a worker or a schoolboy... so many cages,
so many small theatres, in which each
actor is alone, perfectly individualized
and constantly visible. The panoptic
mechanism arranges spatial unities that
make it possible to see constantly and to
recognize immediately. (ibid., p. 194). In the transition from sovereign to
disciplinary societies, different forms of
organization of power coexisted, from the still
prevalent "ancient rights of the monarchy"
to others that had a "preventive, utilitarian,
corrective conception of a right to punish that
belongs to society as a whole". One of these
forms of organization of power involved the
establishment of a "punitive city," Discipline and Punish first presents the
history of the body subjected to torture in
“sovereign societies” (these being societies
that go back deep into human history and are
centered on the figure of a single sovereign). Such societies prevailed up until the Industrial
Revolution, when there was a transition to
‘disciplinary societies’ based on a system
intended to make human bodies docile. Leisure at work Things were no longer the way they were prior
to the Industrial Revolution, when work time 199 199 Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 Simone Jubert, Lúcia Leitão in public squares so as to provide visibility for
the prevailing power structure, in disciplinary
societies, there is an orchestrated division of
representations of power, resulting in control
being more evenly distributed – and exercised
and represented by ‘social cells’. How then did it come about that the
appropriation of leisure time by work is seen as
a cause of satisfaction by some employees? Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 Productivity through
surveillance While,
in sovereign societies, punishment and control
of bodies involved subjecting them to torture [...] a functioning of penal power,
distributed throughout the social
space; present everywhere as scene,
spectacle, sign, discourse; legible like an
open book; operating by a permanent
recodification of the mind of the citizens;
eliminating crime by those obstacles
placed before the idea of crime; acting
invisibly and uselessly on the ‘soft fibres Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 200 “Leisure” spaces within work environments... The control and discipline imposed on
bodies led Foucault to develop the notion of
biopower. In his course at the Collège de France
in 1978, titled Sécurité, territoire, population
(Security, territory, population), Foucault
defines biopower as “the set of mechanisms
through which the basic biological features
of the human species became the object
of a political strategy, of a general strategy
of power” (Foucault, 2008, p. 3). Biopower
generates biopolitics, which, Foucault argues
is “what brought life and its mechanisms into
the realm of explicit calculations and made
knowledge-power an agent of transformation
of human life” (Foucault, 1988, p. 134). Revel
further clarifies the concept of biopolitics to
the effect that “while discipline takes the form
of a political anatomy applied to bodies and is
applied essentially to individuals, biopolitics
represents social medicine on a large scale
applied to the population as a way of governing
life: life thus forms part of the field of power”
(2005, p. 27). of the brain’, as Servan put it. A power
to punish that ran the whole length of
the social network would act at each
of its points, and in the end would
no longer be perceived as a power of
certain individuals over others, but as an
immediate reaction of all in relation to
the individual. (Ibid., p. 129) The kind of surveillance involved in the
idea of the panopticon and punitive power
shared by the whole social fabric molded the
thinking and institutions of the time. Foucault
argues that surveillance for the purpose of
creating docile bodies10 came to pervade the
whole social fabric and all social activities,
taking concrete form in ‘apparatuses’.11
Architecture and urban planning themselves
should thus be seen as tools of surveillance
in the disciplinary society, 12 molding
localities in conformity with the interests of
governmentality and ensuring that bodies are
made docile. Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 The control society ‘dividuals’ and masses become samples,
data, markets, or ‘banks.’ Money, perhaps,
best expresses the difference between
the two kinds of society, since discipline
was always related to molded currencies
containing gold as a numerical standard,
whereas control is based on floating
exchange rates, modulations depending
on a code setting sample percentages for
various currencies. (ibid., p. 222) The technological (r)evolution following the
Second World War and the wave of revolutions
that came in its wake exercised a decisive
influence on the way western societies are
organized. As well as providing new forms of
entertainment, the broad dissemination of
video technology and easier access to it have
expanded the scope of the panopticon. The
disciplinary society now has information and
communications technologies at its disposal
and can use mass communications to make
bodies docile, helping to effect radical changes
in the way urban spaces are occupied, causing
some parts to fall into decline, while others
become more densely populated. The advent
of the Internet simultaneously created a new
and radical network of invisible ties within the
city; the panopticon could now be installed
in the virtual world, and individuals induced
voluntarily to provide the tracks through which
they can be surveilled. The overlapping discourses in
control societies address new relations and
practices regarding space and time, and the
virtualization, which began in disciplinary
societies, reaches new levels. People no longer
need to go to the bank or the post office to
make payments, videogames can now be
played by multiple players at the same time
in various places around the world, political
propaganda is relayed by social networks,
security cameras observe and record the
private lives of individuals and track their
movements and behavior. It is in this control society that we have
come to be monitored by surveillance cameras
at work and in which virtual environments are
extensively used. De Masi expresses a certain
optimism regarding the new urban order effected
by a shift from metropolis to ‘telepolis’ and he
sees the virtualization of processes as something
fresh and new. "Built entirely on the concept of
living and working at a distance, the more the
inhabitants of Telepolis stay at home working
and consuming remotely, blurring the boundaries
between work, home, social life, production,
reproduction, and entertainment, the more the
city bustles with activity" (1999, p. Productivity through
surveillance According to Foucault, we thus
live in a “carceral continuum” (ibid., p. 298), in
which we are led from one prison to another. The concepts of biopolitics and
biopower together help shed light on how the
disciplinary society has been modified and
perfected in relation to its mission of producing
docile bodies, coming eventually to focus on
the individual and a form of surveillance that
is increasingly diffused throughout society, but
also increasingly heavier and more insistently
present. Thus, through a kind of refinement
of the disciplinary society, surveillance would
appear already to be rooted in the subject –,
and, at this point, it can already be seen that
entrenched surveillance is one of the root
causes of the appropriation of non-work time
by work. Bodies can supposedly be made docile in
this way at an early age in school, in communal
spaces, in college, in institutions, or at work
in a factory or an office. The production of
space as a whole in a disciplinary society aims
to control the bodies of individuals in such a
way that spaces are readapted in accordance
with the precepts of the disciplinary system. Hospitals are thus divided into wings,
schools have rows of desks, factories have
workstations. And it because of this need for
discipline that spaces in school, at work, in
hospitals, in army barracks and so forth come
to resemble one another. 201 201 Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 Simone Jubert, Lúcia Leitão [...] the key thing is no longer a signature
or number but a code: codes are
passwords, whereas disciplinary societies
are ruled (when it comes to integration
or resistance) […] Individuals become Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 The control society Above all,
it is marked by the decline of some of
the basic institutions of modern society
– such as the school, the factory, the
prison, and the hospital, including the
home designed to accommodate a
nuclear family on the bourgeois model. On the other hand, complementary to
this, the private company has become a
kind of primary inspiration and example
for all other institutions, including, and
perhaps fundamentally, the most up-to-
date versions of these modern fossils. By infecting them with an omnipresent
"entrepreneurial spirit", they have
forever transformed these organizations
so as to make them increasingly
compatible with the pace and demands
of contemporary society". (Bruno et al.,
2018, pp. 208-209) deploys ‘should’. After a certain point
of productivity, ‘should’ reaches a limit. To increase productivity, it is replaced
by ‘can’. The call for motivation,
initiative, and projects exploits more
effectively than whips and commands. As an entrepreneur of the self, the
achievement-subject is free insofar
as he or she is not subjugated to a
commanding and exploiting Other. However, the subject is still not really
free because he or she now engages
in self-exploitation— and does so
of his or her own free will… Auto-
exploitation proves much more efficient
than allo-exploitation because it is
accompanied by a feeling of liberty. This
makes possible exploitation without
domination. (Han, 2020a, p. 21) The popular idea that being an
entrepreneur is an easy solution or one
associated with freedom aptly expresses the
rationale underlying the performance society. The home office, coworking, hackathons,
design sprints, and other fads of modern
entrepreneurial culture reveal a pattern of
self-exploitation. Modern offices contain pool
tables, hammocks, and videogame machines. There is now a 24/7 regime, in which the
individual can work at any time, on any day, as
they see fit. The control society 216) In 1990, Gilles Deleuze suggested that a
shift was underway from a disciplinary society
to one of control. As he put it, “we're moving
away from disciplinary societies, we've already
left them behind. We're moving toward control
societies that no longer operate by confining
people but through continuous control and
instant communication" (2008, p. 216). Deleuze
argues that “disciplinary societies have two
poles: signatures standing for individuals, and
numbers or places in a register standing for their
position in a mass" while, in "control societies", [...] the key thing is no longer a signature
or number but a code: codes are
passwords, whereas disciplinary societies
are ruled (when it comes to integration
or resistance) […] Individuals become In the shift from the disciplinary society
to the society of control, old disciplinary
structures are being rapidly transformed,
making way for Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 202 “Leisure” spaces within work environments... [...] a system governed by excess
p r o d u c t i o n a n d e x a g g e r a t e d
consumption, by marketing and
publicity, flows of capital in real
time, interconnectivity, and global
communications networks. Above all,
it is marked by the decline of some of
the basic institutions of modern society
– such as the school, the factory, the
prison, and the hospital, including the
home designed to accommodate a
nuclear family on the bourgeois model. On the other hand, complementary to
this, the private company has become a
kind of primary inspiration and example
for all other institutions, including, and
perhaps fundamentally, the most up-to-
date versions of these modern fossils. By infecting them with an omnipresent
"entrepreneurial spirit", they have
forever transformed these organizations
so as to make them increasingly
compatible with the pace and demands
of contemporary society". (Bruno et al.,
2018, pp. 208-209) [...] a system governed by excess
p r o d u c t i o n a n d e x a g g e r a t e d
consumption, by marketing and
publicity, flows of capital in real
time, interconnectivity, and global
communications networks. The performance society More recent phenomena in the fields
of communications, technology, market
economics, and the financial market, suggest
that discourses of power are increasingly
being shaped differently. Such discourses are
seen to pervade social and cultural relations,
redirecting efforts and policy towards a new
social logic, in which the "spirit of enterprise"
holds sway and in which surveillance is finally
inculcated in the very subjectivity of the
individual. This is what Byung-Chul Han has
called the performance society, arguing that
such a society This apparently harks back to the
time when free time and work were closely
intertwined, in so far as they were intrinsically
linked to everyday life and leisure spilt over
into the working day. It can, however, be
seen from the passage cited above that, in
the performance society, leisure and liberty
operate as work in disguise. The existence
within the workplace of spaces and facilities
designed for leisure and use during free time
causes individuals to spend more time engaged [...] is wholly dominated by the modal
verb ‘can’– in contrast to disciplinary
society, which issues prohibitions and Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 203 Simone Jubert, Lúcia Leitão of boundaries. When one is not told what
one “should” do, but only what one “can,”
spaces hold the power to retain us for longer,
with all the potentially negative or positive
consequences this may entail. in productive labor, motivated by the periods
of time during which they believe that they are
not working. In this state of constant production
and utilitarianism, what role does leisure
play? Boundaries are frayed and begin to
break down. Dividing lines are blurred. In the
performance society, every space is a frontier. Everything begins to make itself present as a
possibility. The apparent freedom provided by new
contemporary forms of work, characterized by
informality and the blurring of the difference
between times and spaces set aside for leisure
and those intended for work, is worn as a
badge of distinction by those classes of worker
who have access to such spaces. It provides
employees with an identity that associates
both them and the company they work for with
ideas of creativity and modernity. Now that leisure is understood as a right
that has already been won, it is worth asking
whether time free of work can nowadays be
rightly seen as leisure time. The performance society 191-208, jan/abr 2024 “Leisure” spaces within work environments... The discourse relating to flexibility,
liberty, and informality thus masks continuous
surveillance of the leisure time spent within
the workplace, carried out with the full and
unrestricted cooperation of the employee. life, shaping and regulating, through mass
communications and consumerism (which
exist in a feedback relation to one another),
the desires and identities of individuals. The
identification of workers with their jobs forms
part of a sophisticated technology of power
that has inculcated control by the employer,
or, through self-employment, the very figure
of the employer, into the unconscious of the
employee. Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 The performance society Leisure as a right
won by the working class, as a time set aside
during the day and in the calendar for rest
and for their own private pastimes, no longer
seems to serve the same function. The ideas
of productivity and high performance that
individuals have internalized and on which
their salaries depend are so unrelenting that
the right to time free of work seems no longer
to be guaranteed. The exploitation of labor develops a new
facet, when, in addition to generating
wealth through the productive force of
labor, it also relates "its products" with
the identity of the worker, including the
dynamics of biopolitical exploitation
of labor [...] This new process of
determining value effects a shift from
quantitative economic measurement
to a more subjective approach, since
the value of intangible factors is linked
to factors related to the construction of
ideologies among those operating within
this new mode of exploitation. (Souza,
Avelino, and Silveira, 2018, pp. 108-109) Flexibility has become a byword within
the logic of the performance society, in which
“can” trumps “should”, and this leads to a
blurring of the boundaries between work
and non-work, to the effect that these two
spheres are constantly intertwined and the
right to leisure no longer exists. Although
workers may have a formal contract, the logic
of performance persists and pervades the
culture of working life in the contemporary
world, requiring, sometimes despite
appearances, total dedication and the highest
possible degree of productivity on the part of
employees. The inclusion of leisure spaces in the
workplace thus implies that to heighten productivity, the paradigm of
disciplination is replaced by the paradigm
of achievement, or, in other words, by
the positive scheme of ‘can’; after a
certain level of productivity obtains, the
negativity of prohibition impedes further
expansion. The positivity of ‘can’ is much
more efficient than the negativity of
‘should’ (Han, 2020b). to heighten productivity, the paradigm of
disciplination is replaced by the paradigm
of achievement, or, in other words, by
the positive scheme of ‘can’; after a
certain level of productivity obtains, the
negativity of prohibition impedes further
expansion. The positivity of ‘can’ is much
more efficient than the negativity of
‘should’ (Han, 2020b). Spaces have come to reflect this new
modus operandi, which goes beyond a
conceptual hybrid and amounts to a loosening 204 Cad. Metrop., São Paulo, v. 26, n. 59, pp. [II] https://orcid.org/0000-0003-4935-2077 Universidade Federal de Pernambuco, Departamento de Arquitetura e Urbanismo, Programa de
Pós-Graduação em Desenvolvimento Urbano. Recife, PE/Brasil. lucia.leitao@ufpe.br Translation: this article was translated from Portuguese to English by Peter Ratcliffe, email: peter@
sharingenglish.com.br [I] https://orcid.org/0000-0001-9871-8536 [I] https://orcid.org/0000-0001-9871-8536
Universidade Federal de Pernambuco, Departamento de Arquitetura e Urbanismo, Programa de
Pós-Graduação em Desenvolvimento Urbano. Recife, PE/Brasil. simone.jubert@ufpe.br Universidade Federal de Pernambuco, Departamento de Arquitetura e Urbanismo, Programa de
Pós-Graduação em Desenvolvimento Urbano. Recife, PE/Brasil. simone.jubert@ufpe.br Notes (1) The arguments used by employers to justify the adoption of such spaces include, according to
newspaper articles on the phenomenon in Brazil (Ferreira, 2014; Formiga, 2015; Grego, 2018;
Melo, 2014; Pivetti, 2017), a desire to foster creativity and communication, and to enhance the
well-being and productivity of employees. (1) The arguments used by employers to justify the adoption of such spaces include, according to
newspaper articles on the phenomenon in Brazil (Ferreira, 2014; Formiga, 2015; Grego, 2018;
Melo, 2014; Pivetti, 2017), a desire to foster creativity and communication, and to enhance the
well-being and productivity of employees. (2) An expression coined by Florida (2011) for those who work in the field of software, games, audiovisual
production, music, media, editing, fashion, publicity and so forth. Florida wrote about the growing
role of the creative classes in the contemporary world, believing that cities could benefit from the
promotion of cultural policy, without discussing the gentrification of these areas that may result. (2) An expression coined by Florida (2011) for those who work in the field of software, games, audiovisual
production, music, media, editing, fashion, publicity and so forth. Florida wrote about the growing
role of the creative classes in the contemporary world, believing that cities could benefit from the
promotion of cultural policy, without discussing the gentrification of these areas that may result. (3) In academic work of a conceptual nature, Duerden, Courtright and Widmer (2017) have attempted
to shed light on the phenomenon by understanding the incorporation of leisure into the workplace
as providing a greater opportunity for individuals and groups to fully realize their potential in
organizations. (3) In academic work of a conceptual nature, Duerden, Courtright and Widmer (2017) have attempted
to shed light on the phenomenon by understanding the incorporation of leisure into the workplace
as providing a greater opportunity for individuals and groups to fully realize their potential in
organizations. (3) In academic work of a conceptual nature, Duerden, Courtright and Widmer (2017) have attempted
to shed light on the phenomenon by understanding the incorporation of leisure into the workplace
as providing a greater opportunity for individuals and groups to fully realize their potential in
organizations. Conclusion Spaces dedicated to leisure and free time
exist because of the need for a break from the
world of work, to move from the condition of
an individual as a producer of labor to one of
being a consumer, engaging in cultural activities
or experiencing contact with nature – that non-
-built space that is a relic of times when there
was greater symbiosis with the environment. This not only leads us to question the
appropriation of non-work leisure time by
work and the way individuals are trapped in
the bubbles of mutual coexistence that offices
have increasingly become, but also shows
how the workers involved in this process of
appropriation of free time are alienated from
the city in which they live. This reinforces
practices that distance people from social
contact with a variety of different kinds of
people in the public sphere and demobilizes
individuals in relation to the occupation of
urban space and the demand for the right to
the city. It thereby also demobilizes them in
relation to demands for more and better public
leisure spaces, in which individuals (albeit still
under surveillance) are truly in control of their
own non-work time. The appropriation of non-work time by
work through the production of leisure spaces
within areas designated for work is a dynamic
that involves the participation and collaboration
of the employee. The contemporary stage of
capitalism has been established gradually over
centuries of surveillance and control of bodies,
spreading out slowly through the social fabric. It has thus increasingly come to overlap with
everyday life in all spheres, including private 205 Simone Jubert, Lúcia Leitão Notes (4) Resende (2018) presents the findings of a study of individuals working in companies that have
incorporated leisure spaces into the workplace and shows that workers associate the existence of
such spaces with heightened motivation and increased productivity, as well as a greater sense of
attachment to the goals and values of the company for which they work. (4) Resende (2018) presents the findings of a study of individuals working in companies that have
incorporated leisure spaces into the workplace and shows that workers associate the existence of
such spaces with heightened motivation and increased productivity, as well as a greater sense of
attachment to the goals and values of the company for which they work. (5) Retrieved from https://officesnapshots.com/2014/02/18/inside-walmart-coms-sao-paulo-offices/. Access: July 9, 2019. (5) Retrieved from https://officesnapshots.com/2014/02/18/inside-walmart-coms-sao-paulo-offices/. Access: July 9, 2019. (6) Retrieved from https://exame.abril.com.br/negocios/por-dentro-do-arrojado-laboratorio-de-
inovacao-da-vivo/. Access: July 9, 2019. (6) Retrieved from https://exame.abril.com.br/negocios/por-dentro-do-arrojado-laboratorio-de-
inovacao-da-vivo/. Access: July 9, 2019. (7) Retrieved from https://exame.abril.com.br/negocios/por-dentro-do-arrojado-laboratorio-de-
inovacao-da-vivo/. Access: July 9, 2019. (7) Retrieved from https://exame.abril.com.br/negocios/por-dentro-do-arrojado-laboratorio-de-
inovacao-da-vivo/. Access: July 9, 2019. 206 Cad. Metrop., São Paulo, v. 26, n. 59, pp. 191-208, jan/abr 2024 “Leisure” spaces within work environments... (8) Retrieved from https://www.galeriadaarquitetura.com.br/projetos/referencias-ambientes-c/132/
salas-de-descompressao/. Access: July 9, 2019. (8) Retrieved from https://www.galeriadaarquitetura.com.br/projetos/referencias-ambientes-c/132/
salas-de-descompressao/. Access: July 9, 2019. (9) The Littré French dictionary defines the verb ‘surmener’ as ‘to cause excessive fatigue in a beast
of burden by making it walk too far or for too long’. The word is also used figuratively refer to
extreme exhaustion in humans. The English translation adopted here is ‘overwork’. ‘Burnout’ is
another translation sometimes used. (10) “A body is docile that may be subjected, used, transformed and improved.” (Foucault, 2018, p. 134). (10) “A body is docile that may be subjected, used, transformed and improved.” (Foucault, 2018, p. 134). (11) For Foucault (1980), an apparatus (dispositif) is “a thoroughly heterogeneous ensemble consisting
of discourses, institutions, architectural forms, regulatory decisions, laws, administrative
measures, scientific statements, philosophical, moral and philanthropic propositions– in short, the
said as much as the unsaid” (p. 364). (11) For Foucault (1980), an apparatus (dispositif) is “a thoroughly heterogeneous ensemble consisting
of discourses, institutions, architectural forms, regulatory decisions, laws, administrative
measures, scientific statements, philosophical, moral and philanthropic propositions– in short, the
said as much as the unsaid” (p. 364). (12) See Lima (2017). (9) The Littré French dictionary defines the verb ‘surmener’ as ‘to cause excessive fatigue in a beast
of burden by making it walk too far or for too long’. The word is also used figuratively refer to
extreme exhaustion in humans. The English translation adopted here is ‘overwork’. ‘Burnout’ is
another translation sometimes used. (8) Retrieved from https://www.galeriadaarquitetura.com.br/projetos/referencias-ambientes-c/132/
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A perspectiva dialógica na formação de professores
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http://doi.org/10.7213/1981-416X.20.064.AO08
ISSN 1981-416X
A perspectiva dialógica na formação de professores
The dialogical perspective in teacher training
La perspectiva dialógica en la formación de profesores
LUCICLEIDE ARAÚJO DE SOUSA ALVES
a
LUIZ SÍVERES
b
Resumo
Apresenta caminhos metodológicos na formação de professores, tendo como
eixos norteadores os sete saberes requeridos para o futuro (MORIN, 2010), e como
dinâmica transversal deste percurso, o diálogo. O diálogo sendo compreendido
como um jeito de ser (dialógico), uma maneira de saber (dialético) e uma forma de
agir (dialogicidade). Optou-se pela pesquisa qualitativa, por privilegiar os
conhecimentos significativos, que em diálogo com os sentidos atribuídos pelos
professores em suas vivências e experiências, refletem em atitudes concretas e de
valorização da pluralidade cultural entre os distintos saberes. Vinte docentes
participaram da pesquisa e integram o Grupo de Pesquisa “Comunidade Escolar –
Encontros e Diálogos Educativos”. Eles atuam em disciplinas de áreas específicas e
frequentam o curso de mestrado e doutorado em uma instituição particular, do
Distrito Federal. Utilizou-se dos seguintes procedimentos: a revisão bibliográfica e
a aplicação de dois questionários: o primeiro com uma questão fechada, e o
segundo com uma questão aberta que foi respondida por meio de narrativas.
Produzidos os dados, estes foram analisados e categorizados segundo o processo
de “análise de conteúdo” (BARDIN, 2002), sob o olhar dos sete saberes e baseado
a
Universidade Católica de Brasília (UCB), Brasília, DF, Brasil. Doutora em Psicologia, e-mail:
lucicleide.ead@gmail.com
b
Universidade Católica de Brasília (UCB), Brasília, DF, Brasil. Doutor em Desenvolvimento
Sustentável, e-mail: luiz.siveres@gmail.com
Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020
A perspectiva dialógica na formação de professores
353
no princípio dialógico, segundo Morin (2007). Concluiu-se que a reorganização do
espaço pedagógico, com foco na valorização das histórias de vida dos sujeitos e de
suas relações de aprendizagem, tendo o diálogo como princípio fundante e
regenerador, constitui uma das vias possíveis para propiciar as condições
necessárias de gestão, valorizando-se os pensamentos e ações, para que
possibilitem a concretude do fazer pedagógico revolucionário e transformador.
Palavras-chave: Formação de professores. Sete saberes. Perspectiva dialógica.
Abstract
It presents methodological paths in teacher training, having as its guiding axes the
seven knowledges required for the future (Morin, 2010), and as a transversal dynamic
of this course, dialogue. Dialogue being understood as a way of being (dialogical), a way
of knowing (dialectic) and a way of acting (dialogicity). Qualitative research was chosen
because it privileges the significant knowledge that, in dialogue with the senses
attributed by the teachers in their experiences and experiences, reflects in concrete
attitudes and appreciation of the cultural plurality between the different knowledges.
Twenty teachers participated in the research and are part of the Research Group
“School Community – Educational Meetings and Dialogues”. They work in disciplines of
specific areas and attend the masters and doctorate courses at a private institution in
the Federal District. The following procedures were used: the bibliographic review and
the application of two questionnaires: the first with a closed question, and the second
with an open question that was answered through narratives. These data were
analyzed and categorized according to the "content analysis" process (BARDIN, 2002),
based on the seven knowledge and based on the dialogical principle, according to
Morin (2007). It was concluded that the reorganization of the pedagogical space,
focusing on the valuation of the subjects' life histories and their learning relations,
having dialogue as a founding and regenerating principle, is one of the possible ways to
provide the necessary management conditions, valuing the thoughts and actions, so
that they make possible the concreteness of the revolutionary and transformative
pedagogical doing.
Keywords: Teacher training. Seven knowledge. Dialogical perspective.
Resumen
En el caso de los profesores, los directores de la escuela, los profesores, los profesores,
los alumnos y los alumnos. El diálogo es comprendido como una forma de ser
(dialógico), una manera de saber (dialéctico) y una forma de actuar (dialogicidad). Se
optó por la investigación cualitativa, por privilegiar los conocimientos significativos, que
en diálogo con los sentidos atribuidos por los profesores en sus vivencias y
experiencias, reflejan en actitudes concretas y de valorización de la pluralidad cultural
entre los distintos saberes. Veinte docentes participaron de la investigación e integran el
Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020
354
ARAÚJO, L.; SÍVERES, L.
Grupo de Investigación 'Comunidad Escolar - Encuentros y Diálogos Educativos'. Ellos
actúan en disciplinas de áreas específicas y frecuentan el curso de maestría y
doctorado en una institución particular, del Distrito Federal. Se utilizó de los siguientes
procedimientos: la revisión bibliográfica y la aplicación de dos cuestionarios: el primero
con una cuestión cerrada, y el segundo con una cuestión abierta que fue respondida
por medio de narrativas. En el caso de los países de la Unión Europea, los Estados
miembros de la Unión Europea (UE) y los Estados miembros de la Unión Europea (UE).
Se concluyó que la reorganización del espacio pedagógico, con foco en la valorización
de las historias de vida de los sujetos y de sus relaciones de aprendizaje, teniendo el
diálogo como principio fundante y regenerador, constituye una de las vías posibles
para propiciar las condiciones necesarias de gestión, valorizando -se los pensamientos
y acciones, para que posibiliten la concreción del hacer pedagógico revolucionario y
transformador.
Palabras clave: Formación de profesores. Siete saberes. Perspectiva dialógica.
Introdução
O cenário mundial se apresenta, no alvorecer do novo século, com uma
tendência cada vez mais globalizada, seja pelos grupos econômicos, pelos sistemas
políticos ou pelas redes sociais. Por outro lado, percebe-se uma dinâmica de
fragmentação, seja pela unilateralidade individual, pela polaridade conjuntural ou
pela desigualdade social. Tais características podem ser percebidas, principalmente,
no nível pessoal, educacional e social e, por isso, esta realidade tem se configurado
como um desafio para os pesquisadores contemporâneos, bem como para os
professores em formação, que, dispostos a inovar, têm pensado e repensado suas
jornadas
pedagógicas
e
epistemologias,
mediante
processos
formativos
“autopoiéticos” (MATURANA e VARELA, 2001) no sentido de ato criativo, de
elaborações e reelaborações a partir das próprias vivências de experiências de vida,
na vida educativa, sob a perspectiva dialógica baseada no pensamento complexo.
Estas características, de uma forma ou de outra, têm influenciado todos os
setores da sociedade e, de modo especial, a dinâmica relacional. Isto já foi
constatado por Buber (2001), ao afirmar que a humanidade estaria fortalecendo o
antidiálogo; por Habermas (2012), porque estaríamos instrumentalizando a razão e,
em consequência, instrumentalizando os processos comunicacionais; e por Freire
Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020
A perspectiva dialógica na formação de professores
355
(1987), porque as relações sociais estariam sendo pautadas pelo monólogo. Estes
argumentos revelam, portanto, a necessidade de retomarmos a abordagem teórica, a
reflexão e a prática do diálogo como um dos princípios fundantes e constitutivos
para contribuir com processos educacionais formativos trans-formadores.
Embora existam muitas alternativas de formação, este artigo tem por
objetivo compreender o diálogo como um procedimento transversal, propor alguns
encaminhamentos, tendo como eixos norteadores do percurso os sete saberes
necessários à educação do futuro e do presente propostos por Morin (2010), em
vista de poder contribuir com a formação de docentes para a qualificação
profissional e o compromisso social no contexto da realidade contemporânea.
O diálogo é a base para o processo de cognição em processos de formação
humana e, portanto, urgente e necessário. Neste sentido, estamos sugerindo que o
diálogo possa ser um elemento motivador e articulador do processo de formação de
professores, para se compreender a importância da presença viva do humano na vida e
de sua interferência social, mediada por uma ética responsável para consigo, com o
outro e com o planeta.
Os procedimentos metodológicos aqui pensados foram produzidos no sentido
de garantir possíveis deslocamentos em relação ao papel dos educadores, que nos seus
projetos formativos não foram contemplados para caracterizar o diálogo como um
princípio e uma mediação do processo de ensino e aprendizagem, bem como no
exercício da docência ou na formação continuada. Esta constatação foi percebida,
principalmente, na participação dos autores na Conferência Internacional: “Saberes para
uma cidadania planetária” (UNESCO, 2016), cujo contexto os mobilizou a pensar e
elaborar processos teóricos em sintonia com a práxis, com base nos seguintes
questionamentos: Frente aos contextos educativos atuais, com vistas à necessidade de
uma reforma no pensamento, de que forma professores e alunos poderiam se distanciar
de um pensamento monológico e praticar uma educação baseada na relação dialógica?
Como sair das “gaiolas epistemológicas”, para construir, de forma dialógica, um
conhecimento pertinente? E como reverter o modelo disciplinar, baseado na
fragmentação, e sugerir um conhecimento inter ou transdisciplinar? Em outros termos,
como romper com o espaço enquadrado, o saber engaiolado e o conhecimento
disciplinado no processo de formação de professores?
Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020
356
ARAÚJO, L.; SÍVERES, L.
Para a tessitura desse processo, numa analogia compreendida como fios para
a constituição de uma trança, é mais que requerida a necessidade para esse tecer, de
uma educação permeada pelo diálogo, bem como a constituição de subjetividades e
intersubjetividades a partir de uma constante dialogicidade entre os mundos intra e
interpsíquicos, que se mostram, simultânea e sincronicamente, como princípio e
processo na formação de professores.
Neste sentido, inicialmente será apresentado o diálogo como uma dinâmica
transversal e, na sequência, uma breve síntese sobre os sete saberes e como esses
saberes poderiam se caracterizar como fios que se entrelaçariam para uma formação
de professores, no presente e no futuro. Por fim, sob a iluminação dos saberes
emergentes suscitados a partir da análise das narrativas proferidas pelos professores,
por meio das vivências de suas experiências pedagógicas, será feita uma vinculação
com o pensamento complexo, sob a perspectiva dialógica.
O diálogo como uma dinâmica transversal
O diálogo como uma dinâmica transversal será compreendido como um jeito de
ser (dialógico), uma maneira de saber (dialético) e uma forma de agir (dialogicidade). É
uma proposta pedagógica alternativa que se justifica em função do contexto social e, em
decorrência, do projeto educacional que tem se apresentado mediante acentuadas
características unilaterais, polarizantes e fragmentadas. Por esta razão, será
compreendida tridimensionalmente a partir da relação antropológica, epistemológica e
pedagógica, respectivamente, pois a partir dessas dimensões é possível se transformar
numa sugestão relevante para a formação de professores no contexto contemporâneo.
Considerando, portanto, que o diálogo é um princípio inerente à condição
humana, o mesmo tem contribuído com a constituição histórica de cada pessoa e
com a construção civilizatória da sociedade. Isto é, o diálogo constitui o ser humano
que, por sua vez, é constituinte da sociedade, e nesta dinâmica interrelacional a
humanidade se revela, desenvolve e se projeta por meio do diálogo.
No exercício da dialógica, segundo Buber (2001), todo ser humano articula a
vivência na experiência e esta naquela. Nesta relação de reciprocidade é preciso levar
em conta a plenitude da relação que acontece entre o Eu-Tu e a efemeridade da
Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020
A perspectiva dialógica na formação de professores
357
disposição que se realiza entre o Eu-Isso sob a perspectiva de Buber (2001). A
vivência é tudo que envolve a inteireza do ser, a possibilidade relacional e a dinâmica
de sentido para a vida. A experiência é tudo o que se pode observar, explicar ou
dominar, ainda que provisoriamente. Assim, a vivência do Eu-Tu estaria mais
próxima da singularidade de cada ser, do percurso de sua historicidade e da
finalidade do seu projeto de vida. Enquanto a experiência do Eu-Isso estaria
retratada na familiaridade com a “tecnologia” (LEMOS, 2004), com os contatos
estabelecidos e com as metas que se deseja alcançar.
Nesta perspectiva, o diálogo entre a vivência e a experiência é fundamental
para constituir o ser humano e a sociedade (unitas multiplex), porque por meio dessas
polaridades, não contrárias, mas complementares, explicita-se a condição humana
que expressa suas inúmeras virtualidades, entre elas, como seres de relação, que
estão em constante conexão e em profundo vínculo existencial. Do estabelecimento
dessa dinâmica relacional entre a vivência e a experiência, o ser humano se
constituirá como um ser dialógico, desenvolvendo-se por meio de experiências de
vida mais integrada e polifônica. Esta relação entre a vivência (Eu-Tu) e a
experiência (Eu-Isso), foi, em grande parte, a desencadeadora de toda a reflexão
proposta por Buber (1987) sobre o processo de diálogo nos últimos tempos.
Portanto, para esta proposta de caminho de formação de professores, o diálogo
será compreendido nesta dimensão de dinâmica transversal que acontece mediante a
relação que ocorre entre o sujeito consigo mesmo, com o outro e com o transcendente,
tendo como eixos articuladores os sete saberes necessários para a educação do presente e
do futuro, convergindo, conforme a proposta buberiana, por compreender o ser humano
como um ser social, de relação, com possibilidades de produzir conhecimento e de
atuação consciente na sua comunidade local e na sociedade global.
Os sete saberes necessários à educação do presente e do futuro
Os sete saberes propostos por Morin (2010) serão os eixos-base para esta
formação de professores, entrelaçados pelas sete disposições apresentadas por Nóvoa
(2016) e as vivências de profissionais da área educativa que estão comprometidos com a
transformação da educação, compõem as referências desta reflexão.
Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020
358
ARAÚJO, L.; SÍVERES, L.
Estas três contribuições — compreendidas pela analogia de uma trança —
serão consideradas para identificar cada um dos saberes, bem como suas
emergências durante o exercício da profissão docente, seja no decurso de sua
formação inicial ou continuada. Embora nosso olhar esteja sempre voltado para o
conjunto destes saberes, é oportuno, porém, apresentar uma pequena percepção
sobre cada um destes saberes.
As cegueiras do conhecimento: o erro e a ilusão: É importante o reconhecimento
de que todo conhecimento comporta o risco do erro e da ilusão. Portanto, não há
conhecimento que não esteja ameaçado por esta dupla face. Todo ele é tradução e
reconstrução sob a vista de um ponto, não traduzindo o real como visão única, mas
compreendendo-o a partir de suas múltiplas facetas de verdades provisórias, cuja
interpretação pode ser feita sob os mais diversos olhares e diferentes ângulos, pois a
própria realidade apresenta-se sob os contextos multidimensionais.
Os princípios do conhecimento pertinente: É o conhecimento processado, refletido,
organizado e traduzido na conjunção, permitindo a integração entre o todo
(sociedade) e as partes (indivíduo), imbricados com o processo de vida. Este
conhecimento é demarcado por um tempo e lugar e, em um momento histórico,
com vistas a manter a dinâmica sócio-histórica-cultural da humanidade caminhante.
Ele busca estabelecer os elos necessários entre os mais diferentes saberes,
permitindo a percepção da realidade sob a visão de conjunto e não fragmentada,
pois recoloca o conhecimento aprendido no contexto.
Ensinar a condição humana: Este é um dos principais saberes necessários para o
desfrute de uma vida em plenitude. Ensinar a condição humana é compreender a
unidade (humano) na diversidade (cultura) humana e esta naquela, para o
desenvolvimento
conjunto
das
autonomias
individuais,
das
participações
comunitárias e do sentimento de pertencimento do sujeito, posicionando-se no
mundo. É conhecer o humano, situando-o no contexto e não fora de sua realidade.
Ensinar a identidade terrena: Ensinar a identidade terrena é perceber-se parte
constitutiva dessa sociedade complexa para ser capaz de enfrentar com autonomia
as suas incertezas. Já dizia Pascal, citado por Morin (2008, p. 25): “considero
impossível conhecer as partes sem conhecer o todo, assim como conhecer o todo
sem conhecer, particularmente, as partes [...]”.
Rev. Diálogo Educ., Curitiba, v. 20, n. 64, p. 352-378, jan./mar. 2020
A perspectiva dialógica na formação de professores
359
Enfrentar as incertezas: É importante que estejamos prontos a enfrentar as
incertezas do conhecimento, pois toda tradução é um ponto de vista (BOFF, 1997),
que possivelmente pressupõe erro. No entanto, erro este que pressupõe ponto de
partida para novos acertos e erros, possibilitando um caminhar que não se encerra
em si mesmo, mas que estará sempre renascendo, mantendo o sujeito em sua
dinâmica processual construtiva de conhecimento, aprendendo a conviver com as
certezas do conhecimento, ainda que provisórias.
Ensinar a compreensão: “Educar para a compreensão humana”, traduz-se em
colocar-se no lugar do outro e não desejar para o outro o que não se desejaria para
si mesmo. Ou seja, uma compreensão que parte do eu (de si mesmo) para o tu (o
outro) legitimando-o.
A ética do gênero humano: A consciência de nossa identidade como seres
pertencentes a um todo se embasaria na ética da compreensão caracterizada pelo
“pensar bem”, vinculado a ética da compreensão entre as pessoas à ética da
era planetária.
Repensar a formação de professores, sob o olhar dos sete saberes propostos
por Edgar Morin, pode contribuir para a ressignificação dos atuais contextos
educativos, com vistas à superação da prática pedagógica voltada exclusivamente
para o ensino, prevalecendo a perspectiva positivista, para a complexa, que privilegia
a religação entre a cultura científica e a humanística.
Metodologia
Na perspectiva de avançarmos na formação de professores, tendo o diálogo
como o fio transversal e entrelaçado pelos sete saberes, as sete disposições e
vivências de professores, fizemos opção pela pesquisa qualitativa, por esta privilegiar
os conhecimentos significativos que, em diálogo com os sentidos atribuídos pelos
professores em suas vivências e experiências, refletem em atitudes concretas e de
valorização da pluralidade cultural entre os distintos saberes. Este tipo de pesquisa
vai além da visão positivista e de seus parâmetros epistemológicos, por trazer de
volta ao seu fazer pedagógico o sujeito e sua subjetividade, ao dar ênfase na
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construção de processos formativos, mediante às representações, os significados e o
sentido existencial elaborado, segundo Lemos (2004).
Os docentes participantes da pesquisa integram o Grupo de Pesquisa
“Comunidade Escolar — Encontros e Diálogos Educativos”.
Eles atuam em
disciplinas de áreas específicas e frequentam o curso de mestrado e doutorado
em uma instituição particular, de caráter confessional e comunitário, do
Distrito Federal.
Para a produção dos dados, utilizamo-nos dos seguintes procedimentos: a
revisão bibliográfica e a aplicação de dois questionários: o primeiro com uma
questão fechada, e o segundo com uma questão aberta que foi respondida por meio
de narrativas.
O primeiro questionário foi encaminhado aos 20 participan9tes do Grupo de
Pesquisa com a seguinte questão: Considerando a sua compreensão e sua prática
educativa, com qual dos sete saberes, propostos por Morin, você mais se identifica?
Indique em ordem de prioridade, de 1 a 3, os três saberes que estariam mais
próximos da sua experiência de vida.
Do total dos 20 participantes do grupo, 16 responderam ao questionamento
acima, cujos dados seguem descritos na sequência:
Gráfico 1 - Questionário
Fonte: Acervo dos autores.
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Segundo o Gráfico 1, todos os saberes receberam algum tipo de menção,
exceto o “ensinar a identidade terrena”. O conjunto dos dados revela que os três
saberes mais apontados, incluindo a indicação de alguma prioridade, foram: ensinar
a condição humana, enfrentar as incertezas e ensinar a compreensão. Assim,
no ensinar a condição humana, 5 indicaram como prioridade 1, 4 a prioridade 2 e 3
a prioridade 3, totalizando 12 manifestações. No saber enfrentar as incertezas, 3
responderam como prioridade 1, 4 a prioridade 2 e 6 como prioridade a 3. Por fim,
no ensinar a compreensão, 4 respondentes informaram como prioridade 1, 4 como
prioridade 2 e 5 como prioridade 3, resultando também em 13 indicações.
A partir da aplicação do primeiro questionário, os dados gerados revelaram
que alguns saberes são prioritários em relação aos demais. Por isso, com base na
resposta ao primeiro questionário, optou-se por aprofundar, na análise e discussão,
apenas os três saberes mais reconhecidos, a saber: ensinar a condição humana, enfrentar
as incertezas e ensinar a compreensão.
Após tomada essa decisão, foi encaminhado o segundo questionário aos 12
participantes que indicaram como primeira prioridade um dos três saberes mais
recorrentes. Assim, foi encaminhada a seguinte questão: Com base na sua resposta,
gostaríamos de convidá-lo novamente para relatar uma experiência educativa. Para o
primeiro grupo foi solicitado uma experiência de “como ensinar a condição
humana”, para o segundo, “como enfrentar as incertezas”, e ao terceiro, “como
ensinar a compreensão”.
Produzidos os dados nessa segunda etapa, estes foram categorizados e
analisados, segundo o processo de análise de conteúdo baseado em Bardin (2002),
sob o olhar dos sete saberes e na perspectiva baseada no princípio dialógico,
segundo Morin (2000, 2007). Partiu-se da leitura de todo o material, na busca por
significados que emergissem das falas como atribuições dos sujeitos a suas próprias
ações, de forma a auxiliar nas interpretações a serem discutidas teoricamente. Os
temas foram interpretados no seu conjunto, estabelecendo-se relações com o
referencial teórico adotado, mas foram selecionados apenas três depoimentos,
vinculados aos três saberes mais evidenciados.
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Resultados e discussões
No sentido de contribuir com o enfrentamento dos desafios da educação
contemporânea, por meio de ações concretas que possam incorporar os saberes na
prática formativa de professores, foram acolhidas as manifestações de três
participantes do Grupo de Pesquisa, ora identificados com nomes de uma família
bíblica: Marta, Maria e Lázaro, com o objetivo de resguardar a sua identidade.
Assim, foi acolhido o relato de Maria: Ensinar a condição humana, de Lázaro:
Enfrentar as incertezas, e Marta: Ensinar a compreensão. Segue a descrição das
narrativas:
Como ensinar a condição humana? Eis a reflexão de Maria:
O presente relato versa sobre um trabalho da disciplina Gestão das Instituições
Educativas e Esportivas na Licenciatura em Educação. [...]. Como parte da citada
disciplina, os estudantes de Educação Física visitaram, durante um semestre uma
escola, realizando observações e entrevistas com professores e alunos. Após as
primeiras visitas, identificaram que o público atendido reunia alunos de diferentes
setores sociais (inclusive alguns moravam em abrigos prévios à espera de adoção),
religiões e de diversas nacionalidades (17 países, incluindo República Dominicana,
Cuba, Venezuela, Síria, México, Bulgária e outras nações limítrofes). Paralelamente,
observaram que a instituição carecia de jogos ou espaços recreativos no pátio escolar,
totalmente pintado de cinza. As impressões registradas durante os recreios eram de que
os alunos permaneciam isolados. A não existência de áreas recreativas – árvores,
gramados, bancos, brinquedos – deixava a instituição muito similar a um cárcere [...]. A
partir desse diagnóstico, decidiu-se, de forma coletiva, pela elaboração de um projeto
de criação/pintura de jogos, que também contribuiria para fortalecer o senso de
pertencimento à instituição escolar – até então pouco consolidado. Os alunos da escola
foram indagados sobre as atividades recreativas mais frequentes em seus países de
origem, capazes de serem recriadas no pátio escolar. Uma vez realizada a seleção de
jogos (amarelinhas, labirintos, redes para esportes coletivos, xadrez, minhocas), os
professores de Artes e Recreação foram convidados para colaborar no desenho e
execução do projeto batizado de “Pintada na Escola nº 3”. Uma semana depois que a
ornamentação da área de lazer foi concluída, realizou-se uma quermesse de
inauguração dos espaços recreativos do pátio, reunindo a comunidade escolar mais os
estudantes de Educação Física. Música, cores, animação e integração entre os alunos e
estudantes mostraram a relevância da aproximação com o outro: escutar suas vozes,
identificar suas necessidades. Assim, uma disciplina, em princípio teórica, se
transformou numa ágora de integração, não apenas de saberes (gestão, artes,
recreação), como também de integração da docência, da pesquisa e da extensão. E,
sobretudo, de culturas, biografias e esperanças de que naquela escola é possível
aprender e resgatar o sentimento de pertencer à própria comunidade escolar.
Como enfrentar as incertezas? Segue o depoimento de Lázaro:
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O cenário que ora se apresenta é aquele no qual as tecnologias funcionam quase
como um dos membros do corpo humano. Aliado a isso, a motivação dos
estudantes passou a ser um dos desafios para o processo de ensino e
aprendizagem. [...]. Fui tomado pela incerteza sobre qual método de ensino seria
considerado o ideal para o alcance dos objetivos propostos a partir de uma
experiência vivenciada no início de um semestre letivo. Naquela oportunidade,
escutei de alguns dos alunos relatos do tipo “nem mesmo eu sei o que estou
fazendo aqui”; enquanto outro dizia: “vou ficar aqui para ver o que dá…”; teve
ainda: “não sei se vou aguentar até o fim”. Diante dessa situação de incerteza
acerca de quais eram os objetivos de parte daquele grupo, procurei refletir na
tentativa de responder à seguinte questão: como aplicar técnicas de ensino que
pudessem motivar esses estudantes, face às incertezas e quanto à possível eficácia
ou fracasso da estratégia por mim escolhida.
Como ensinar a compreensão? Segue o relato de Marta:
Esse relato de experiência versa sobre um processo interventivo de Gestão Escolar,
constituído num esforço educativo de abrir possibilidades de humanização,
cooperação, e reflexão do papel do gestor e da comunidade escolar em busca do
fazer coletivo e democrático. Teve como espaço uma instituição de Educação
Básica no Distrito Federal entre os anos de 2011 a 2016, localizada em área urbana.
O diagnóstico e a análise das situações vivenciadas pela comunidade escolar
indicaram serem urgentes mediar e auxiliar o coletivo da escola, a compreenderem
e refletirem sobre os valores da comunidade, e acreditarem na capacidade de seus
alunos e de seus pares. Por outro lado, as fragilidades que culminaram na vinda de
um interventor à escola permitiram ao grupo redimensionar o pensar,
reformulando suas ações pela compreensão do que a comunidade escolar
(entendida aqui os alunos, pais, professores, equipe pedagógica, direção,
funcionários) pode fazer para estimular a função social da escola. [...]. O
levantamento da situação revelou muitos problemas estruturais, necessidade de
diversas reformas e reorganização dos espaços pedagógicos, nova abordagem à
Proposta Pedagógica que promovesse a melhoria nos altos índices de reprovação e
evasão, e também, a falta de apoio da comunidade. Sendo a atuação do gestor
escolar uma tarefa que perpassa várias dimensões, sejam elas administrativas,
pedagógicas, políticas, sociais é urgente estar atento ao ato de compreender-se
gestor e a partir dessa sensibilidade conduzir coletivamente a formulação do
projeto pedagógico, agir na coordenação, acompanhamento e cumprimento das
responsabilidades compartilhadas, ouvindo e dando sentido ao que a comunidade
escolar tem a oferecer. [...] O momento era tenso e as relações pessoais estavam
abaladas. Iniciamos investigando as causas dos conflitos, os valores da escola, as
lideranças do grupo, a comunidade, entre outros. Iniciamos realizando um
diagnóstico com análise da situação, os objetivos a alcançar, ajustar estratégias de
receptividade ao grupo, melhorar o ambiente, criar empatia, pois não
pertencíamos ao grupo, e construir um planejamento juntamente com toda a
comunidade escolar para resolver os problemas. [...] Após o planejamento
partimos para a ação. A experiência pautou-se na reconstrução do Projeto
Pedagógico nas reuniões coletivas da escola, com encontros semanais. A cada
semana a fala inicial era dada ao grupo para que narrassem suas expectativas,
seus projetos, seu trabalho, e a partir da reflexão e da compreensão da história de
cada um, constituiu-se um conjunto de ações, que compartilhados, uniram às
metas e aos objetivos coletivos, mas também às responsabilidades individuais, a
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tarefa dada ao gestor foi articular responsabilidades individuais e coletivas de
modo que todos tenham consciência de seu papel dentro da instituição de ensino,
o que exigiu mais que aprender a compreender o outro, ensinar a importância de
fazer-se compreendido.
Saberes trançados por três fios entrelaçados
A formação de professores, por muito tempo, e até hoje, continua se dissipando
numa abordagem disciplinar, organizada em arcabouços epistemológicos engaiolados e
sendo exercida continuamente a partir do quadro-negro. Totalmente desconexa da
realidade ao dar ênfase exacerbada à teoria, com pouca preocupação de como esta
reflete em cada realidade e em conformidade com o seu contexto cultural.
Conduzir os futuros professores e/ou em formação continuada, mediante o
conhecimento dos fundamentos da profissão baseada nos sete saberes, por meio de
observação da realidade, escuta sensível, pesquisa em profundidade, pensamento
reflexivo, para intervir na realidade com ações concretas, projetos de vida, por
soluções de caminhos alternativos decididos no coletivo, tendo em vista o
restabelecimento de relações mais amistosas, a contínua troca de experiências,
mediada por processos dialógicos capazes de integrar as partes ao todo e o todo às
partes, é a condição humana a ser ensinada nos espaços formativos.
A condição humana é um dos fios dos sete saberes a serem trançados,
entrelaçados com outros, para práticas formativas mais atuantes e menos passivas
frente aos problemas sociais da vida. O saber “ensinar a condição humana” foi
indicado por Marta, Lázaro e Maria como primeira prioridade entre os 7 saberes, e
será, pois, o fio de análise da construção de um texto, cujo contexto da trama se
desenvolve a partir dos fios entrelaçados.
Fio 1. Ensinar a condição humana
No exercício da práxis do saber “Ensinar a condição humana”, Morin (1999,
2007) alerta sobre a necessidade da consciência do conhecimento sobre o próprio
conhecimento, pois este sempre comporta erro e ilusão, bem como, de um
pensamento abrangente e mais articulado para uma compreensão mais adequada e
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consciente sobre os processos formativos e suas necessidades inerentes, mediante
constantes releituras da realidade em conjunto com os pares. Além disso, importa a
atuação dos sujeitos envolvidos na dinâmica da formação, por meio de um agir, em
existência, com toda a inteireza, utilizando-se dos conhecimentos e das experiências
apreendidas durante o viver/conviver.
Compreende-se que dessa tessitura construída e constituída nos mais diversos
espaços de vida, de formação inicial e/ou continuada, perpassados, vividos e
experienciados por sujeitos dispostos que desejam exercer a cidadania, podem emergir
ações concretas, com transformações bem-sucedidas, pelo resgate dos sujeitos por meio
de práticas mais dialógicas. Ou seja, um fazer desembocar ações particulares na direção
do universal, com atitudes concretas. Assim como bem procedeu Marta, após ter se
apropriado de novas epistemologias em espaços formativos, vivência na prática o saber
“Ensinar a condição humana”, ao levar os seus estudantes a uma imersão na realidade
profissional futura, para conhecerem as suas condições reais e intervirem sem
descontextualizar os sujeitos de sua cultura (LARAIA, 1986), numa tomada de ações
em conformidade com as circunstâncias locais.
De forma também semelhante agiu Maria após ter elaborado diagnóstico
para análise das situações vivenciadas pela comunidade escolar, mediado pelo
exercício do diálogo para a compreensão dos problemas e reflexões no coletivo
sobre os valores aparentemente “perdidos”, visando ao resgate da credibilidade na
capacidade de seus alunos e de seus pares e ao retorno destes como membros
participantes e ativos na transformação da própria comunidade de aprendizagem.
A consciência epistemológica adequada do real, para a tomada de atitudes,
consoante a escolhas políticas e estratégicas, como as de Marta e Maria, foram
decisões-chave para a busca de vias de ação e de transformação nos espaços
educativos considerados encarcerados, desacreditados, em verdadeiras ágoras. E o
efetivo restabelecimento das relações comunicativas entre os membros da
comunidade, com vistas à apropriação dos bens culturais da humanidade, mediante
o resgate da convivência harmoniosa e humana entre os sujeitos.
Ao reintegrar os sujeitos da comunidade escolar (alunos, pais, professores,
equipe pedagógica, administrativa e diretiva), a função social da escola é exercitada e
reforçada pela participação ativa de cada um de seus membros, atribuída pelo
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sentido da importância do pertencimento, ao se colocar as experiências e vivências
na reconstituição e humanização do todo, que se encontravam desconectados e
fragilizados entre si, enquanto comunidade.
É fato que os espaços formativos não podem se “[...] constituir similares a
um cárcere”, como analogia intensificada e proferida metaforicamente pelo
depoimento de Marta. Tampouco serem geridos e gestados à revelia, sem qualquer
compromisso com o desenvolvimento humano a partir da compreensão de seus
problemas singulares inerentes. A capacidade de pensar conjuntamente os
problemas locais e globais, mediante a criação de espaços para o exercício da relação
dialógica, que possibilitem os caminhos e as circunstâncias necessários para os
sujeitos em formação abrirem-se ao inesperado, com consciência formada por meio
de epistemologias mais abertas, para um agir em coletividade, é procedimento cada
vez mais urgente e necessário, em função dos tempos atuais, fluidos e carentes de
relações comunicativas mais humanizantes e humanizadoras.
Neste sentido, humanizar a convivência com o outro é da ordem do
humano, e esta acontece na e pela cultura, perpassada pelo diálogo consigo, com o
outro e com a vida. Do contrário, contribuiremos muito mais para a disjunção do
que para a integração nos espaços formativos, reforçando a prática de ações
antidialógicas, improdutivas, mutilantes e mutiladoras.
Reunir a comunidade escolar para uma escuta sensível sobre o que vivencia
na dinâmica de sua comunidade de aprendizagem e de vida, cotidianamente, criando
espaços dialógicos, conforme indicado pela narrativa de Maria, só demonstra e
reafirma a “relevância da aproximação com o outro: escutar suas vozes, identificar
suas necessidades”, conforme assertiva por ela mesma proferida.
Relacionar-se para sentir-se integrado, portanto, é uma condição humana
necessária para a prática dialógica que sugere abertura para o compartilhamento,
troca de ideias e a construção de projetos interventivos de vida, com vistas à
transformação da própria realidade numa simbiose homem/sociedade/natureza.
Neste sentido, o conhecimento sobre os saberes da profissão professor (NÓVOA,
2016) interpelados e construídos no exercício de uma prática educativa concebida
por meio da dialogicidade para perceber a realidade e nela intervir, requer ações em
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conformidade com as necessidades e circunstâncias de cada localidade, em conjunto
com os seus pares, integrantes daquele microssistema.
A consciência da necessidade de uma escuta sensível do outro, de suas
necessidades e sonhos, foi percepção fundamental, como reforçado também por
Lázaro em sua narrativa, ao fazer opção por ouvir atentamente o que os seus alunos
apresentavam como expectativas em relação à disciplina e quais eram os seus
objetivos, como também o demonstrou Marta, ao ouvir atentamente os membros da
comunidade escolar por meio da realização de diagnóstico para análise das situações
vivenciadas, visando encontrar soluções conjuntas mediadas pelo diálogo.
Evidencia-se, assim, o quanto a comunicação intersubjetiva e os
relacionamentos são condições humanas necessárias para o fortalecimento do
sentimento de pertencimento e integração a uma comunidade para o convívio e
desenvolvimento humano saudável.
Cada uma das iniciativas propostas por Maria, Marta e Lázaro para a
reconstrução de processos de ensino e aprendizagem formativos — conforme propõe
Nóvoa (2016), — contribuem para o exercício de práticas formativas baseadas na
perspectiva dialógica, suscitando perguntas e respostas em conjunto e sob os mais
diferenciados olhares, interpeladas, sobretudo pelo sentimento de esperança por
soluções quanto aos problemas existentes, propulsionando saltos qualitativos à
comunidade, ao se ter conseguido resgatar a participação ativa dos sujeitos.
A consciência epistemológica apreendida, exercitada pela prática do princípio
da dialogicidade, como mediador da “costura” entre os saberes científicos e
humanísticos, foi mister e significativa para o ensinar a condição humana (Fio 1),
pois este saber requer o respeito às ideias contrárias e às certezas provisórias, cujas
perspectivas e óticas em relação à vida são plurais e multidimensionais, quando se
lida com grupos de visões heterogêneas.
E como assegura Morin (1999), nos momentos em que não é possível
superar as contradições, vencer os antagonismos e ultrapassar os paradoxos, é
preciso aprender a lidar e a conviver com os opostos, compreendendo-os, pois é
nesse emaranhado da trama que emerge a complexidade, pois do mesmo lugar que
surgem os problemas nascem também as suas soluções. E “o entrechoque de ideias,
opiniões e comportamentos é uma das principais fontes de inspiração para a
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criatividade e a resolução dos problemas de convivência”, como assegura Mariotti
(2007, p. 151), para o enfrentamento das incertezas (Fio 2), mediante o ensinamento
da compreensão (Fio 3).
Fio 2: Enfrentar as incertezas
A consciência sobre a própria ação pressupõe a consciência de um fazer
compreendido em constante movimento de estado de evolução, alimentado por um
pensar complexo e práticas pedagógicas congruentes com os pressupostos
metodológicos menos tecnicistas e mais complexos, plurais e multidimensionais,
com vistas à superação do distanciamento do professor do quadro negro (NÓVOA,
2016) e a imersão no mundo em rede, pela necessidade de que “é preciso aprender a
conviver com as incertezas ligadas ao conhecimento” (MORIN, 2010, p. 82).
Porém, sem descontextualizar-se do mundo, tampouco de suas inovações e
bens científico-culturais, mas buscando compreendê-los em sua própria dinâmica. É
o que buscam traduzir as iniciativas de Maria, Lázaro e Marta, pois práxis é
confronto. E confrontar é estar disposto a pensar o real para transformá-lo,
confrontando-se o vivido com o pensado, pois a certeza do risco de sua incerteza é
inerente a cada ação. Por isso é sempre desafio, e eliminá-la é impossível, pois “a
imprevisibilidade reside no próprio cerne do determinismo” (MORIN, 2015, p. 41),
requerendo, portanto, um constante negociar com a incerteza, pelo recurso à
reflexão, combinando riscos e estratégias de precaução, mediante uma escuta
vigiada, para propulsionar o movimento de aprendizagem e o seu desenvolvimento
processual e contínuo.
E como assegura Morin (2015, p. 51), “é preciso aprender a navegar em um
oceano de incertezas, através de arquipélagos de certezas”. Para tanto, o exercício do
diálogo intra e intersubjetivo é uma via para a possibilidade de se compreender o
processo de formação dos sujeitos, mediante uma ética comprometida, valores
concebidos e pelo respeito ao repertório cultural que constroem e trazem consigo
durante a vida.
O pressuposto do diálogo não consiste em convencer o outro, mas abrir
possibilidades para partilhar significados construtivos de conhecimentos, durante o
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viver e o conviver, para então se direcionar ao agir. Este agir, no sentido de criar
ações interativas, pela percepção e conhecimento da realidade, mesmo mediante
contextos complexos de diferenciados posicionamentos e ideias, pois estes e estas,
ao serem articulados com os saberes apreendidos ao longo da vida, por seus
diversificados membros em processos dialógicos, podem suscitar outras ideias
criativas e vias de caminhos antes não pensados.
Dessa forma, a atitude dialógica possibilita emergir a comunicação, sem
autoridade hierarquicamente estabelecida, numa perspectiva contínua e evolutiva de
solidariedade e compartilhamento, por meio da construção de pensamentos
baseados em saberes interconectados e integrados. Em um processo relacionalmente
favorável, na busca por inovadoras possibilidades de conhecimento e apreensão da
realidade, pela prática do diálogo, com flexibilidade e respeito às ideias do outro, de
modo a assegurar a manutenção dos processos de fluxos dialógicos contínuos,
circulares, e de constantes recursividades, como foi possível notar nos três relatos
das experiências vivenciadas por Maria, Lázaro e Marta.
A reflexão sobre os fatos passados, remetendo para o futuro, os olhares dos
sujeitos no presente, mesmo sem os fundamentos teóricos fortemente consolidados,
mas já desabrochados — pela necessidade de um fazer pedagógico diferente —
retrata a necessidade da consciência sobre o próprio pensar e fazer, dos
fundamentos implícitos a cada sujeito, orientadores de suas ações, reforçando-se
assim o dito por Freire (1996), de que não há uma prática sem teoria e nem esta sem
aquela. Ambas se imbricam, revelando o sujeito e suas epistemologias, trazendo-as à
consciência para revê-las e ressignificá-las no presente, com posturas mais
conscientes e fundamentadas, no sentido de se dar uma nova ordem ao que se
mostra desorganizado e desconexo do real.
A partir do momento em que os professores são munidos por novos
fundamentos e perspectivas metodológicas, como proposto pelos “Saberes
Necessários à Educação do Futuro”, no presente, espera-se que eles possam
encontrar novas vias pedagógicas para além do quadro-negro, dos conteúdos
engessados, e processos antidialéticos nos espaços formativos.
Distanciar-se de um processo formativo mais estático, compreendido numa
perspectiva de meros repetidores de informações, como sugere Nóvoa (2016),
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ARAÚJO, L.; SÍVERES, L.
requer por parte dos professores, atitude de autonomia e autoria para criar e
protagonizar nos espaços educativos e para além destes, tendo em vista oportunizar
aos membros da comunidade educativa saírem das próprias gaiolas epistemológicas,
consideradas por D’Ambrósio (2009). Tal atitude foi bem encaminhada por Maria,
ao levar os estudantes para conhecerem outras realidades, para além da sala de aula,
e se apropriarem de conhecimentos provindos da realidade, mais pertinentes e
munidos de sentido e significado, numa tentativa esperançosa de reverter a lógica
reprodutivista e de passividade dos sujeitos nos espaços formativos, com vistas a
avançar para uma perspectiva de formação com protagonismo docente.
Coadunar os momentos de teoria com o seu exercício na prática, pela
vivência no campo profissional, permite aos sujeitos em formação pensar e repensar
sobre a profissão, pela pesquisa, elaboração e desenvolvimento de projetos, para o
acesso e maior proximidade com a cultura e seu repertório contemporâneo,
apreendendo-a, pela observação, pelo diálogo e ações viáveis que possam revitalizar
os espaços aparentemente “cinzentos” (sem vida), como dito por Maria.
Nas ações propostas por Maria e Marta, a exigência de novos itinerários
pedagógicos formativos, a reaproximação dos sujeitos com a sociedade do século
XXI — a partir de ações mais integradoras e coletivas — foi atingida pelo
sentimento de pertencimento. Ao sentir-se pertencente a uma comunidade de
diferentes fins, o sujeito passa a se relacionar, e, consequentemente, a agir,
aprendendo, reaprendendo e se desenvolvendo em comunhão com os seus pares,
em prol do bem viver, reafirmando assim que é na relação com o outro que nos
constituímos, aprendemos e nos transformamos mediante as trocas de experiências
vivenciadas no decurso do viver/conviver — pela condição de sujeito social que
somos — mediados pelo diálogo. Dessa forma, nos transformamos conforme
transformamos o mundo, e isso requer disposição, mesmo diante dos incertos e
desafiadores processos educativos e de vida.
Paralelas à coragem e às “certezas” de possíveis transformações, emergem
também a insegurança e as incertezas. No relato de Lázaro, por exemplo, urge a
incerteza quanto aos conhecimentos de sua profissão que poderiam propiciar a
motivação necessária para o atendimento aos interesses da turma, bem como quanto
à escolha do método, e a insegurança quanto à sua efetividade, e se aquela seria a
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A perspectiva dialógica na formação de professores
371
melhor via a ser trilhada. Destaca-se também a desconstrução da própria prática
pedagógica, sinalizado por Lázaro ao expressar não se sentir preparado para inovar
diante do inesperado.
Diante deste contexto, é fundamental aprender a aprender a lidar com os
paradoxos: certeza e incerteza, ordem e desordem, segurança e insegurança,
primando-se pelo equilíbrio e o restabelecimento da ordem, para novas desordens e
reorganizações
em
espaços
formativos.
Desse
movimento
de
relações
aparentemente contraditórias, porém complementares, é que a vida mantém o seu
ciclo em processo ativo de desenvolvimento e de produção, construindo-se,
desconstruindo-se e reconstruindo-se o tempo todo. O reconhecimento do risco
permite aos sujeitos um olhar mais profundo, com maior consciência sobre os
problemas da vida tanto pessoal quanto socialmente, pelo processo de
autoconhecimento (o aprender a ser), pelas experiências vivenciadas durante o viver,
com vistas a enfrentar as incertezas presentes na dinâmica da vida (o aprender a
conviver), para então se colocar o agir (o aprender a fazer).
Dessa forma, “Ensinar a condição humana” como primeira prioridade em
espaços formativos é uma necessidade vital, pois requer a legitimação do outro, bem
como o exercício da autopercepção enquanto pessoa, profissional, sujeito construtor da
própria história, na relação com o outro, reconhecendo, de forma equilibrada, os
próprios limites humanos das incertezas por suas ações, pois “em toda ação há escolha
e risco” (MORIN, 2010, p. 42). Neste sentido, é mister “conceber a unidade que
assegure e favoreça a diversidade, a diversidade que se inscreve na unidade” (MORIN,
2010, p. 55), pelo ensinamento da condição humana, entrelaçado pelo da compreensão
para enfrentar as inúmeras incertezas em relação ao conhecimento e a vida.
Fio 3: Ensinar a compreensão
A consciência sobre a pluralidade cultural e multifacetada das instituições,
que engloba, como frisa Marta, “diversas realidades socioeconômicas, emocionais e
culturais; um ambiente heterogêneo por princípio”, leva a valorizar as trajetórias de
histórias de vida construídas e constituídas pelos sujeitos que compõem diferentes
comunidades e suas diferenciadas culturas. É fazer com que os processos subjetivos
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ARAÚJO, L.; SÍVERES, L.
se façam presentes e sejam centrais — para transformações por meio da
manifestação dos potenciais adormecidos e antidialéticos — mediante o
restabelecimento do diálogo, na coletividade, para ações locais concretas.
Uma disciplina em princípio teórica, segundo Maria, se transformou numa
ágora de integração de saberes (gestão, artes, recreação), como também de
integração da docência, da pesquisa e da extensão. E, sobretudo, “de culturas,
biografias e esperanças de que naquela escola é possível aprender e resgatar o
sentimento de pertencer à própria comunidade escolar”.
E uma intervenção, segundo Marta, que inicialmente poderia ter sido
marcada por autoritarismos, posturas unilaterais e apartadas da trajetória da
comunidade, constituiu-se em um movimento de aprender e, sobretudo, de ensinar
a compreensão, enfrentando as incertezas provindas das soluções estabelecidas em
conjunto. Isto porque toda a ação é imbuída de retroações, ou seja, de riscos que
fogem ao controle humano. No entanto, sem correr riscos será impossível
compreender o “problema importante (MORIN, 2007)”, pois só se pode
compreendê-lo, entregando-se ao aleatório, ao imprevisível, pois são estes os
processos emergentes que possibilitam o crescimento e a evolução das relações
comunitárias, na medida em que enfrentam junto o inesperado, pelo ensinamento da
compreensão, arcando com as incertezas inerentes, em conformidade com as
condições humanas e de respeito às realidades singulares.
No relato de Marta, “Ensinar a compreensão” tornou-se, por conseguinte, a
base de todo o processo de intervenção à medida que, ao perceber o outro, abriu-se
a possibilidade de atribuir novos significados à história da comunidade a partir da
história de cada sujeito. Visto que todos nós trazemos diferentes repertórios
culturais, conhecimentos incorporados, desejos e sonhos, e nessa escrita da própria
história de vida, significá-la e ressignificá-la são o roteiro a ser considerado e
respeitado para a apreensão de “conhecimentos pertinentes” (MORIN, 2010), a
partir da releitura dos problemas emergentes.
Quando o sujeito é constantemente desafiado a intervir em sua comunidade,
a partir da compreensão dos seus problemas inerentes, por um agir fortalecido pelo
diálogo com o outro e outros, mediante consciências despertas, o diálogo e a ação
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A perspectiva dialógica na formação de professores
373
em conjunto se tornam fundamentos necessários para o exercício de uma cidadania
planetária civilizatória.
Bohm (2005, p. 77) assegura que “o compartilhamento de consciências é
mais importante do que o conteúdo das opiniões”. Acrescenta, ainda, que “a própria
estrutura de um pressuposto ou opinião implica que eles devem estar abertos à
demonstração de que podem não estar corretos”. Por isso, o exercício do diálogo é
atitude a ser desenvolvida nos espaços destinados à formação docente, para que no
exercício futuro da profissão, os sujeitos do ensino possam exercê-la, e com
protagonismo gestar soluções aos problemas emergentes, movidos sempre por uma
prática fundamentada e desenvolvida na perspectiva dialógica. Para tanto, o agir
pressupõe disposição ao risco, coragem para enfrentar as suas incertezas, e dar um
salto na indeterminação, pela superação do pensamento unilateral, em um deverfazer irresistível em coletividade.
Assim, é fundamental a criação de espaços pedagógicos dialógicos, de escuta
sensível ao outro, às suas ideias e opiniões, às suas histórias e trajetórias, colocando
em suspenso todo e qualquer tipo de julgamento, em vista da possibilidade de
abertura de novos caminhos de diálogo, visto que não há caminho único para a
busca da verdade, pois todas as trilhas apontam para se chegar ao “não-caminho”
(BOHM, 2005).
Vida e conhecimento, portanto, são indissociáveis no processo de aprender e
de conhecer a realidade em quaisquer espaços formativos. Separar a história das
ações biológicas e sociais do sujeito de seu contexto torna-se inviável, pois o sujeito
se constrói nas relações dialógicas. É, pois, por meio desse imbricamento que o
mundo aparece, à própria imagem e semelhança, como assim asseguram Maturana e
Varela (2001).
Para tanto, é válido reforçar a importância do reconhecimento da diversidade
das condições humanas presentes nos ambientes formativos e o respeito às suas
diferentes histórias de vida. Desta forma, ao se perceberem reconhecidos, os sujeitos
da dinâmica educativa poderão sentir-se parte de um todo bem maior que os
engloba, aprendendo e ensinando na relação uns com os outros, mediados pelas
trocas intersubjetivas de conhecimentos, na tentativa de enfrentar as incertezas
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ARAÚJO, L.; SÍVERES, L.
presentes na vida para evoluir psicológica, afetiva e socialmente, pela compreensão
uns dos outros.
Enfim, Ensinar a condição humana, enfrentar as incertezas e ensinar a compreensão é
fazer com que o sujeito se perceba como parte constitutiva dessa sociedade
complexa, que não cabe em uma gaiola, capacitando o sujeito a enfrentar com
autonomia suas incertezas a partir de um olhar para dentro de si mesmo,
reconhecendo-se, tanto os professores quanto os estudantes, como protagonistas na
dinâmica que envolve o viver e o aprender nos espaços de formação formalmente
reconhecidos. É condição necessária para a construção da identidade profissional
docente, humana e terrena, mantida e transformada na alteridade, possibilitada pelo
diálogo (BUBER, 1987, 2001), no reconhecimento de si mesmo e do outro, de sua
identidade a partir de sua construção histórico-cultural como sujeito pertencente a
essa humanidade caminhante.
É importante reforçar que o exercício da dialogicidade vai além do encontro
do sujeito com os saberes apreendidos nos espaços educativos formais. Constitui-se
em um processo de retomada da voz interior dos sujeitos, do respeito aos seus
valores construídos em comunidade e aos seus saberes produzidos, pois o diálogo é
o propulsor e a conexão para o estabelecimento do elo entre o sujeito e a vida. De
acordo com Freire (1987), é a essência constitutiva da educação como prática da
liberdade, com responsabilidade, mesmo que relativa.
A criação e o fortalecimento de espaços para o exercício da dialogicidade
ampliam as possibilidades de os sujeitos vivenciarem momentos de aprendizagens
com mais solidariedade, constituindo-se e desenvolvendo-se humanamente, de
forma respeitosa na dinâmica da formação de professores. Este processo é
fundamental para a ocorrência de momentos de construção, desconstrução e
reconstrução de significância e ressignificação, de produção de novas zonas de
sentido, mediante relacionamentos saudáveis com o outro e os outros, legitimados
pela presença do sujeito e da percepção de sua importância para a constituição de si
mesmo, na coletividade, a ser processada e desenvolvida autopoieticamente.
A formação de professores compreendida e desenvolvida nessa perspectiva
da dialogicidade reveste-se da autopoiese, cujo pressuposto de sua dinâmica visa
aproximar relacionalmente sujeito e vida, pois no processo construtivo do
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A perspectiva dialógica na formação de professores
375
conhecimento, o sujeito precisa estar em interação permanente consigo, com o
outro e com o meio cultural. Em plena congruência e coerência com as próprias
ideias, sem deixar perder seus valores inerentes.
As formas de sentir e ser (dialógico), pensar e saber (dialético) e de atuar e
agir (dialogicidade) do sujeito perante a vida, pelo próprio jeito de apreender a
realidade e de transformar o fazer, ao mesmo tempo em que o ser vai se
reconstruindo, é processo que envolve um conjunto de relações complexas
apreendidas e ressignificadas cotidianamente no decurso do viver/conviver. Estas
atitudes são expressões da subjetividade, traduzidas como a síntese da singularidade
e da individualidade humana construída e constituída no decurso da vida social e
cultural, da dialogicidade estabelecida entre a subjetividade social (saberes
intersubjetivos) e a subjetividade individual (saberes intra-subjetivos). Esta seria a
fonte criativa em que fluem as ideias, as emoções, a identidade do sujeito, sua marca
singular que o identifica e que o faz ser igual em meio às diferenças, e ser respeitado
em sua pluralidade cultural e singular, enquanto cidadão participante de sua
comunidade local/global.
Considerações finais
As instituições formadoras (públicas e privadas) ainda são os espaços
formativos favoráveis para os atuais e futuros professores aprenderem e se
desenvolverem em níveis de conhecimento, construírem e se constituírem mediante
a apropriação da cultura em circulação. No entanto, requer dos próprios membros
dessas comunidades que sejam sujeitos ativos, e em conjunto com seus pares,
busquem as condições necessárias para que os processos de desenvolvimento
ocorram na relação uns com os outros, respeitando-se a realidade situada. Engajados
nas ações educativas, professores e estudantes, pais, direção, etc., permanentemente
em relação, sintam-se corresponsáveis pelo crescimento de sua comunidade e
coautores da cultura que os constituirá.
Nos espaços formativos compreendidos dessa forma, as relações e os
processos educativos se alternam e se alteram, pois o aprender e o ensinar requerem
envolvimento, motivos para um agir dos docentes, dos discentes e de todos os
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ARAÚJO, L.; SÍVERES, L.
envolvidos, direta ou indiretamente, no convívio educacional. Além disso, é
necessário estar atento aos interesses dos professores em formação, para que estes
também estejam atentos aos interesses dos seus estudantes, identificando os seus
problemas pertinentes e seus desejos. Isto, que exige sensibilidade por parte,
também, dos responsáveis pelos processos formativos, sensibilidade para com a
percepção do outro ao olhar o mundo, ao seu agir, pois dependendo das
circunstâncias, estas podem limitar ou potencializar os sujeitos do ensino e da
aprendizagem a seguirem em busca de alternativas que lhe possibilite criar para
transpor o problema emergente.
Daí a importância de o(a) professor(a) — com consciência epistemológica —
ser capaz de criar as condições necessárias para os sujeitos ascenderem em termos
de conhecimento, abrindo-lhes possibilidades de caminhos, brechas para a
apropriação dos bens culturais em relação ao zeitgeist (espírito de época), e de ajudálos a transformar os saberes (as informações apreendidas) em conhecimentos
pertinentes, com significância e significado, e a construir e reconstruir novas “zonas
de produção de sentidos” em relação ao conhecimento, para que possam alcançar as
melhores soluções e alternativas para a superação dos problemas de sua comunidade
educativa, em coletividade.
Mudar a cultura dos cursos de formação de professores, há tempos habituada
com as formas convencionais de monólogos e discurso unilateral impregnados há
séculos, não é tão simples assim. Requer coragem e muita disposição por parte de todo
o corpo gestor escolar para superar a repetição de modelos e práticas já conhecidos.
A reorganização do espaço pedagógico, com foco na valorização das histórias de
vida dos sujeitos e de suas relações de aprendizagem, tendo o diálogo como princípio
fundante e regenerador, constitui uma das vias possíveis para propiciar as condições
necessárias de gestão, valorizando-se os pensamentos e ações, para que possibilitem a
concretude do fazer pedagógico revolucionário e transformador.
Assim, uma caminhada educativa construída e demarcada por um constante
processo de reflexão, de escuta e compartilhamento de significados, por meio de
ações concretas, ainda que certo do risco que a incerteza das escolhas pode
despertar e desenvolver os princípios fundantes do diálogo: o ouvir, o compartilhar
e o agir.
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A perspectiva dialógica na formação de professores
377
Portanto, estar articulado pelos três fios da trança dos saberes que envolvem
o ensinar a condição humana, o enfrentamento das incertezas e o ensinamento da
compreensão —
compreendidos a
partir das dimensões: antropológica,
epistemológica e pedagógica — suscita a condição de criar e recriar as utopias,
abertura de possibilidades de produção de conhecimentos pertinentes, e percorrer
caminhos inovadores na docência. Todo este procedimento mesclado pelo diálogo
pode possibilitar relações humanas mais significativas, e promover processos de
formação de professores numa perspectiva mais dialógica e solidária, mais saudável
e humanizadora.
Referências
BARDIN, L. Análise de conteúdo. Lisboa: Edições 70, 2002.
BOFF, L. A águia e a galinha: uma metáfora da condição humana. Petrópolis: Vozes, 1997.
BOHM, D. Diálogo: comunicação e redes de convivência. São Paulo: Palas Athena, 2005.
BUBER, M. Sobre comunidade. São Paulo: Perspectiva, 1987.
BUBER, M. Eu e Tu. 8. ed. São Paulo: Centauro, 2001.
D'AMBRÓSIO, U. Transdisciplinaridade. 2. ed. São Paulo: Palas Athena, 2009.
FREIRE, P. Pedagogia do Oprimido. 17. ed. Rio de Janeiro: Paz e Terra, 1987.
FREIRE, P. Pedagogia da Autonomia: saberes necessários à prática educativa. 3. ed. São
Paulo: Paz e Terra, 1996.
HABERMAS, J. Teoria do agir comunicativo. Racionalidade da ação e racionalização social.
Trad. Paulo Astor Soethe. São Paulo: WMF Martins Fontes, 2012.
LARAIA, R. Cultura: um conceito antropológico. Rio de Janeiro: Zahar, 1986.
LEMOS, A. Cibercultura, tecnologias e vida social na cultura contemporânea. Porto Alegre: Sulina, 2004.
MARIOTTI, H. Pensamento complexo: suas aplicações à liderança, à aprendizagem e ao
desenvolvimento sustentável. São Paulo: Atlas, 2007.
MATURANA, H.; VARELA, F. A árvore do conhecimento: as bases biológicas da
compreensão humana. 5. ed. São Paulo: Palas Athena, 2001.
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MORIN, E. Da necessidade de um pensamento complexo. In: MARTINS, F. M; SILVA, J.
M. Para navegar no século XXI: tecnologias do imaginário e cibercultura. Porto Alegre:
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MORIN, E. Introdução ao pensamento complexo. 3. ed. Porto Alegre: Sulina, 2007.
MORIN, E. A cabeça bem-feita: repensar a reforma, reformar o pensamento. 15. ed. Rio de
Janeiro: Bertrand Brasil, 2008.
MORIN, E. Os sete saberes necessários à educação do futuro. 2. ed. Brasília: Unesco, 2010.
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NÓVOA, A. Sete disposições necessárias ao educador do presente. In: Conferência
Internacional: Saberes para uma cidadania planetária. (palestra proferida), Fortaleza (CE), 27 de
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em: <http://www.unesco.org/new/pt/brasilia/about-this-office/singleview/news/international_conference_discusses_education_humanism_ethi/>. Acesso em:
23 maio 2016.
RECEBIDO: 21/06/2019
APROVADO: 05/02/2020
RECEIVED: 06/21/2019
APPROVED: 02/05/2020
RECIBIDO: 21/06/2019
APROBADO: 05/02/2020
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https://ccforum.biomedcentral.com/track/pdf/10.1186/cc8093
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T-cell-specific peroxisome proliferator-activated receptor gamma depletion inhibits T-cell apoptosis and improves survival of septic mice via an IL-2-dependent mechanism
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Critical care
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cc-by
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P2 Methods Pneumonia was induced by intranasal instillation of
bacteria (104 CFU) while sepsis was developed by placing the
fibrin–thrombin clot containing a known amount of bacteria
(102 CFU) into the peritoneal cavity of animals. Various cytokine
(TNFα and IL-1α) levels were estimated using ELISA and the
degree of lung inflammation (that is, inflammatory cell infiltration)
was evaluated by histopathological analysis. The other markers of
inflammation (that is, nitric oxide (NO), malondialdehyde (MDA)
and myeloperoxidase (MPO)) were estimated by standard
biochemical methods. Introduction Urokinase receptor (uPAR, CD87), a glycosylphos-
phatidylinositol-anchored protein, is considered to play an
important role in inflammation and fibrinolysis. The Gram-negative
bacterium Burkholderia pseudomallei is able to survive and
replicate within leukocytes and causes melioidosis, an important
cause of pneumonia-derived community-acquired sepsis in South-
east Asia. We here investigated the expression and function of
uPAR both in patients with septic melioidosis and in a murine
model of experimental melioidosis. Introduction Urokinase receptor (uPAR, CD87), a glycosylphos-
phatidylinositol-anchored protein, is considered to play an
important role in inflammation and fibrinolysis. The Gram-negative
bacterium Burkholderia pseudomallei is able to survive and
replicate within leukocytes and causes melioidosis, an important
cause of pneumonia-derived community-acquired sepsis in South-
east Asia. We here investigated the expression and function of
uPAR both in patients with septic melioidosis and in a murine
model of experimental melioidosis. Results Mice with sepsis showed 100% mortality within 5 post
infection days whereas all the animals with pneumonia survived. In
animals suffering from K. pneumoniae B5055-induced pneumonia
all the inflammatory parameters (TNFα, IL-1α, MPO, MDA and NO)
were found to be maximum until the third post infection day, after
that a decline was observed, whereas in septic animals all the
above-mentioned markers of inflammation kept on increasing until
death of the animals. Histopathological study showed that
inflammatory damage to the lungs in pneumonia was not very
severe as lesser neutrophil infiltration and pulmonary damage (that
is, alveolitis, bronchiolitis, endothelitis and perivascular congestion)
was seen as compared with lungs taken from septic animals. This
can be further strengthened by the presence of alternatively
activated alveolar macrophages (AAMacs) or foam cells in lungs of
mice with pneumonia after the third post infection day and their
number kept on increasing until the seventh post infection day,
which might have contributed to the induction of resolution of
inflammation and clearance of the infection. P2 Urokinase receptor is necessary for bacterial defense
against Gram-negative sepsis (melioidosis) by facilitating
phagoctytosis A comparison of acute lung inflammation in Klebsiella
pneumoniae B5055-induced pneumonia and sepsis in
BALB/c mice phagoctytosis
W Joost Wiersinga1,2, JWR Hovius1,2, GJW van der Windt1,2,
JCM Meijers3, JJ Roelofs4, A Dondorp5, M Levi1, NP Day5,6,
SJ Peacock5,6, T van der Poll1,2
1Center for Infection and Immunity Amsterdam, 2Center for
Experimental and Molecular Medicine, 3Department of Vascular
Medicine and 4Department of Pathology, Academic Medical
Center, University of Amsterdam, Amsterdam, the Netherlands;
5Mahidol-Oxford Tropical Medicine Research Unit, Faculty of
Tropical Medicine, Mahidol University, Bangkok, Thailand; 6Center
for Clinical Vaccinology and Tropical Medicine, Nuffield Department
of Clinical Medicine, University of Oxford, UK
Critical Care 2009, 13(Suppl 4):P1 (doi: 10.1186/cc8057) W Joost Wiersinga1,2, JWR Hovius1,2, GJW van der Windt1,2,
JCM Meijers3, JJ Roelofs4, A Dondorp5, M Levi1, NP Day5,6,
SJ Peacock5,6, T van der Poll1,2
1Center for Infection and Immunity Amsterdam, 2Center for
Experimental and Molecular Medicine, 3Department of Vascular
Medicine and 4Department of Pathology, Academic Medical
Center, University of Amsterdam, Amsterdam, the Netherlands;
5Mahidol-Oxford Tropical Medicine Research Unit, Faculty of
Tropical Medicine, Mahidol University, Bangkok, Thailand; 6Center
for Clinical Vaccinology and Tropical Medicine, Nuffield Department
of Clinical Medicine, University of Oxford, UK
Critical Care 2009, 13(Suppl 4):P1 (doi: 10.1186/cc8057) V Kumar, S Chibber
Department of Microbiology, Panjab University, Chandigarh, India
Critical Care 2009, 13(Suppl 4):P2 (doi: 10.1186/cc8058) W Joost Wiersinga1,2, JWR Hovius1,2, GJW van der Windt1,2,
JCM Meijers3, JJ Roelofs4, A Dondorp5, M Levi1, NP Day5,6,
SJ Peacock5,6, T van der Poll1,2
1Center for Infection and Immunity Amsterdam, 2Center for
Experimental and Molecular Medicine, 3Department of Vascular
Medicine and 4Department of Pathology, Academic Medical
Center, University of Amsterdam, Amsterdam, the Netherlands;
5Mahidol-Oxford Tropical Medicine Research Unit, Faculty of
Tropical Medicine, Mahidol University, Bangkok, Thailand; 6Center
for Clinical Vaccinology and Tropical Medicine, Nuffield Department
of Clinical Medicine, University of Oxford, UK
Critical Care 2009, 13(Suppl 4):P1 (doi: 10.1186/cc8057) Introduction Lungs play an important role in the body’s defense
against a variety of pathogens, but this network of immune system
mediated defense can be deregulated during acute pulmonary
infections. Objective The present study compares the acute lung inflam-
mation (ALI) occurring during Klebsiella pneumoniae B5055-
induced pneumonia and sepsis in BALB/c mice. Available online http://ccforum.com/supplements/13/S4 Available online http://ccforum.com/supplements/13/S4 Amsterdam, the Netherlands, 11–14 November 2009 Published online: 11 November 2009
These abstracts are available online at http://ccforum.com/supplements/13/S4
© 2009 BioMed Central Ltd Published online: 11 November 2009
These abstracts are available online at http://ccforum.com/supplements/13/S4
© 2009 BioMed Central Ltd P1
Urokinase receptor is necessary for bacterial defense
against Gram-negative sepsis (melioidosis) by facilitating
phagoctytosis
W Joost Wiersinga1,2, JWR Hovius1,2, GJW van der Windt1,2,
JCM Meijers3, JJ Roelofs4, A Dondorp5, M Levi1, NP Day5,6,
SJ Peacock5,6, T van der Poll1,2
1Center for Infection and Immunity Amsterdam, 2Center for
Experimental and Molecular Medicine, 3Department of Vascular
Medicine and 4Department of Pathology, Academic Medical
Center, University of Amsterdam, Amsterdam, the Netherlands;
5Mahidol-Oxford Tropical Medicine Research Unit, Faculty of
Tropical Medicine, Mahidol University, Bangkok, Thailand; 6Center
for Clinical Vaccinology and Tropical Medicine, Nuffield Department
of Clinical Medicine, University of Oxford, UK
Critical Care 2009, 13(Suppl 4):P1 (doi: 10.1186/cc8057) P3
Performance evaluation and further development of the
PCR and microarray-based Prove-it™ Sepsis assay P3 Methods DNA was extracted from blood cultures using the
automated DNA extraction instrument easyMAG (bioMérieux) prior
to the Prove-it™ Sepsis assay. Conventional blood culture was
conducted in parallel and results were only revealed for
comparison at the statistical analysis stage. Discordant results
were studied by DNA sequencing and case-by-case review of
original microbiology laboratory data. P Tissari1, E Tarkka1, S Mero1, L Savolainen1, M Vaara1,
A Zumla2, J Huggett2, C Carder2, V Gant2, A Aittakorpi3,
S Laakso3, M Lindfors3, P Piiparinen3, N Kumlin3, H Piiparinen3,
M Mäki3 P Tissari1, E Tarkka1, S Mero1, L Savolainen1, M Vaara1,
A Zumla2, J Huggett2, C Carder2, V Gant2, A Aittakorpi3,
S Laakso3, M Lindfors3, P Piiparinen3, N Kumlin3, H Piiparinen3,
M Mäki3 Results Of the analyzed 3,318 blood cultures, 2,107 yielded a
pathogen by conventional techniques. Of these, 302 samples
contained microbes not covered by Prove-it™ Sepsis, and an
additional 137 cultures contained more than one organism. Sensitivity and specificity for Prove-it™ Sepsis were 94.7% and
98.7%, respectively. Of particular significance was the assay’s
faultless ability to differentiate MRSA from MSSA and from CNS. Furthermore, it provided results on average 1 day earlier than
reference methods. Introduction The Prove-it™ Sepsis assay is a rapid, broad-range
PCR and microarray-based assay designed to identify the majority
of sepsis-causing bacteria from positive blood cultures. The
pathogen panel covers 50 Gram-negative and Gram-positive
bacterial species (Table 1). It also reports methicillin resistance by
detecting the mecA gene. The assay time is 3 hours. Our objective
was to conduct a performance evaluation study for Prove-it™
Sepsis according to the EN 13612-standard (Performance
evaluation of in vitro diagnostic medical devices) and to compare
obtained results with those of current culture-based diagnostics. We evaluated the sensitivity, specificity and time to result of Prove-
it™ Sepsis in two major teaching hospitals in Helsinki and London. Materials A total of 3,318 blood samples from patients with
suspected sepsis were collected. Blood culture bottles of
BacT/ALERT 3D (bioMérieux) and BACTEC 924 (Becton Dickinson)
were incubated for a total of 6 days or until flagged as positive. Conclusions Prove-it™ Sepsis was considered to be a fast, robust,
and high-performance diagnostic platform, which is easily
implemented into everyday laboratory workflow. Both study sites
identified cases where timely information provided by Prove-it™
Sepsis would have significantly improved patient management. P3
Performance evaluation and further development of the
PCR and microarray-based Prove-it™ Sepsis assay Examples include more rational management and antibiotic choice
subsequent to earlier differentiation of Gram-positive cocci in
clumps into MRSA, MSSA, or CNS, and earlier speciation of
Gram-negative organisms. Prove-it™ Sepsis is further configured
for detection of Candida spp. and new bacterial targets. The assay
now identifies 60 out of the 302 samples not covered during the
evaluation, increasing the pathogen coverage from 86% to 89%. The earlier speciation provided by Prove-it™ Sepsis could
contribute to faster, more evidence-based patient management
and, thus, positive outcomes. Methicillin resistance marker mecA P2 But no such AAMacs
or foam cells were seen in lungs of septic mice on histo-
pathological examination, lungs were seen to be infiltrated with
only neutrophils on all experimental days. Methods Using a translational approach we conducted a patient
study in patients with culture-confirmed sepsis caused by
B. pseudomallei, in vitro experiments using wild-type (WT) and
uPAR knockout (KO) cells, and mouse studies using WT and
uPAR KO mice inoculated with B. pseudomallei. Results uPAR mRNA and surface expression was increased in
patients with septic melioidosis in/on both peripheral blood
monocytes and granulocytes as well as in the pulmonary
compartment during experimental pneumonia-derived melioidosis
in
mice. uPAR-deficient
mice
intranasally
infected
with
B. pseudomallei showed an enhanced growth and dissemination
of B. pseudomallei when compared with WT mice, corresponding
with increased pulmonary and hepatic inflammation. uPAR KO
mice demonstrated significantly reduced neutrophil migration
towards the pulmonary compartment after inoculation with
B. pseudomallei. Further in vitro experiments showed that uPAR-
deficient macrophages and granulocytes display a markedly
impaired phagocytosis of B. pseudomallei. Additional studies
showed that uPAR deficiency did not influence hemostatic and
fibrinolytic responses during severe melioidosis. Conclusions These data suggest that uPAR is crucially involved in
the host defense against sepsis caused by B. pseudomallei by
facilitating the migration of neutrophils towards the primary site of
infection and subsequently facilitating the phagocytosis of
B. pseudomallei. Conclusions Hence, during pneumonia controlled activation of
AAMacs or foam cells led to the resolution of inflammation and
infection as well. S1 Critical Care November 2009 Vol 13 Suppl 4
Sepsis 2009 P3
Performance evaluation and further development of the
PCR and microarray-based Prove-it™ Sepsis assay
P Tissari1, E Tarkka1, S Mero1, L Savolainen1, M Vaara1,
A Zumla2, J Huggett2, C Carder2, V Gant2, A Aittakorpi3,
S Laakso3, M Lindfors3, P Piiparinen3, N Kumlin3, H Piiparinen3,
M Mäki3
1Division of Clinical Microbiology, Helsinki University Hospital
Laboratory HUSLAB, Helsinki, Finland; 2Department of Clinical
Microbiology, University College London Hospitals NHS
Foundation Trust, and University College London Medical School,
Centre for Infectious Diseases and International Health, London,
UK; 3Mobidiag Ltd, Helsinki, Finland
Critical Care 2009, 13(Suppl 4):P3 (doi: 10.1186/cc8059) P5 Use of a screening authorization and randomization center
for severe sepsis patient qualification and real-time
enrollment in a phase 2 trial of eritoran tetrasodium
(E5564), a TLR4 antagonist Objective The aim of this study was to evaluate the efficacy of
eritoran for interaction effects with baseline illness severity. Methods Prospective covariates from a randomized, double-blind,
placebo-controlled, phase 2 trial were analyzed for treatment
interaction measured by 28-day mortality. Breslow–Day and
multiple logistic regression (LR) were used to assess categorical
(CAT) and continuous (CONT) treatment by severity-of-illness
interactions. J Schentag1, S Opal2, M Lynn3, A Wittek3, J Wheeler3
1University at Buffalo School of Pharmacy and Pharmaceutical
Sciences, Buffalo, NY, USA; 2Brown University Medical School,
Providence, RI, USA; 3Eisai Medical Research, Inc., Ridgefield
Park, NJ, USA
Critical Care 2009, 13(Suppl 4):P5 (doi: 10.1186/cc8061) Results Modified intent-to-treat population (n = 292) all-cause 28-
day mortality was: placebo, 33.3% (32/96); total eritoran 45
mg/105 mg, 29.6% (58/196). LR analysis identified Acute
Physiology and Chronic Health Evaluation (APACHE) II scores,
Predicted Risk of Mortality (PROM) scores, IL-6, age, sex, race,
and eritoran as associated with survival outcomes. Significant
treatment interactions were observed (eritoran vs. placebo) for
baseline covariates: APACHE II (CAT, P = 0.059; CONT, P = 0.035);
PROM scores (CAT, P = 0.028; CONT, P = 0.008); number of
organ failures (CAT, P = 0.079); international normalized ratio
(CAT, P = 0.05); and acute physiology score (CONT, P = 0.039). No significant treatment interactions were observed with age, sex,
shock, DAA use, infection site, microorganism type, platelets, IL-6,
or endotoxin levels. Interaction results were similar for eritoran
105 mg only versus placebo. Introduction Trials of many promising sepsis modifiers have often
failed to demonstrate benefits because of, among other reasons,
poor characterization of enrolled patients. Materials Advantages of utilizing a screening authorization and
randomization center (SAC) method to characterize patients in
trials for sepsis modifiers are presented. Methods A central SAC on call 24 hours per day was employed in
a phase 2, double-blind, randomized comparison of eritoran 45
and 105 mg versus placebo. SAC activities were conducted
(January 2002 to April 2005) by six clinical pharmacists. Severe
sepsis was defined with at least three systemic inflammatory
response syndrome criteria within 12 hours before onset of ≥1
new organ dysfunction. Patients were randomized and treated
within 8 to 12 hours. Nandrolone abuse aggravates septic shock YF Hsu1, C Lin1,2, D-R Chen1,2
1Department of Research, and 2Department of Surgery, Changhua
Christian Hospital, Changhua, Taiwan
Critical Care 2009, 13(Suppl 4):P4 (doi: 10.1186/cc8060) Results The SAC screened 1,025 patients from 78 sites; 300
patients from 54 sites were randomized, and 293 were treated. The Independent Clinical Evaluation Committee subsequently
qualified 229/293 (78%). Calls to the SAC averaged 24 patients/
month; eight patients/month were randomized (33%). Enrollments
ranged from 0 to 39 patients/site; 35 sites randomized ≥3 patients;
nine sites randomized ≥10 patients. Of 35 sites with ≥3 patients,
6 to 100% led to randomization. Introduction One million individuals in the United States alone are
estimated to be current or past users of anabolic-androgenic
steroid. Methods To investigate the effects of nandrolone, an anabolic-
androgenic steroid, 108 6-week-old male BALB/c inbred mice
were used (minimal lethal dose n = 30, high-dose vs. low-dose
n = 24, screening surrogates n = 6, and surrogates n = 48). Mice
were sacrificed at 0, 3 or 6 hours after septic shock induction. The
serum levels of malondialdehyde, liver TNFα and spleen IFNγ in
mice with septic shock were analyzed. The gene expression of
insulin-like growth factor 1, insulin-like growth factor type 1
receptor and insulin-like growth factor binding proteins was also
studied. Conclusions Advantages of utilizing the SAC included a high
evaluable rate of enrolled patients, correct Predicted Risk of
Mortality calculations, timing of qualifying organ failures, drug
preparation advice, verification of key clinical data, and eligibility
with sponsor prior to enrollment. SAC activities ensured an
informative phase 2 trial and will be utilized for the phase 3 trial. Results Nandrolone significantly increased serum malondialde-
hyde at 0, 3 and 6 hours (P = 0.004, 0.006 and 0.004), and liver
TNFα at 0 and 6 hours (P = 0.04 and 0.016). It also increased the
spleen IFNγ level at 0 and 6 hours (P = 0.031 and 0.01). Com-
pared with 0 hours, the data indicated that nandrolone increases
lung insulin-like growth factor type 1 receptor, insulin-like growth
factor binding protein 1 and insulin-like growth factor binding
protein 2 mRNA expression at 6 hours (P <0.05). These indicated
changes due to nandrolone. Available online http://ccforum.com/supplements/13/S4 Available online http://ccforum.com/supplements/13/S4 Available online http://ccforum.com/supplements/13/S4 based on Acute Physiology and Chronic Health Evaluation
(APACHE) II predicted mortality to yield a balanced allocation of
high and low APACHE II predicted mortality within the three
treatment groups: one sequence for subjects with APACHE II
predicted mortality 20 to 50%, and another for subjects with
predicted mortality 51 to 80% as calculated by the SAC. Results The SAC screened 1,025 patients from 78 sites; 300
patients from 54 sites were randomized, and 293 were treated. The Independent Clinical Evaluation Committee subsequently
qualified 229/293 (78%). Calls to the SAC averaged 24 patients/
month; eight patients/month were randomized (33%). Enrollments
ranged from 0 to 39 patients/site; 35 sites randomized ≥3 patients;
nine sites randomized ≥10 patients. Of 35 sites with ≥3 patients,
6 to 100% led to randomization. Conclusions Advantages of utilizing the SAC included a high
evaluable rate of enrolled patients, correct Predicted Risk of
Mortality calculations, timing of qualifying organ failures, drug
preparation advice, verification of key clinical data, and eligibility
with sponsor prior to enrollment. SAC activities ensured an
informative phase 2 trial and will be utilized for the phase 3 trial. P6
Influence of severity of illness on the effects of eritoran
tetrasodium (E5564), a TLR4 antagonist, in patients with
severe sepsis
SM Opal1, AC Kalil2, SP LaRosa1, J Gogate3, M Lynn3,
AE Wittek3, and the Eritoran Sepsis Study Group
1Brown University, Providence, RI, USA; 2University of Nebraska
Medical Center, Omaha, NE, USA; 3Eisai Medical Research, Inc.,
Ridgefield Park, NJ, USA
Critical Care 2009, 13(Suppl 4):P6 (doi: 10.1186/cc8062) P4 based on Acute Physiology and Chronic Health Evaluation
(APACHE) II predicted mortality to yield a balanced allocation of
high and low APACHE II predicted mortality within the three
treatment groups: one sequence for subjects with APACHE II
predicted mortality 20 to 50%, and another for subjects with
predicted mortality 51 to 80% as calculated by the SAC. based on Acute Physiology and Chronic Health Evaluation
(APACHE) II predicted mortality to yield a balanced allocation of
high and low APACHE II predicted mortality within the three
treatment groups: one sequence for subjects with APACHE II
predicted mortality 20 to 50%, and another for subjects with
predicted mortality 51 to 80% as calculated by the SAC. Nandrolone abuse aggravates septic shock P6 Influence of severity of illness on the effects of eritoran
tetrasodium (E5564), a TLR4 antagonist, in patients with
severe sepsis
SM Opal1, AC Kalil2, SP LaRosa1, J Gogate3, M Lynn3,
AE Wittek3, and the Eritoran Sepsis Study Group
1Brown University, Providence, RI, USA; 2University of Nebraska
Medical Center, Omaha, NE, USA; 3Eisai Medical Research, Inc.,
Ridgefield Park, NJ, USA
Critical Care 2009, 13(Suppl 4):P6 (doi: 10.1186/cc8062) Influence of severity of illness on the effects of eritoran
tetrasodium (E5564), a TLR4 antagonist, in patients with
severe sepsis Influence of severity of illness on the effects of eritoran
tetrasodium (E5564), a TLR4 antagonist, in patients with
severe sepsis
SM Opal1, AC Kalil2, SP LaRosa1, J Gogate3, M Lynn3,
AE Wittek3, and the Eritoran Sepsis Study Group
1Brown University, Providence, RI, USA; 2University of Nebraska
Medical Center, Omaha, NE, USA; 3Eisai Medical Research, Inc.,
Ridgefield Park, NJ, USA
Critical Care 2009, 13(Suppl 4):P6 (doi: 10.1186/cc8062) Conclusions Nandrolone abuse hastens mortality due to septic
shock and increases serum malondialdehyde, liver TNFα, spleen
IFNγ level and lung insulin-like growth factor type 1 receptor
mRNA, as well as lung insulin-like growth factor binding proteins. Nandrolone abuse may aggravate septic shock. Introduction Disease severity varies widely in patients with severe
sepsis. Previous trials (IL-1RA, TNF-sR p55, antithrombin, and
drotrecogin alfa activated (DAA)) suggest that more severely ill
patients benefit most from treatment. Table 1 (abstract P3) Bacteria and an antibacterial resistance marker identified by the Prove-it™ Sepsis assay
Gram-negative
Gram-positive
Antibacterial resistance
Neisseria meningitidis
Staphylococcus aureus
Methicillin resistance marker mecA
Enterobacter aerogenes
Staphylococcus epidermidis
Enterobacter cloacae
Coagulase-negative Staphylococcusd
Escherichia coli
Streptococcus pyogenes
Klebsiella oxytoca
Streptococcus agalactiae
Klebsiella pneumoniae
Streptococcus dysgalactiae subsp. equisimilis
Proteus mirabilis
Streptococcus pneumoniae
Proteus vulgaris
Enterococcus faecalis
Salmonella enterica subsp. entericaa
Enterococcus faecium
Serratia marcescens
Listeria monocytogenes
Enterobacteriaceae familyb
Clostridium perfringens
Acinetobacter baumannii
Pseudomonas aeruginosa
Stenotrophomonas maltophilia
Haemophilus influenzae
Campylobacter jejuni/coli
Bacteroides fragilis groupc
aSalmonella enterica subsp. enterica covers at least the following serovars: Enteritidis, Oranienburg, Othmarschen, Paratyphi, Stanley, Typhi, Typhimurium,
Virchow, Group A, B, C, D. bEnterobacteriaceae covers at least the following species: Citrobacter amalonaticus, Citrobacter freundii, Citrobacter koseri,
Citrobacter braakii, Enterobacter hormaechei, Enterobacter sakazakii, Kluyvera intermedia, Morganella morganii, Pantoea agglomerans, Providencia rettgeri,
Providencia stuartii, Yersinia enterocolitica, Yersinia pseudotuberculosis. cBacteroides fragilis covers at least the following species: B. fragilis, B. vulgatus,
B. thetaiotaomicron. dCoagulase-negative Staphylococcus covers at least the following species: S. capitis, S. lugdunensis, S. haemolyticus, S. hominis, S. saprophyticus, S. warneri, S. xylosus. Bacteria and an antibacterial resistance marker identified by the Prove-it™ Sepsis assay aSalmonella enterica subsp. enterica covers at least the following serovars: Enteritidis, Oranienburg, Othmarschen, Paratyphi, Stanley, Typhi, Typhimurium,
Virchow, Group A, B, C, D. bEnterobacteriaceae covers at least the following species: Citrobacter amalonaticus, Citrobacter freundii, Citrobacter koseri,
Citrobacter braakii, Enterobacter hormaechei, Enterobacter sakazakii, Kluyvera intermedia, Morganella morganii, Pantoea agglomerans, Providencia rettgeri,
Providencia stuartii, Yersinia enterocolitica, Yersinia pseudotuberculosis. cBacteroides fragilis covers at least the following species: B. fragilis, B. vulgatus,
B. thetaiotaomicron. dCoagulase-negative Staphylococcus covers at least the following species: S. capitis, S. lugdunensis, S. haemolyticus, S. hominis, S. saprophyticus, S. warneri, S. xylosus. S2 P7 Introduction Unregulated elevated levels of serum TNFα have
been associated with proinflammatory cytokine cascades that are
characteristic in diseases such as septic shock. Endotoxic shock,
which has a poorer prognosis than found with other forms of septic
shock, is mediated by lipopolysaccharide (LPS), a molecule that is
released from the outer membrane of Gram-negative bacteria. LPS
is a potent stimulator of TNFα secretion by serum monocytes and
tissue macrophages. Whilst the use of monotherapeutic TNFα
antagonists has been trialed, none have been registered for use in
patients with sepsis. Introduction Unregulated elevated levels of serum TNFα have
been associated with proinflammatory cytokine cascades that are
characteristic in diseases such as septic shock. Endotoxic shock,
which has a poorer prognosis than found with other forms of septic
shock, is mediated by lipopolysaccharide (LPS), a molecule that is
released from the outer membrane of Gram-negative bacteria. LPS
is a potent stimulator of TNFα secretion by serum monocytes and
tissue macrophages. Whilst the use of monotherapeutic TNFα
antagonists has been trialed, none have been registered for use in
patients with sepsis. Faster differentiation of Staphylococcus aureus versus
coagulase-negative Staphylococci from blood culture
material: a comparison of different bacterial DNA isolation
methods AJM Loonen1, WLJ Hansen1, A Jansz2, H Kreeftenberg2,
CA Bruggeman1, PFG Wolffs1, AJC van den Brule3
1Department of Medical Microbiology, Maastricht University Medical
Center, Maastricht, the Netherlands; 2Department of Intensive Care,
Catharina Hospital, Eindhoven, the Netherlands; 3Department of
Molecular Diagnostics, Catharina Hospital, Eindhoven, the Netherlands
Critical Care 2009, 13(Suppl 4):P7 (doi: 10.1186/cc8063) Objective The purpose of this study was to test the effect of
canine hyperimmune frozen plasma (HFP), which is known to
contain elevated levels of soluble TNFα receptor 1 (sTNFR1), on
TNFα and inflammatory cell levels in a LPS-mediated rat air pouch
model of inflammation. Introduction Frequent usage of medical devices, such as
intravenous lines, often results in sepsis, which is characterized by
high morbidity and mortality. Rapid and reliable detection and
differentiation between Staphylococcus aureus and coagulase-
negative Staphylococci (CNS) is therefore clinically relevant to be
able to provide adequate early treatment. Blood culture is still the
gold standard method in identifying these pathogens but is time
consuming. Molecular diagnostics might be a promising alternative
to reduce this time-to-result delay. Introduction Frequent usage of medical devices, such as
intravenous lines, often results in sepsis, which is characterized by
high morbidity and mortality. Clinical impact of a PCR-based assay for pathogen
detection in critically ill patients with evidence of infection F Bloos1, A Kortgen1, S Sachse2, M Lehmann3, E Straube2,
K Reinhart1, M Bauer1
1Department of Anesthesiology and Intensive Care Medicine,
University Hospital Jena, Germany; 2University Hospital Jena,
Institute of Medical Microbiology, Jena, Germany; 3SIRS-Lab
GmbH, Jena, Germany
Critical Care 2009, 13(Suppl 4):P9 (doi: 10.1186/cc8065) Conclusions A sensitive RT-PCR was developed for detection and
differentiation of S. aureus versus CNS. Bacterial DNA isolation
from Bact/ALERT blood culture material seems to show better
reproducibility compared with isolation from BACTEC blood
culture material. In this preliminary study the EasyMAG performed
better when compared with MolYsis Plus and the MagNA Pure
system. In future work this method will be further evaluated with
reduced culture times. Introduction Blood cultures are often negative even in patients
with clinical signs of severe sepsis. Furthermore, the long time to
result of culture-based methods does not allow the results to guide
empiric antimicrobial therapy. PCR-based pathogen detection
promises a higher rate of positivity and a faster time to result. Introduction Blood cultures are often negative even in patients
with clinical signs of severe sepsis. Furthermore, the long time to
result of culture-based methods does not allow the results to guide
empiric antimicrobial therapy. PCR-based pathogen detection
promises a higher rate of positivity and a faster time to result. Objective To report the performance of PCR-based pathogen
detection compared with blood culture in ICU patients with evidence
of infection, and the impact of this test on the antimicrobial therapy. Methods Patients treated on an interdisciplinary ICU were
included into this observational study if a blood culture (BC) was Introduction Blood cultures are often negative even in patients
with clinical signs of severe sepsis. Furthermore, the long time to
result of culture-based methods does not allow the results to guide
empiric antimicrobial therapy. PCR-based pathogen detection
promises a higher rate of positivity and a faster time to result. Objective To report the performance of PCR-based pathogen
detection compared with blood culture in ICU patients with evidence
of infection, and the impact of this test on the antimicrobial therapy. Methods Patients treated on an interdisciplinary ICU were
included into this observational study if a blood culture (BC) was P7 The
data suggest that canine HFP, which has been demonstrated to
contain elevated levels of sTNFR1 compared with FFP, has a direct
effect on depressing TNFα levels and neutrophil sequestration in
the rat air pouch model of inflammation. These data suggest that
HFP may be worthy of further investigation to determine whether
such preparations have a therapeutic potential for treatment of
acute inflammatory diseases in which TNFα is implicated. Results The best Tuf RT-PCR method appeared to have a
sensitivity of 100 CFU/ml. Approximately 50 positive blood cultures
containing Gram-positive cocci in clusters were tested in the Tuf
RT-PCR and all were identified correctly. Bacterial DNA isolation,
from spiked blood culture material, with the EasyMAG showed the
highest analytical performance with a detection limit of 103 CFU/ml
in Bact/ALERT material, whereas using BACTEC resulted in a
detection limit of 104 CFU/ml. Hand-on time, for 26 samples, was
lowest for the EasyMAG (10 minutes) and highest for the manual kit
of MolZyme (2 hours). Total handling time was highest for the
MolYsis Plus kit (3.5 hours) and lowest for the automated extractor
EasyMAG (50 minutes). Clinical impact of a PCR-based assay for pathogen
detection in critically ill patients with evidence of infection Clinical impact of a PCR-based assay for pathogen
detection in critically ill patients with evidence of infection P5 The 8-hour to 12-hour window for qualifi-
cation and start of treatment was the primary challenge to the SAC
and study sites. Each site used two sequences of drug assignment Conclusions Potential survival benefits of eritoran in severe sepsis
patients may be associated with high severity of illness. Treatment
by disease severity interaction will be further explored in a phase 3
trial. S3 Critical Care November 2009 Vol 13 Suppl 4
Sepsis 2009 P7 Rapid and reliable detection and
differentiation between Staphylococcus aureus and coagulase-
negative Staphylococci (CNS) is therefore clinically relevant to be
able to provide adequate early treatment. Blood culture is still the
gold standard method in identifying these pathogens but is time
consuming. Molecular diagnostics might be a promising alternative
to reduce this time-to-result delay. Methods A dorsal air pouch in 175 to 200 g Sprague–Dawley rats
was formed by 20 ml subcutaneous infusions of sterile air. Prophylactic subcutaneous injections of canine HFP, canine fresh
frozen plasma (FFP) or carprofen were administered daily for
3 days into the lateral flank of the right foreleg at doses recom-
mended by the manufacturers (n = 10 for each treatment group). Pouch fluid was harvested by syringe at 1, 6, 12, 24 and 48 hours
post LPS administration and subjected to histological and
cytokine/cytokine receptor analysis. TNFα and sTNFR1 levels were
determined by ELISA and an immunofluorescent dot blot assay. Objective This study aims to compare different DNA extraction
methods from two commonly used blood culture materials, BACTEC
(BD) and Bact/ALERT (Biomerieux), to accelerate differentiation
between S. aureus and CNS. Results Pouch fluid analysis: maximal effects were detected at
6 hours post LPS administration. TNFα levels were significantly
depressed in animals dosed with HFP, but not in animals treated
with FFP or carprofen (P <0.05). sTNFR1 levels were significantly
elevated in HFP, but not in FFP or carprofen dosed animals
(P <0.05). Neutrophil numbers were significantly depressed in
HFP dosed but not in FFP or carprofen treated animals (P <0.05). Methods Two fast real-time PCR duplex test assays, targeting the
Tuf gene, to differentiate S. aureus from CNS, were developed in
order to select the most sensitive one. This Tuf RT-PCR was used
to compare three different DNA isolation methods on two different
blood culture systems. Negative blood culture material was spiked
with S. aureus; bacterial DNA was isolated with: automated
extractor EasyMAG (Biomerieux), automated extractor MagNA
Pure (Roche), and a manual kit MolYsis Plus (MolZyme). Conclusions There appears to be a correlation between elevated
levels of sTNFR1 and depression of TNFα and neutrophil levels in
the pouch fluid of HFP dosed rats (r = –0.73, P <0.0001). P13 Comparison of commercial DNA extraction kits for the
detection of bacterial genomic DNA from whole-blood
samples using a broad-range PCR B Krulova, E Nemcova, B Zaloudikova, P Nemec, T Freiberger
Centre for Cardiovascular Surgery and Transplantation, Molecular
Genetic Laboratory, Brno, Czech Republic
Critical Care 2009, 13(Suppl 4):P13 (doi: 10.1186/cc8069) Figure 2 (abstract P9) Introduction Blood culture is still considered a gold standard for
diagnosis of bloodstream infections. Early pathogen detection is a
prerequisite for the successful treatment. Nucleic acid based
techniques offer a rapid and sensitive option particularly in blood
culture negative samples. Efficient bacterial DNA extraction is
crucial for following PCR assays. The aim of this study was to
determine the detection limit of bacterial genomic DNA using
different extraction protocols. Methods We evaluated five commercially available kits for the
extraction of bacterial genomic DNA from whole-blood samples
(QIAamp DNA Blood Mini kit, UltraClean DNA BloodSpin Kit,
Chemagic DNA Blood Kit, ZR Genomic DNAII Kit, NucliSens
miniMAG). Whole-blood samples were spiked with Escherichia
coli CCM 3988 and Staphylococcus aureus CCM 7111. Tenfold
dilution series containing concentrations from 107 to 100 CFU/ml
were prepared under sterile conditions and immediately used. A
broad-range 16S rDNA end-point PCR was performed for the
detection of E. coli and S. aureus DNA. drawn on discretion of the treating physician. Blood cultures and
EDTA-blood were taken by sterile venous puncture. The EDTA-
blood was processed with a PCR-based assay (VYOO®; SIRS-
Lab GmbH, Jena, Germany), which detects a panel of 34 bacterial
and six fungal pathogens as well as five antibiotic resistances. Data
are given as median and interquartile range. Results The sensitivity of each kit was determined as a minimum
rate of CFU providing the positive result in the PCR assay. Our
results showed the extraction by the QIAamp DNA Blood Mini Kit
(supplemented with enzymatic pre-treatment) as the most efficient
and sensitive method. This extraction protocol allowed the
reproducible detection of E. coli and S. aureus at concentrations
of 103 CFU/ml. All kits showed positive results in samples at
concentrations from 107 to 105 CFU/ml. Results Sixty-three patients were included into this study. Age was
68.0 (55.5 to 74.0) years, APACHE II score was 17 (13 to 23),
SOFA score at study inclusion was 10.0 (7.50 to 11.0), and ICU
mortality was 33.3%. In 54 patients (84.3%) infection was either
microbiologically confirmed or clinically proven. P13 The baseline
procalcitonin was 2.4 (0.8 to 8.0) ng/ml. Eighty-two pairs of BCs
and PCRs have been drawn. Ten (12%) BCs and 30 (36.6%)
PCRs were positive (P
<0.001). Time to test result was
significantly shorter in the PCR than in the BC (Figure 1). Twelve
positive PCR results (Figure 2, grey areas) prompted a change in
antibiotic or antimicrobial therapy. Conclusions Extraction kits should be capable to recover nucleic
acids and remove inhibitors from diverse clinical materials
simultaneously. All of the tested kits were able to recover bacterial
genomic DNA from whole-blood samples, but the sensitivity of PCR-
based detection depends on the DNA extraction protocol used. P8 Effect of canine hyperimmune plasma on TNFα and
inflammatory cell levels in a lipopolysaccharide-mediated
rat air pouch model of inflammation Objective To report the performance of PCR-based pathogen
detection compared with blood culture in ICU patients with evidence
of infection, and the impact of this test on the antimicrobial therapy. Methods Patients treated on an interdisciplinary ICU were B Essien, M Kotiw, H Buttler, D Strunin
Centre for Systems Biology, University of Southern Queensland,
Toowoomba, Queensland, Australia
Critical Care 2009, 13(Suppl 4):P8 (doi: 10.1186/cc8064) B Essien, M Kotiw, H Buttler, D Strunin
Centre for Systems Biology, University of Southern Queensland,
Toowoomba, Queensland, Australia
Critical Care 2009, 13(Suppl 4):P8 (doi: 10.1186/cc8064) S4 Available online http://ccforum.com/supplements/13/S4 Figure 1 (abstract P9) P11 P11 Abstract withdrawn Figure 2 (abstract P9) Toll-like receptor 9-dependent gene expression in vivo is
regulated by inductive and suppressive networks Toll-like receptor 9-dependent gene expression in vivo is
regulated by inductive and suppressive networks Conclusions The PCR-based assay resulted in a considerably
higher amount of positive results within a shorter time to result than
the BC in this group of high-risk patients with evidence of infection. These data demonstrate that the shorter time to result may guide
adjustment of antimicrobial therapy earlier than culture-based
methods. Further studies are necessary to prospectively investi-
gate the impact of the PCR technique on antimicrobial therapy and
infection control. S Klaschik1,2, D Tross2, DM Klinman2
1Department of Anesthesiology and Intensive Care Medicine,
University of Bonn, Germany; 2Cancer and Inflammation Program,
NCI Frederick, MD, USA
Critical Care 2009, 13(Suppl 4):P14 (doi: 10.1186/cc8070) Introduction Synthetic oligodeoxynucleotides (ODN) expressing
CpG motifs mimic the immunostimulatory activity of bacterial DNA. CpG ODN interact with Toll-like receptor 9 to stimulate an innate
immune response characterized by the production of Th1 and
proinflammatory cytokines, and the functional maturation of P10 y
y
g
g
p
Materials cDNA was generated from total RNA isolated from
spleen cells of mice 30 minutes to 3 days after in vivo treatment
with 400 μg CpG or control ODN. Methods Dual-color hybridizations (Biomicro) were performed on
murine genome microarrays (NCI). Analyses were conducted on
four independently derived RNA samples for each time point. R2
was 0.90 ± 0.04 for all matched samples. Network analysis was
performed using Ingenuity Pathway Analysis. Results Differential gene activation (P <0.00001) was observed
within 30 minutes of CpG ODN treatment (25 genes), peaked at
3 hours (529 upregulated genes), and fell to near background
levels after 72 hours. TNFα, IL-1β, NF-κB and IFNγ played central
roles in upregulating the early expression of immune-related genes. Of interest, two distinct patterns of gene expression were
observed. One subset of genes was activated shortly after CpG
ODN administration and remained upregulated for a prolonged
period, while unique subsets of additional genes were activated at
specific time points, and were rapidly downregulated. Several
genes responsible for this downregulation (MYC, IL1RN, SOCS1)
were identified. 1. Engel C, Brunkhorst FM, Bone HG, Brunkhorst R, Gerlach H,
Grond S, Gruendling M, Huhle G, Jaschinski U, John S, Mayer
K, Oppert M, Olthoff D, Quintel M, Ragaller M, Rossaint R,
Stuber F, Weiler N, Welte T, Bogatsch H, Hartog C, Loeffler
M, Reinhart K: Epidemiology of sepsis in Germany: results
from a national prospective multicenter study. Intensive
Care Med 2007, 33:606-618. Conclusions This analysis identifies two distinct patterns of gene
regulation associated with CpG-induced activation of the innate
immune system of mice. A small number of regulatory genes
triggers the patterned upregulation of immune related genes from
30 minutes through 72 hours. A separate set of downregulatory
genes subsequently dampens what would otherwise be a
continuous positive feedback loop. P16 Identification of cathepsin G in the generation of elastase-
resistant fragment of vascular endocan: involvement in the
regulation of LFA-1-dependent cascade N De Freitas Caires1,2,3, M Barrier1,2,3, S Sarrazin4,
F Depontieu1,2,3, H Ghamlouch1,2,3, W Morelle5, H Drobecq6,
M Delehedde1,2,3, H Lortat-Jacob4, C Duez1,2,3,
A Scherpereel1,2,3,7, P Lassalle1,2,3
1Inserm, U774, Lille, France; 2Institut Pasteur de Lille, France;
3Université Lille Nord de France, Lille, France; 4CNRS-CEA-UJF,
Institut de Biologie Structurale, Grenoble, France; 5UMR-CNRS
8576, Villeneuve Ascq, France; 6UMR-CNRS 8161, Lille, France;
7CHRU Lille, Hôpital Calmette, Lille, France
Critical Care 2009, 13(Suppl 4):P16 (doi: 10.1186/cc8072) N De Freitas Caires1,2,3, M Barrier1,2,3, S Sarrazin4,
F Depontieu1,2,3, H Ghamlouch1,2,3, W Morelle5, H Drobecq6,
M Delehedde1,2,3, H Lortat-Jacob4, C Duez1,2,3,
A Scherpereel1,2,3,7, P Lassalle1,2,3
1Inserm, U774, Lille, France; 2Institut Pasteur de Lille, France;
3Université Lille Nord de France, Lille, France; 4CNRS-CEA-UJF,
Institut de Biologie Structurale, Grenoble, France; 5UMR-CNRS
8576, Villeneuve Ascq, France; 6UMR-CNRS 8161, Lille, France;
7CHRU Lille, Hôpital Calmette, Lille, France
Critical Care 2009, 13(Suppl 4):P16 (doi: 10.1186/cc8072) Fluid therapy in severe sepsis: results from a
representative survey of German ICUs Fluid therapy in severe sepsis: results from a
representative survey of German ICUs C Hartog1, FM Brunkhorst1, F Bloos1, C Engel2, H Bogatsch3,
K Reinhart1, K Sengebusch4, M Ragaller4
1Department of Anesthesiology and Intensive Care Medicine, Jena
University Hospital, Germany; 2Institute for Medical Informatics,
Statistics and Epidemiology, University of Leipzig, Germany;
3Clinical Trial Centre Leipzig, University of Leipzig, Germany;
4Department for Anesthesiology and Intensive Care Therapy,
University Hospital Carl Gustav Carus, Medical Faculty, TU
Dresden, Germany
Critical Care 2009, 13(Suppl 4):P15 (doi: 10.1186/cc8071) Introduction The migration of polymorphonuclear neutrophils
(PMN) into inflamed tissue requires fine interactions with the
endothelial cell surface. The PMN serine proteases cathepsin G
(CG), neutrophil elastase (NE) and proteinase 3 (PR3) were
originally thought to play a role by the cleavage of endothelial cell
proteins that control the PMN firm adhesion and the
transendothelial cell migration. However, how these proteases
participate in leukocyte adhesion and transmigration remains
controversial. Vascular endocan, also called esm1, is a restricted
endothelial cell-secreted proteoglycan constituted by a protein
core of 20 kDa and by a unique glycosaminoglycan chain of
dermatan sulphate (DS). Endocan is preferentially expressed in
lung and kidney tissues. Endocan binds to its leukocytic receptor,
the LFA-1 integrin, with an affinity of 18 nM, and inhibits the
LFA-1–ICAM-1 interactions. Its expression is upregulated by the
proinflammatory mediators TNF, IL-1, and lipopolysaccharide. In
human sepsis, the serum endocan increases from fivefold to
30-fold the normal value and correlates with bad prognosis. Here,
we examined the role of PMN-derived proteases in the degradation
of endocan. Introduction Fluid resuscitation is a mainstay of sepsis
management. Objective To describe the practice of fluid therapy in German
ICUs. Methods Analysis of data derived from the SepNet cross-sectional
1-day point-prevalence study of patients with sepsis [1]. ICU
directors were asked about their fluid preferences. Data on days
after start of sepsis were adjusted to account for overestimation of
long-stayers in a point-prevalence survey. SPSS 15.0.1 (SPSS,
Chicago, IL, USA) was used; the chi-square test or Kruskal–Wallis
H test was applied where appropriate. Results On operative ICUs, more patients received synthetic
colloids (41.4 vs. 21.2%, P <0.001) and crystalloids (74.6 vs. 65.0%, P <0.05), while albumin use did not differ. ICU directors of
operative ICUs stated using synthetic colloids more frequently and
albumins less frequently than their colleagues from nonoperative Results On operative ICUs, more patients received synthetic
colloids (41.4 vs. P10 P10 P10
Abstract withdrawn Abstract withdrawn S5 Critical Care November 2009 Vol 13 Suppl 4
Sepsis 2009 ICUs. Stated and actual fluid use did not differ by hospital size. Of
415 patients with severe sepsis, 71.6% received crystalloids. 35.2% synthetic colloids (HES; gelatin, dextran), 16.4% received
5% glucose, and 4.1% albumin solutions. HES was the most
frequently used colloid. It was administered to 29.4% of patients
as HES 10% in 10.4% (mean dose, 787.8 ± 420.0 ml/24 hours)
and as HES 6% in 20.7% (mean dose, 769.1 ± 403.1 ml/24 hours). Patients receiving HES had a higher mean SOFA score than
patients without HES (9.92 ± 4.12 vs. 8.00 ± 4.07, P <0.001),
tended to more frequent acute renal failure (ARF) (defined as serum
creatinine >1.5 mg/dl (132.6 μmol/l) and diuresis ≤500 ml/day, no
chronic replacement therapy, RRT), more RRT, lower thrombocyte
counts and more frequently received RBCs, but this was non-
significant. In total, 29.5% of patients with ARF and 30.3% of
patients with RRT received HES. HES was administered to 115
patients between day 0 and day 63 after the start of severe sepsis. Conclusions Fluid therapy in septic patients varied by type of ICU. About 30% of patients received HES regardless of renal
dysfunction. HES was applied for many days after the start of
severe sepsis. immune cells. Changes in gene expression mediated by the in vivo
administration of CpG ODN were identified using microarrays. immune cells. Changes in gene expression mediated by the in vivo
administration of CpG ODN were identified using microarrays. immune cells. Changes in gene expression mediated by the in vivo
administration of CpG ODN were identified using microarrays. Objective We predicted that microarrays could be used to identify
reproducible changes in gene expression induced by CpG ODN
activation in mice treated in vivo over time, and that network
analysis would allow us to identify regulators of gene expression. Materials cDNA was generated from total RNA isolated from
spleen cells of mice 30 minutes to 3 days after in vivo treatment
with 400 μg CpG or control ODN. Objective We predicted that microarrays could be used to identify
reproducible changes in gene expression induced by CpG ODN
activation in mice treated in vivo over time, and that network
analysis would allow us to identify regulators of gene expression. Fluid therapy in severe sepsis: results from a
representative survey of German ICUs 21.2%, P <0.001) and crystalloids (74.6 vs. 65.0%, P <0.05), while albumin use did not differ. ICU directors of
operative ICUs stated using synthetic colloids more frequently and
albumins less frequently than their colleagues from nonoperative S6 Available online http://ccforum.com/supplements/13/S4 Figure 1 (abstract P17)
Age distribution of patients in WCNN intensive therapy unit. Figure 2 (abstract P17)
Distribution of central venous catheters in WCNN intensive therapy
unit. Figure 3 (abstract P17)
Average duration of central venous catheters in WCNN intensive
therapy unit. Figure 1 (abstract P17)
Age distribution of patients in WCNN intensive therapy unit. Methods Human endocan is produced by overexpressing HEK
293 cell lines and purified by anion exchange and affinity
chromatographies. Proteolysis is performed by addition of endocan
with PMA-activated PMN supernatants, purified CG, NE, or PR3. Endocan degradation is evaluated by ELISA, western blot and
MALDI-TOF mass spectrometry. Binding assay on Jurkat cells is
performed
as
described
previously. Endocan-derived
p14
fragments in human serum were detected by immunoprecipitation. Results We demonstrate that CG, NE but not PR3 degrade
endocan. We show that the degradation profiles of endocan by
CG or NE are different. We interestingly identify a novel peptide
fragment of endocan of 14 kDa, named p14, which represents the
main endocan degradation product. This p14 results from the
specific cleavage of the full-length endocan by CG. The generation
of this particular fragment strictly required the presence of the DS
chain on full-length endocan. Furthermore, this p14 fragment
becomes resistant to NE. We also demonstrate that p14 inhibits
the binding of endocan to Jurkat cells. Finally, we present evidence
that p14 could be detected in patient serum suffering from an
acute PMN-mediated disease like sepsis. Conclusions The results suggest that the expression of CG by
PMN shortly after their activation, by modifying the micro-
environment in degrading vascular endocan, may participate in the
complex network that controls tissue infiltration of leukocytes
during sepsis. P17
A review of central venous catheter-related infections in
neurointensive care patients in a tertiary referral centre
C-H Tan, P Nair, A Sule, M Rathbone
Walton Centre for Neurology & Neurosurgery (WCNN), Liverpool,
UK
Critical Care 2009, 13(Suppl 4):P17 (doi: 10.1186/cc8073) Figure 1 (abstract P17) Methods Human endocan is produced by overexpressing HEK
293 cell lines and purified by anion exchange and affinity
chromatographies. Proteolysis is performed by addition of endocan
with PMA-activated PMN supernatants, purified CG, NE, or PR3. Fluid therapy in severe sepsis: results from a
representative survey of German ICUs Endocan degradation is evaluated by ELISA, western blot and
MALDI-TOF mass spectrometry. Binding assay on Jurkat cells is
performed
as
described
previously. Endocan-derived
p14
fragments in human serum were detected by immunoprecipitation. Results We demonstrate that CG, NE but not PR3 degrade
endocan. We show that the degradation profiles of endocan by
CG or NE are different. We interestingly identify a novel peptide
fragment of endocan of 14 kDa, named p14, which represents the
main endocan degradation product. This p14 results from the
specific cleavage of the full-length endocan by CG. The generation
of this particular fragment strictly required the presence of the DS
chain on full-length endocan. Furthermore, this p14 fragment
becomes resistant to NE. We also demonstrate that p14 inhibits
the binding of endocan to Jurkat cells. Finally, we present evidence
that p14 could be detected in patient serum suffering from an
acute PMN-mediated disease like sepsis. Results We demonstrate that CG, NE but not PR3 degrade
endocan. We show that the degradation profiles of endocan by
CG or NE are different. We interestingly identify a novel peptide
fragment of endocan of 14 kDa, named p14, which represents the
main endocan degradation product. This p14 results from the
specific cleavage of the full-length endocan by CG. The generation
of this particular fragment strictly required the presence of the DS
chain on full-length endocan. Furthermore, this p14 fragment
becomes resistant to NE. We also demonstrate that p14 inhibits
the binding of endocan to Jurkat cells. Finally, we present evidence
that p14 could be detected in patient serum suffering from an
acute PMN-mediated disease like sepsis. Age distribution of patients in WCNN intensive therapy unit. Figure 2 (abstract P17) Figure 2 (abstract P17)
Distribution of central venous catheters in WCNN intensive therapy
unit. Conclusions The results suggest that the expression of CG by
PMN shortly after their activation, by modifying the micro-
environment in degrading vascular endocan, may participate in the
complex network that controls tissue infiltration of leukocytes
during sepsis. A review of central venous catheter-related infections in
neurointensive care patients in a tertiary referral centre C-H Tan, P Nair, A Sule, M Rathbone
Walton Centre for Neurology & Neurosurgery (WCNN), Liverpool,
UK
Critical Care 2009, 13(Suppl 4):P17 (doi: 10.1186/cc8073) C-H Tan, P Nair, A Sule, M Rathbone Distribution of central venous catheters in WCNN intensive therapy
unit. Introduction Intravenous catheter-related bloodstream infections
(ICR-BSI) are a major contributing factor to in-hospital mortality
and morbidity extending inpatient stay by 10 days and expenditure
per patient by £2,000 to £30,000 [1]. Figure 3 (abstract P17)
Average duration of central venous catheters in WCNN intensive
therapy unit. Figure 3 (abstract P17)
Average duration of central venous catheters in WCNN intensive
therapy unit. Figure 3 (abstract P17) Figure 3 (abstract P17) Objective A prospective survey was conducted in our unit on all
patients with central venous catheters to ascertain the incidence of
ICR-BSI, identify the organisms and determine the occurrence of
infection from the various sites – femoral, internal jugular and
subclavian lines. Methods The survey was carried out over a period of 13 weeks. Data collected from patients’ case notes included site of central
line insertion, length of line in situ, reason for line removal and
positive blood culture reports. Results During the study period, 104 patients were treated on the
unit. Fifty-two central venous lines were inserted in 36 patients
(Figure 1): 63.5% femoral (n = 33), 32.7% internal jugular (n = 17)
and 3.9% subclavian lines (n = 2) (Figure 2). The lines were
reviewed daily and removed if indicated clinically (pyrexia or raised
white cell count) or if not required. A total 51.5% of femoral lines
(n = 17) were removed due to clinical indications, as were 29%
(n = 5) of internal jugular and 50% (n = 1) of subclavian lines. The
average duration of a line remaining in situ was 4.5 days for
femoral, 6 days for internal jugular and 5 days for subclavian lines
(Figure 3). Blood cultures were taken at the time of line removal. These yielded positive results in eight femoral, seven internal
jugulars and one subclavian line (Figure 4). Our survey indicated
that the incidence of ICR-BSI in our unit is 30.8%, (of this 62.5%
coagulase-negative staphylococci (CNS), 12.5% Escherichia coli
and Pseudomonas each, and 6.25% MSSA and MRSA each)
(Figure 5). Average duration of central venous catheters in WCNN intensive
therapy unit. Conclusions
The
distribution
of
microorganisms
causing
bacteraemia is broadly similar in our unit to that in other teaching
hospitals in the UK [2], in that CNS was the commonest organism
isolated. However, E. coli and Pseudomonas were the next
common organisms, unlike other units where Staphylococcus
aureus was the second most prevalent organism. The incidence of
bacteraemia from femoral lines (53.7/1,000 catheter-days) was
lower than that from internal jugular lines (68.6/1,000 catheter-
days) possibly due to a higher index of suspicion in the case of
femoral lines and earlier removal (Figure 6). Heart rate variability, cytokine, and brain responses to
infection: insights from a mouse model K Fairchild, R Gaykema, L Goehler
Department of Pediatrics, University of Virginia School of Medicine,
Charlottesville, VA, USA
Critical Care 2009, 13(Suppl 4):P18 (doi: 10.1186/cc8074) Introduction Continuous heart rate variability (HRV) monitoring
can detect early sepsis in certain high-risk patient populations, but
the mechanisms by which sepsis depresses HRV are not well
understood. Our prior studies in rodents have shown that endo-
toxin causes cytokine-related depression of HRV and activation of
specific neuronal networks. The aim of the current studies was to
identify pathogen-specific patterns of cytokine expression, HRV
changes, and activation of central autonomic pathways in mice. Distribution of ICR-BSI in WCNN intensive therapy unit. Figure 5 (abstract P17)
Distribution of ICR-BSI organisms in WCNN intensive therapy unit. Figure 6 (abstract P17)
Incidence of ICR-BSI in WCNN intensive therapy unit. Distribution of ICR-BSI in WCNN intensive therapy unit. g
p
y
Materials and methods Adult male C57BL/6 mice implanted with
radiotelemetry probes for continuous ECG and temperature
monitoring were inoculated intraperitoneally with Klebsiella pneumo-
niae
(KP, n = 21), methicillin-resistant Staphylococcus aureus
(MRSA-COL, n = 9) or Candida albicans (CA-SC5314, n = 2). Heart
rate variability (standard deviation of RR intervals) was measured
continuously for up to 3 days in K. pneumoniae and MRSA mice and
up to 3 weeks in CA mice. Blood was obtained at two or more time
points for culture and measurement of G-CSF, KC, MIP-1β, IFNγ,
TNFα, IL-6, and IL-10. Neuronal activation was assessed in multiple
brain regions of K. pneumoniae-infected mice by c-Fos staining. g
p
y
g
Results Compared with sham-treated mice, infected mice had
increases in multiple cytokines at 18 hours and 42 hours post
inoculation. Cytokine profiles were similar among the three
organisms except that CA-infected mice expressed less KC. Bacteria-inoculated mice with adverse outcome (positive blood
culture and/or death, n = 8 of 21 K. pneumoniae and 3 of 9
MRSA) had significantly higher levels of all cytokines compared
with mice with good outcome. Substantial depression of HRV was
seen in all 11 bacteria-infected mice with adverse outcome and in
only one of 19 mice with good outcome. Levels of G-CSF and IL-6
were negatively correlated with HRV (Spearman correlation
coefficient = –0.36 and –0.37, respectively, P = 0.05 for each)
and there was a trend toward a correlation with KC (–0.35,
P = 0.07). Immunohistochemical studies revealed that, compared
with sham-treated controls (n = 2), K. Heart rate variability, cytokine, and brain responses to
infection: insights from a mouse model pneumoniae infection (n = 3)
was associated with c-Fos induction in the dorsal vagal complex
and ventrolateral medulla, paraventricular hypothalamic nucleus,
preoptic area, subfornical organ, bed nucleus of the stria terminalis,
and medial prefrontal and insular cortices. c-Fos immunoreactivity
also occurred in ventricular ependymal cells and in cells associated
with large blood vessels. Distribution of ICR-BSI organisms in WCNN intensive therapy unit. Conclusions Infection with Gram-positive or Gram-negative
bacteria invokes similar changes in cytokines and HRV in mice,
whereas preliminary studies suggest Candida infection results in
different patterns. K. pneumoniae infection causes widespread
neuronal activation within the central autonomic network. Incidence of ICR-BSI in WCNN intensive therapy unit. the fact that femoral lines, which are often the safest option for
unstable patients with head injury, can be effectively managed with
strict adherence to guidelines to reduce ICR-BSI. References
1.
Maki DG, Kluger DM, Crnich CJ: The risk of bloodstream
infection in adults with different intravascular devices: a
systematic review of 200 published prospective studies.
Mayo Clin Proc 2006, 81: 1159-1171. Figure 3 (abstract P17) Our study highlights S7 Critical Care November 2009 Vol 13 Suppl 4
Sepsis 2009 the fact that femoral lines, which are often the safest option for
unstable patients with head injury, can be effectively managed with
strict adherence to guidelines to reduce ICR-BSI. References
1. Maki DG, Kluger DM, Crnich CJ: The risk of bloodstream
infection in adults with different intravascular devices: a
systematic review of 200 published prospective studies. Mayo Clin Proc 2006, 81: 1159-1171. 2. Coello R, Charlett A, Ward V, et al.: Device-related sources
f b
t
i
i
E
li h h
it l
t
iti
f
th
Figure 4 (abstract P17)
Distribution of ICR-BSI in WCNN intensive therapy unit. Figure 5 (abstract P17)
Distribution of ICR-BSI organisms in WCNN intensive therapy unit. Figure 6 (abstract P17)
Incidence of ICR-BSI in WCNN intensive therapy unit. Figure 4 (abstract P17)
Distribution of ICR-BSI in WCNN intensive therapy unit. Figure 4 (abstract P17) P18 Central venous catheter-related infection: a cohort study
evaluating dedicated central venous catheter packs Central venous catheter-related infection: a cohort study
evaluating dedicated central venous catheter packs P-Y Chu, D-Z Hsu, M-Y Liu
Department of Environmental and Occupational Health, National
Cheng Kung University Medical College, Tainan, Taiwan
Critical Care 2009, 13(Suppl 4):P20 (doi: 10.1186/cc8076) S Mukerji, R Daniels, K Maung, A Mattin
Good Hope Hospital, Worcester, UK
Critical Care 2009, 13(Suppl 4):P22 (doi: 10.1186/cc8078) S Mukerji, R Daniels, K Maung, A Mattin
Good Hope Hospital, Worcester, UK Introduction Hypotension is well relative to the high mortality of
sepsis. Sesamol increases the survival rate of septic mice. However,
the effect of sesamol on septic hypotension after the onset of
systemic inflammation has never been studied. The aim of the study
is to investigate the effect of sesamol on septic hypotension. Introduction Central venous catheter (CVC)-related bloodstream
infections (CRBSI) are the third most common healthcare-
associated infection (HAI) in ICUs, associated with significant
morbidity, mortality, increased length of stay and costs [1,2]. Several care bundle studies have suggested that utilising various
strategies together (such as training, regular line monitoring and
using dedicated line insertion trolleys) can have a positive impact
on CRBSI rates [3-6]. However, the impact solely attributable to
the provision of a dedicated, stand-alone CVC insertion pack has
not been evaluated. We therefore investigated the impact of a new
EPIC2 compliant CVC pack, introduced in Good Hope Hospital in
2007, on CVC tip colonisation rates. Materials Wistar rats, lipopolysaccharide (LPS) (derived from Escher-
ichia coli, serotype O55:B5), and sesamol were used in this study. Methods Hypotension was induced by injecting LPS intravenously. Mean arterial pressure was measured using an invasive blood
pressure system. Serum nitrite and cytokine levels were deter-
mined using the Griess reaction and ELISA, respectively. Peroxisome proliferator-activated receptor (PPAR) activation was
measured using a PPAR assay kit. Results LPS administration significantly increased the serum
TNFα level at 1 hour. Sesamol treated 1 hour after LPS adminis-
tration inhibited the LPS-associated blood pressure decrease. Sesamol failed to decrease the LPS-induced nitrite production, but
decreased the LPS-induced TNFα and IL-1β production after the
onset of systemic inflammation. Sesamol enhanced the IL-10
production in serum and the PPAR activation in white blood cells. Methods Data were collected prospectively between June 2007
and December 2008. Patients were divided into two cohorts:
patients whose CVCs were inserted using the CVC packs (B), and
those receiving CVCs prior to the introduction of the packs (nB). P20 Sesamol attenuates septic hypotension through
peroxisome proliferator-activated receptor activation after
the onset of systemic inflammatory response Central venous catheter-related infection: a cohort study
evaluating dedicated central venous catheter packs Data were collated from questionnaires as well as patients’ notes
on: patient’s age and sex; type and site of CVC inserted; location at
the time of insertion; the grade of practitioner; and the duration
CVCs remained in situ. Using the hospital’s patient information
system and patient notes, data on CVC tip cultures were obtained. Data are presented as percentages and analysed using multivariate
analysis. Conclusions Sesamol may attenuate septic hypotension through
alternating cytokine production by PPAR activation after the onset
of systemic inflammatory response. Available online http://ccforum.com/supplements/13/S4 models. However, its mechanism is still unclear. The aim of this
study was to determine the effects of muscimol, a GABAA
receptor agonist, on lipopolysaccharide-induced mortality and
inflammation in mice. the pathogenesis and development of sepsis. 3,4-Methylene-
dioxyphenol (sesamol), one of the lignans in sesame oil, protects
against endotoxin-induced oxidative stress and organ failure. However, the effects of sesamol on systemic inflammation and
oxidative stress in septic rats have never been investigated. Materials C57BL6 mice, lipopolysaccharide (derived from Escher-
ichia coli, serotype O55:B5), and muscimol were used in this study. Methods Mice endotoxemia was induced by 10 mg/kg lipopoly-
saccharide intraperitoneally. Muscimol ranging from 0 to 3 mg/kg
was given subcutaneously 30 minutes before lipopolysaccharide
administration. Serum TNFα, IL-1β, IL-10, and IL-12 were
determined using ELISA. Materials C57BL6 mice, lipopolysaccharide (derived from Escher-
ichia coli, serotype O55:B5), and muscimol were used in this study. Objective To investigate the effects of sesamol on systemic inflam-
mation and oxidative stress in septic rats. Methods Septic rats were induced by cecal ligation and puncture
(CLP). Rats received sesamol (10 mg/kg, subcutaneously) 0 and
6 hours after CLP. IL-1β, lipid peroxidation, hydroxyl radical, super-
oxide anion, xanthine oxidase activity, and nitrite levels in blood
were determined 12 hours after CLP. Results Muscimol significantly increased the survival rate in sub-
lethal dose of lipopolysaccharide-treated mice (from 7% to 100%)
(P < 0.0001) within 72 hours. Muscimol inhibited serum TNFα,
IL-1β, and IL-12 production in a dose-dependent manner. Further-
more, muscimol significantly increased serum IL-10 levels
(P < 0.001) in lipopolysaccharide-treated mice. Results IL-1β, lipid peroxidation, hydroxyl radical, superoxide anion,
xanthine oxidase activity, and nitrite levels were significantly
increased in CLP-treated rats compared with those in the sham-
operation group (all P <0.05). Sesamol significantly reduced IL-1β,
lipid peroxidation, hydroxyl radical, superoxide anion, xanthine
oxidase activity, and nitrite levels compared with the saline group in
CLP-treated rats (all P <0.05). Conclusions Muscimol potently increased the survival rate and
inhibited inflammatory response in endotoxemic mice. Conclusions Sesamol might attenuate systemic inflammation and
oxidative stress by inhibiting proinflammatory cytokine and reactive
oxygen species generation in septic rats. Muscimol increases the survival rate and inhibits the
inflammatory response in endotoxemic mice 1. Maki DG, Kluger DM, Crnich CJ: The risk of bloodstream
infection in adults with different intravascular devices: a
systematic review of 200 published prospective studies. Mayo Clin Proc 2006, 81: 1159-1171. D-Z Hsu, Y-H Li, P-Y Chu, M-Y Liu
Department of Environmental and Occupational Health, National
Cheng Kung University Medical College, Tainan, Taiwan
Critical Care 2009, 13(Suppl 4):P19 (doi: 10.1186/cc8075) 2. Coello R, Charlett A, Ward V, et al.: Device-related sources
of bacteraemia in English hospitals – opportunities for the
prevention of hospital-acquired bacteraemia. J Hosp Infect
2003, 53:46-57. Introduction Affecting the γ-amino butyric acid (GABA) pathway
results in an alteration of inflammatory response in various animal Introduction Affecting the γ-amino butyric acid (GABA) pathway
results in an alteration of inflammatory response in various animal S8 P23 TLR4 on hematopoietic cells is crucial for host defense
against Klebsiella pneumonia but TLR2 is needed when
bacterial numbers are high C Wieland1,2,3, MH van Lieshout3,4, AJ Hogendijk3,4, T van der Poll3,4
1Laboratory of Experimental Intensive Care and Anesthesiology,
2Department of Intensive Care, 3Center of Experimental and
Molecular Medicine, and 4Center of Infection and Immunity,
Academic Medical Center Amsterdam, the Netherlands
Critical Care 2009, 13(Suppl 4):P23 (doi: 10.1186/cc8079) Introduction Klebsiella species are opportunistic pathogens that
can give rise to severe infections including pneumonia and sepsis. Typically, Klebsiella infections are nosocomial and mainly caused
by Klebsiella pneumoniae, the medically most important species of
the genus. Number of days CVCs were left in situ in both cohorts. Number of days CVCs were left in situ in both cohorts. g
Objective We set out to validate and extend our previous data using
C3H/HeJ mice that demonstrated an important role for TLR4 in K. pneumoniae pneumonia. Moreover, we were interested in the relative
roles of cells from hematopoietic origin and parenchymal cells. Methods Using TLR2 and TLR4 single and TLR2x4 double
knockout (KO) mice on a C57BL/6 background, the roles of TLR2
and TLR4 were investigated independently and together. We
intranasally inoculated C57BL/6 wild-type (WT) and KO mice with
K. pneumoniae (4 x 10–3 CFU per mouse) and studied host
defense. Moreover, we performed bone marrow transplantation
(BMT) experiments in which we transplanted KO bone marrow into
irradiated WT mice and vice versa. Objective We set out to validate and extend our previous data using
C3H/HeJ mice that demonstrated an important role for TLR4 in K. pneumoniae pneumonia. Moreover, we were interested in the relative
roles of cells from hematopoietic origin and parenchymal cells. Figure 2 (abstract P22)
Differences in colony growth in the two cohorts. *P <0.05. Figure 2 (abstract P22)
Differences in colony growth in the two cohorts. *P <0.05. Methods Using TLR2 and TLR4 single and TLR2x4 double
knockout (KO) mice on a C57BL/6 background, the roles of TLR2
and TLR4 were investigated independently and together. We
intranasally inoculated C57BL/6 wild-type (WT) and KO mice with
K. pneumoniae (4 x 10–3 CFU per mouse) and studied host
defense. Moreover, we performed bone marrow transplantation
(BMT) experiments in which we transplanted KO bone marrow into
irradiated WT mice and vice versa. Results Shortly after infection, both TLR4 and TLR2x4 KO mice
demonstrated an attenuated proinflammatory response in the lungs. Introduction The pandemic by the novel H1N1 virus has created
the need to study any probable effects of that infection in the
immune system of the host. P23 This was associated with higher bacterial counts 24 hours after
infection in the lungs, liver and spleens of both TLR4 and TLR2x4
KO animals. Interestingly, although no differences in antibacterial
host defense of TLR2 KO animals were observed, TLR2x4 KO
animals were more susceptible to K. pneumoniae infection than the
single TLR4 KO mice: after 44 hours of infection, 0/8 WT, 0/8 TLR2
KO mice, 5/8 TLR4 KO mice and 8/8 TLR2x4 KO mice had
succumbed. Moreover, when infecting all strains with a high dose of
K. pneumoniae (10–4 CFU), no differences in outgrowth were
detected between WT, TLR2 and TLR4 KO animals, whereas
double KO animals suffered from higher bacterial burdens in the
lungs, liver, spleen and blood. BMT of WT bone marrow into irradiated
TLR2x4 KO mice resulted in a reversed phenotype with similar
bacterial growth compared with syngenic transplanted WT mice. Differences in colony growth in the two cohorts. *P <0.05. (Figure 2). The bundle cohort had no MRSA growth compared with
four incidences in the nB group. Conclusions Our results indicate that use of dedicated CVC packs
was associated with a significant reduction in the colonisation rate
of CVCs, despite lines being left in situ for longer periods and the
more frequent use of quin-lumen catheters in the intervention group. There was also a trend toward prevention of MRSA colonisation. 1. Blake M: Update: catheter-related bloodstream infection
rates in relation to clinical practice and needleless device
type. Can J Infect Control 2008, 23:156-160, 162. Conclusions These data confirm our previous research that,
during low-dose infections, TLR4 is of primary importance in host
defense against K. pneumoniae. Nevertheless, when high numbers
of bacteria are present, TLR2 acts together with TLR4 to
orchestrate the immune response, a protective effect that is
primarily mediated by hematopoietic cells. 2. Tacconelli E, Smith G, Hieke K, Lafuma A, Bastide P: Epi-
demiology, medical outcomes and costs of catheter-
related bloodstream infections in intensive care units of
four European countries: literature and registry based
estimates. J Hosp Infect 2009, 72:97-103. Introduction The pandemic by the novel H1N1 virus has created
the need to study any probable effects of that infection in the
immune system of the host. P24 3. Mermel LA: Prevention of central venous catheter-related
infections: what works other than impregnated or coated
catheters? J Hosp Infect 2007, 65(Suppl 2):30-33. Effect of the novel influenza A (H1N1) virus in the human
immune system A Antonopoulou, M Raftogiannis, F Baziaka, P Koutoukas,
A Savva, T Kanni, M Georgitsi, A Pistiki, EJ Giamarellos-Bourboulis
4th Department of Internal Medicine, University of Athens, Medical
School, Athens, Greece
Critical Care 2009, 13(Suppl 4):P24 (doi: 10.1186/cc8080) P21 Results Complete data were obtained for 347 patients: 246
patients in group nB, 101 patients in group B. Male/female ratio,
average age, site of insertion, clinical area of insertion and grade of
practitioner were similar in both cohorts. There was a significantly
higher number of 5-lumen catheters inserted in group B compared
with group nB (81% compared with 44%, P <0.05), reflecting
change in hospital practice. More B catheters (51%) were left in
situ longer, for 6 to 10 days, compared with nB catheters (31%)
(Figure 1). Thirty-one per cent of nB tips grew colonies of at least
one pathogen. There was a significant reduction in the number of
B tips growing colonies (12% compared with 31%, P <0.05) 3,4-Methylenedioxyphenol attenuates systemic
inflammation and oxidative stress in septic rats Y-H Li, D-Z Hsu, M-Y Liu
Department of Environmental and Occupational Health, National
Cheng Kung University Medical College, Tainan, Taiwan
Critical Care 2009, 13(Suppl 4):P21 (doi: 10.1186/cc8077) Y-H Li, D-Z Hsu, M-Y Liu
Department of Environmental and Occupational Health, National
Cheng Kung University Medical College, Tainan, Taiwan
Critical Care 2009, 13(Suppl 4):P21 (doi: 10.1186/cc8077) Introduction Sepsis is one of the major causes of mortality in
ICUs. Systemic inflammation and oxidative stress are involved in S9 Critical Care November 2009 Vol 13 Suppl 4
Sepsis 2009 Figure 1 (abstract P22)
Number of days CVCs were left in situ in both cohorts. Figure 2 (abstract P22)
Differences in colony growth in the two cohorts. *P <0.05. Figure 1 (abstract P22)
Number of days CVCs were left in situ in both cohorts. P23 Effect of the novel influenza A (H1N1) virus in the human
immune system 4. Berenholtz SM, Pronovost PJ, Lipsett PA, et al.: Eliminating
catheter-related bloodstream infections in the intensive
care unit. Crit Care Med 2004, 32:2014-2020. A Antonopoulou, M Raftogiannis, F Baziaka, P Koutoukas,
A Savva, T Kanni, M Georgitsi, A Pistiki, EJ Giamarellos-Bourboulis
4th Department of Internal Medicine, University of Athens, Medical
School, Athens, Greece
Critical Care 2009, 13(Suppl 4):P24 (doi: 10.1186/cc8080) 5. Anonymous: Reduction in central line-associated blood-
stream infections among patients in intensive care units
Pennsylvania, April 2001 March 2005. MMWR Recomm
Rep 2005, 54:1013-1016. 6. Pronovost P, Needham D, Berenholtz S, et al.: An interven-
tion to decrease catheter-related bloodstream infections
in the ICU. N Engl J Med 2006, 355:2725-2732. 6. Pronovost P, Needham D, Berenholtz S, et al.: An interven-
tion to decrease catheter-related bloodstream infections
in the ICU. N Engl J Med 2006, 355:2725-2732. S10 Available online http://ccforum.com/supplements/13/S4 Available online http://ccforum.com/supplements/13/S4 Available online http://ccforum.com/supplements/13/S4 Methods Blood was sampled within the first 2 days of the
presentation of signs of infection from 10 healthy volunteers; from
18 patients of flu-like syndrome (FLS); and from 30 patients of
infection by H1N1 confirmed by reverse RT-PCR. Absolute counts
of subtypes of monocytes and of lymphocytes were determined
after staining with monoclonal antibodies and analysis by flow
cytometry. Peripheral blood mononuclear cells (PBMCs) were
isolated and stimulated with various bacterial stimuli. Concen-
trations of TNFα, of IL-1β and of IFNγ were estimated in super-
natants by an enzyme immunoassay. compared with FLS). No effect of other stimuli was shown on
release of TNFα and of IFNγ. Release of IL-1β was not affected. Conclusions Infection by the H1N1 virus is accompanied by a
characteristic impairment of the innate immune responses charac-
terized by defective cytokine responses to S. pneumoniae. Alterations of the adaptive immune responses are predominated by
an increase of Tregs. These findings signify a predisposition for
pneumococcal infections after infection by H1N1 influenza. P25 y
y
y
Results Mean absolute counts of CD14 monocytes of healthy
volunteers, of FLS and of patients infected by the H1N1 virus were
271.1, 464.4 and 607.2%, respectively (P <0.0001 compared
with other groups). Respective values of CD4+/CD25+/CD127–
cells were 2.0, 4.7 and 9.0% (P <0.0001 compared with other
groups). Respective values of CD19 cells were 297.9, 151 and
137.9%. No differences between the three groups were found
regarding CD3/CD4 lymphocytes, CD3/CD8 lymphocytes, natural
killer (NK) cells and NKT cells. No differences were also found
regarding the rate of apoptosis of the above subtypes. Six patients
had H1N1-related pneumonia. Mean T-regulatory cells (Tregs) of
H1N1-infected patients without pneumonia and with pneumonia
were 6.7 and 17.8%, respectively (P = 0.034). Mean release of
TNFα
by
phytohemagglutin-stimulated
PBMCs
of
healthy
volunteers, of FLS and of patients infected by H1N1 was 3,658.5,
1,877.3 and 874.4 pg/ml, respectively (P <0.0001 compared with
other groups). Respective release of TNFα by Streptococcus
pneumoniae-stimulated
PBMCs
was
1,836.9,
949.9
and
478.0 pg/ml (P <0.0001 compared with other groups). Mean
respective release of IFNγ by PHA-stimulated PBMCs was
1,651.8, 1,235.1 and 1,114.3 pg/ml (P = 0.010 compared with
FLS). Mean respective release of IFNγ by S. pneumoniae-stimu-
lated PBMCs was 1,085.7, 748.1 and 709.7 pg/ml (P = 0.024 Clarithromycin reverses sepsis-induced immunoparalysis
of monocytes M Raftogiannis, A Antonopoulou, F Baziaka, P Koutoukas,
T Tsaganos, A Pelekanou, A Spyridaki, M Mouktaroudi,
EJ Giamarellos-Bourboulis
4th Department of Internal Medicine, University of Athens, Medical
School, Athens, Greece
Critical Care 2009, 13(Suppl 4):P25 (doi: 10.1186/cc8081) Introduction In a recently published double-blind, randomized trial
conducted by our study group, clarithromycin was intravenously
administered in patients with ventilator-associated pneumonia
(VAP) and sepsis for three consecutive days [1]. An earlier
resolution of VAP and a fivefold decrease of the risk for death by
septic shock and multiple organ failure (MODS) compared with
placebo were shown. p
Objective To investigate the mode of action of clarithromycin. Objective To investigate the mode of action of clarithromycin. Methods Blood was sampled before administration of the
investigational product and on six consecutive days. Peripheral
blood mononuclear cells (PBMCs) were isolated after gradient
centrifugation over Ficoll. PBMCs were incubated and adherent
monocytes were harvested and stimulated for 24 hours with Figure 1 (abstract P25) P26 Four groups of mice (n = 8) were treated intra-
peritoneally with (1) placebo, (2) rh-TFPI every 8 hours, (3)
ceftriaxone twice daily or (4) rh-TFPI in combination with
ceftriaxone. Early (8 hours) and late (24 hours) initiated treatments
were evaluated. Bronchoalveolar lavage fluid (BALF), lungs and
plasma were obtained 24 hours (for groups in which treatment was
started after 8 hours) or 48 hours (treatment started after 24 hours)
after infection. Statistical analysis was performed by Mann–Whitney
U test. Introduction Pneumonia is characterized by local activation of
coagulation leading to alveolar fibrin deposition. Lung injurious
mechanical ventilation (LI-MV) with conventional tidal volumes (VT)
and no positive end-expiratory pressure (PEEP) aggravates
pulmonary coagulopathy. We hypothesized administration of anti-
thrombin (AT), a natural anticoagulant, to attenuate ventilator-
induced coagulopathy and inflammation in a rat model of Strepto-
coccus pneumoniae pneumonia. Methods Rats challenged intratracheally with bacteria were
ventilated 40 hours later (VT = 12 ml/kg/no PEEP) after systemic
administration of plasma-derived human AT (250 U/kg) or placebo
for 3 hours. Endpoints: BALF levels of thrombin–antithrombin
complexes (TATc), fibrin degradation products (FDP), AT,
plasminogen activator activity (PAA), plasminogen activator
inhibitor-1 (PAI-1), pulmonary cytokines and blood cultures. Data
are presented as the mean ± SD. Statistics: one-way ANOVA with
Dunn’s multiple comparison test. Results Pneumonia resulted in local and systemic activation of
coagulation (as reflected by increased thrombin–antithrombin
complexes) and inhibition of fibrinolysis (as reflected by increased
plasminogen activator inhibitor-1 and decreased plasminogen
activator activity). Both early and late treatment with rh-TFPI
reduced pneumonia-induced coagulation in lungs and plasma; rh-
TFPI given with ceftriaxone further attenuated coagulation relative
to ceftriaxone only. No effects of rh-TFPI on pneumonia-inhibited
fibrinolysis were observed. Cell recruitment in BALF did not differ
between groups. Remarkably, rh-TFPI reduced levels of several
cytokines and chemokines not only in lung homogenates, but also
in BALF (IL-6, IFNγ, MCP-1, LIX) and plasma (IL-6, TNFα, IFNγ). The attenuated host inflammatory response was not reflected by
differences in total histopathology scores between treatment
groups. In mice not treated with ceftriaxone, rh-TFPI decreased
bacterial loads in lung homogenates ~10-fold (P <0.01 vs. placebo) at 48 hours, while leaving bacterial loads in BALF and the
systemic compartment unaltered. Results S. pneumoniae
pneumonia was characterized by
activation of coagulation (TATc: in pneumonia vs. healthy control,
3.7 ± 0.3 vs. 1.2 ± 0.5 ng/ml; FDP: 291 ± 40 vs. 15 ± 5 ng/ml;
AT: 3.7 ± 0.3 vs. P26 Plasma-derived human antithrombin attenuates ventilator-
induced coagulopathy in a Streptococcus pneumoniae
pneumonia model in rats Introduction Streptococcus pneumoniae is the most common
causative pathogen in community-acquired pneumonia and a major
cause of sepsis. Pneumonia elicits a procoagulant state in the lung
resulting from activation of coagulation, downregulation of
anticoagulant pathways and concurrent inhibition of fibrinolysis. Tissue factor is the main initiator of coagulation. Recombinant
human tissue factor pathway inhibitor (rh-TFPI) attenuates sepsis-
induced coagulation and has been evaluated in clinical trials
involving patients with sepsis and community-acquired pneumonia. Objective To examine the effect of rh-TFPI on coagulation,
inflammation and bacterial outgrowth in S. pneumoniae pneumonia
in mice, either alone or with concurrent antibiotic treatment. Methods Pneumonia was induced by intranasal inoculation with S. pneumoniae. Four groups of mice (n = 8) were treated intra-
peritoneally with (1) placebo, (2) rh-TFPI every 8 hours, (3)
ceftriaxone twice daily or (4) rh-TFPI in combination with
ceftriaxone. Early (8 hours) and late (24 hours) initiated treatments
were evaluated. Bronchoalveolar lavage fluid (BALF), lungs and
plasma were obtained 24 hours (for groups in which treatment was
started after 8 hours) or 48 hours (treatment started after 24 hours)
after infection. Statistical analysis was performed by Mann–Whitney
U test. Introduction Streptococcus pneumoniae is the most common
causative pathogen in community-acquired pneumonia and a major
cause of sepsis. Pneumonia elicits a procoagulant state in the lung
resulting from activation of coagulation, downregulation of
anticoagulant pathways and concurrent inhibition of fibrinolysis. Tissue factor is the main initiator of coagulation. Recombinant
human tissue factor pathway inhibitor (rh-TFPI) attenuates sepsis-
induced coagulation and has been evaluated in clinical trials
involving patients with sepsis and community-acquired pneumonia. Objective To examine the effect of rh-TFPI on coagulation,
inflammation and bacterial outgrowth in S. pneumoniae pneumonia
in mice, either alone or with concurrent antibiotic treatment. M th d
P
i
i d
d b i
l i
l i
i h S H Aslami1,2, JJ Haitsma1, JJ Hoffstra2, M Levi2, H Zhang1,
AS Slutsky1, MJ Schultz1
1Interdepartmental Division of Critical Care Medicine, Keenan
Research Center, Li Ka Shing Knowledge Institute, St Michael’s
Hospital, Toronto, Ontario, Canada; 2Departments of Intensive Care
and Internal Medicine, Laboratory of Experimental Intensive Care
and Anesthesiology, Academic Medical Center, University of
Amsterdam, the Netherlands
Critical Care 2009, 13(Suppl 4):P26 (doi: 10.1186/cc8082) AS Slutsky1, MJ Schultz1 Methods Pneumonia was induced by intranasal inoculation with S. pneumoniae. P27 Conclusions Administration of clarithromycin was accompanied by
a considerable improvement of the response of monocytes to ex
vivo stimulation with the release of TNFα and IL-6. These results
signify that clarithromycin effectively reverses sepsis-induced
immunoparalysis of monocytes. R f Recombinant human tissue factor pathway inhibitor exerts
anticoagulant, anti-inflammatory and antibacterial effects
in murine pneumococcal pneumonia F van den Boogaard1,2,3, X Brands1,2, M Schultz3, M Levi4,
J Roelofs5, C van ‘t Veer1,2, T van der Poll1,2
1Center for Experimental and Molecular Medicine, 2Center for
Infection and Immunity Amsterdam, 3Laboratory of Experimental
Intensive Care and Anesthesiology, 4Department of Internal
Medicine, and 5Department of Pathology, Academic Medical
Center, University of Amsterdam, the Netherlands
Critical Care 2009, 13(Suppl 4):P27 (doi: 10.1186/cc8083) References 1. Giamarellos-Bourboulis EJ, Pechère JC, Routsi C, et al.:
Effect of clarithromycin in patients with sepsis and ventila-
tor-associated pneumonia. Clin Infect Dis 2008, 46:1157-
1164. 1. Giamarellos-Bourboulis EJ, Pechère JC, Routsi C, et al.:
Effect of clarithromycin in patients with sepsis and ventila-
tor-associated pneumonia. Clin Infect Dis 2008, 46:1157-
1164. Figure 1 (abstract P25) Figure 1 (abstract P25) Figure 1 (abstract P25)
*Statistically significant differences between groups at the indicated time intervals. Day 0 corresponds to time before start of the investigational
drug. *Statistically significant differences between groups at the indicated time intervals. Day 0 corresponds to time before start of the investigational
drug *Statistically significant differences between groups at the indicated time intervals. Day 0 corresponds to time before start of the investigational
drug. S11 Critical Care November 2009 Vol 13 Suppl 4
Sepsis 2009 10 ng/ml LPS of Escherichia coli O55:B5. Concentrations of
TNFα and of IL-6 were estimated in supernatants by an enzyme
immunoassay. – P = NS). The infected animals showed similar numbers of
bacteria in blood samples at the start of the experiment, which did
not change during the experiment. Results One hundred patients were treated with placebo and
another 100 patients with clarithromycin. Median concentrations of
TNFα and of IL-6 in monocyte supernatants of all patients and
separately of those with septic shock and MODS are shown in
Figure 1. Conclusions Systemic administration of AT attenuated ventilator-
induced coagulopathy but not inflammation. P26 Recognition of fungi such as Candida albicans is
mediated through receptors of the innate immune system, such as
Toll-like receptors (TLRs), that in turn activate innate immune
system and antifungal defense. Objective To assess whether polymorphisms in fungal innate immune
receptors such as TLRs and dectin-1 influence susceptibility to
invasive candidiasis. Methods Frequencies of mostly nonsynonymous polymorphisms in
several innate immune receptors were genotyped in a total of 331
patients that developed invasive candidiasis and compared with a
total of 341 matched control patients that had the same
predisposing factors. These included neutropenia, mucosal barrier
injury and treatment with immunosuppressive regimens. Further-
more, in vitro studies with healthy volunteers were conducted to
assess the functional consequences of these polymorphisms
regarding cytokine responses. Results Genotyping for polymorphisms in innate immune receptor
genes revealed a higher frequency of three independent non-
synonymous TLR1 polymorphisms in the affected group patients
that developed invasive candidiasis. These polymorphisms were
also demonstrated to be associated with impaired cytokine
responses upon stimulations of immune cells, including IL-1β, IL-6
and IL-8. tions. The SCR was introduced in the emergency and acute
assessment units in our teaching hospital setting within central
London, accompanied by a seminar-based educational programme
for medical and nursing staff. Methods Two months after its introduction, the use of the SCR
form was audited in all acute medical admissions who met the
clinical criteria for sepsis. One hundred sequential patients were
assessed in a 6-week period over the winter. Conclusions Polymorphisms in TLR1, which is known to dimerize
with TLR2 and TLR6, are associated with impaired immune
recognition through these receptors and predispose to invasive
candidiasis in humans. Results One-half of the audit sample had SCR forms completed. Specificity of the sepsis criteria was good, with <10% of patients
subsequently judged not to have had sepsis. The patients with and
without audit forms had comparable demographics, severity of
illness and microbiology (Figure 1). Frequency of abnormal
temperature was significantly higher in patients with the SCR,
suggesting fever remains an important prompt for physicians to
consider sepsis. The use of the SCR was also associated with
significantly improved assessment of GCS, lactate, travel history
and the need for isolation, as well as significantly greater number of
specialist consultations (Table 1), albeit still inadequate, ~10%
(without SCR) to ~20% (with SCR). P26 The SCR had no effect on
frequency of clinical review by senior resident physicians,
recording of FiO2, antibiotic guidelines compliance or blood/urine
culture requests, all evident in 40 to 60% of patients. Approxi-
mately 75% of all patients received antibiotics within 2 to 6 hours,
and a trend for earlier antibiotics was associated with use of the
SCR (Figure 2). P26 20.0 ± 5.2 IU/ml – all P <0.05) and inhibition of
fibrinolysis (PAA: 99.2 ± 5.9 vs. 73.3 ± 5.7% of normal, P <0.05;
PAI-1: 1.9 ± 0.6 vs. 10.0 ± 1.4 ng/ml, P <0.05). Systemic
administration of AT resulted in supraphysiologic levels of BALF AT
levels (25.4 ± 4.9 vs. 5.3 ± 1.0 IU/ml) and prevented further
activation of coagulation by MV (TATc: in LI-MV with AT vs. placebo, 3.9 ± 0.3 vs. 6.5 ± 0.8 ng/ml – P <0.05). No changes in
pulmonary cytokines were observed between the infected and
mechanically ventilated animals (TNF: LI-MV with AT vs. placebo,
1.6 ± 0.4 vs. 1.9 ± 1.3 ng/ml and IL-6: 2.5 ± 1.3 vs. 2.4 ± 1.4 ng/ml Conclusions rh-TFPI attenuates local and systemic coagulopathy,
the local and systemic inflammatory response and pulmonary
bacterial growth during S. pneumoniae pneumonia in mice. S12 Available online http://ccforum.com/supplements/13/S4 Figure 1 (abstract P29) Figure 1 (abstract P29) P28 Toll like receptor 1 polymorphisms and susceptibility to
invasive candidiasis Toll like receptor 1 polymorphisms and susceptibility to
invasive candidiasis T Plantinga1, M Johnsson2, B Scott3, E van de Vosse4, D Velez3,
JWM van der Meer1, J van Dissel4, J Perfect2, B-J Kullberg1,
MG Netea1
1Radboud University Nijmegen, the Netherlands; 2Duke University
Medical Center, Durham, NC, USA; 3University of Miami Miller
School of Medicine, Miami, FL, USA; 4Leiden University Medical
Center, Leiden, the Netherlands
Critical Care 2009, 13(Suppl 4):P28 (doi: 10.1186/cc8084) T Plantinga1, M Johnsson2, B Scott3, E van de Vosse4, D Velez3,
JWM van der Meer1, J van Dissel4, J Perfect2, B-J Kullberg1,
MG Netea1 Introduction Invasive candidiasis is a severe systemic fungal
infection with Candida spp. affecting immunocompromised hosts,
which is responsible for the highest mortality rate of all nosocomial
infections. Although several clinical predisposing factors are
known, the individual risk for developing invasive candidiasis varies
significantly. Recognition of fungi such as Candida albicans is
mediated through receptors of the innate immune system, such as
Toll-like receptors (TLRs), that in turn activate innate immune
system and antifungal defense. Introduction Invasive candidiasis is a severe systemic fungal
infection with Candida spp. affecting immunocompromised hosts,
which is responsible for the highest mortality rate of all nosocomial
infections. Although several clinical predisposing factors are
known, the individual risk for developing invasive candidiasis varies
significantly. Differential effects of IL-17 pathway in disseminated
candidiasis and zymosan-induced multiple organ failure Differential effects of IL-17 pathway in disseminated
candidiasis and zymosan-induced multiple organ failure FL van de Veerdonk1,2, BJ Kullberg1,2, IC Verschueren1,2,
T Hendriks3, JWM van der Meer1,2, LAB Joosten1,2,4,
MG Netea1,2
1Department of Medicine, Radboud University Nijmegen Medical
Centre, Nijmegen, the Netherlands; 2Nijmegen Institute for
Infection, Inflammation and Immunity, Nijmegen, the Netherlands;
3Department of Surgery, Radboud University Nijmegen Medical
Centre, Nijmegen, the Netherlands; 4Department of Rheumatology,
Radboud University Nijmegen Medical Centre, Nijmegen, the
Netherlands
Critical Care 2009, 13(Suppl 4):P30 (doi: 10.1186/cc8086) FL van de Veerdonk1,2, BJ Kullberg1,2, IC Verschueren1,2,
T Hendriks3, JWM van der Meer1,2, LAB Joosten1,2,4,
MG Netea1,2 FL van de Veerdonk1,2, BJ Kullberg1,2, IC Verschueren1,2,
T Hendriks3, JWM van der Meer1,2, LAB Joosten1,2,4,
MG Netea1,2 Methods Mice defective in caspase-1, ASC, NLRP3 or P2X7 were
infected intravenously with Candida albicans. Survival, fungal
outgrowth in the organs, histology, and cytokine production were
compared in these mouse strains with the wild-type C57/Bl6
control mice. PBMCs from healthy volunteers with or without
reactive oxygen species (ROS) inhibitor and PBMCs from patients
with chronic granulomatous disease (CGD) that are deficient in
ROS production were stimulated with C. albicans. Results Caspase-1–/– mice and ASC–/– mice had a decreased
survival during disseminated candidiasis (50%) compared with the
control mice (100%). Caspase-1–/– mice had a 100-fold increase
in fungal loads in the kidneys of the deficient animals (P <0.05) Methods Mice defective in caspase-1, ASC, NLRP3 or P2X7 were
infected intravenously with Candida albicans. Survival, fungal
outgrowth in the organs, histology, and cytokine production were
compared in these mouse strains with the wild-type C57/Bl6
control mice. PBMCs from healthy volunteers with or without
reactive oxygen species (ROS) inhibitor and PBMCs from patients
with chronic granulomatous disease (CGD) that are deficient in
ROS production were stimulated with C. albicans. 1Department of Medicine, Radboud University Nijmegen Medical
Centre, Nijmegen, the Netherlands; 2Nijmegen Institute for
Infection, Inflammation and Immunity, Nijmegen, the Netherlands;
3Department of Surgery, Radboud University Nijmegen Medical
Centre, Nijmegen, the Netherlands; 4Department of Rheumatology,
Radboud University Nijmegen Medical Centre, Nijmegen, the
Netherlands
C iti
l C
2009 13(S
l 4) P30 (d i 10 1186/
8086) cal Care 2009, 13(Suppl 4):P30 (doi: 10.1186/cc8086 Results Caspase-1–/– mice and ASC–/– mice had a decreased
survival during disseminated candidiasis (50%) compared with the
control mice (100%). Challenges to implementation of sepsis guidelines S Patel1, E Wise2, J Hartin1, D Walker1, M Noursadeghi2,3
1Critical Care, 2Acute Assessment Unit, and 3Infection and
Immunity, University College London Hospitals NHS Foundation
Trust, London, UK
Critical Care 2009, 13(Suppl 4):P29 (doi: 10.1186/cc8085) Introduction International surviving sepsis guidelines identified an
important role for acute medicine in early management of severe
sepsis, but local and multicentre international audits show poor
adherence to these guidelines. Introduction International surviving sepsis guidelines identified an
important role for acute medicine in early management of severe
sepsis, but local and multicentre international audits show poor
adherence to these guidelines. Materials We evaluated the use of a Sepsis Case Record (SCR)
supported by a systematic educational programme to improve
standards. A one-page SCR was derived from surviving sepsis
guidelines, to prompt recognition of sepsis syndromes, compre-
hensive secondary assessment, initiation of resuscitation and
antibiotic treatment bundles, and appropriate specialist consulta- Conclusions The SCR was well received but not used
consistently. The lack of abnormal temperature may contribute to
this. The use of the SCR did improve early management of sepsis, S13 Critical Care November 2009 Vol 13 Suppl 4
Sepsis 2009 Table 1 (abstract P29)
Parameter
With Forms
No Forms
P value
SpR review
24/50
25/50
ns
FiO2
29/50
25/50
ns
Blood culture
42/50
35/50
ns
Urine culture
29/50
23/50
ns
Abx guide compliant
31/50
25/50
ns
ID consult
9/50
8/50
ns
ITU consult
11/50
6/50
ns
GCS
40/50
29/50
<0.05
Lactate
45/50
35/50
<0.05
Micro consult
6/50
0/50
<0.05
Any consult
20/50
8/50
<0.05
Travel history
23/50
9/50
<0.05
Need for isolation
18/50
2/50
<0.05
ns, not significant. Table 1 (abstract P29) studies reported that IL-17 may contribute to inflammatory
pathology and worsening of fungal disease. To address these
discrepancies, we assessed the differential role of IL-17 pathway in
two models of fungal sepsis: intravenous infection with live
Candida albicans, in which fungal growth is the main cause of
mortality, and zymosan-induced multiple organ failure in which the
inflammatory pathology drives the mortality. Methods IL-17 receptor-deficient (IL-17RA–/–) and control mice
were intravenously infected with 2 x 105 CFU live C. albicans
UC820 per mouse. Mortality, fungal loads in the kidneys,
neutrophil recruitment and phagocytosis and killing were assessed. IL-17RA–/– and control mice were also assessed for mortality in a
multiorgan failure sepsis model induced by the fungal component
zymosan. Challenges to implementation of sepsis guidelines Results On the one hand, IL-17RA–/– mice showed increased
mortality and higher fungal loads in the kidneys in the model of
disseminated candidiasis. On the other hand, the absence of
IL-17RA in the knockout mice did not protect the mice against the
multiorgan failure induced by zymosan. Furthermore, no reduction
in neutrophil recruitment and defects in phagocytosis and killing in
the first few hours of Candida infection were found. A significantly
lower TNF production in response to Candida in cells from
IL-17RA–/– mice was observed. Conclusions These data demonstrate that the IL-17 pathway does
not have a major contribution to the inflammatory pathology leading
to organ failure in fungal sepsis, and support the concept that the
IL-17 pathway is protective during fungal sepsis. In addition, IL-17
deficiency does not appear to reflect a pure innate defect, since it
did not result in loss of neutrophil recruitment and function during
the first few hours of fungal sepsis. Furthermore, the lower TNF
production in response to Candida in cells from IL-17RA–/– mice
could contribute to susceptibility to disseminated candidiasis. Figure 2 (abstract P29) P31 Caspase-1 and ASC but not NLRP3 mediate antifungal
defense in candidiasis sepsis Caspase-1 and ASC but not NLRP3 mediate antifungal
defense in candidiasis sepsis F van de Veerdonk1,2, LAB Joosten2, P Shaw1, S Smeekens2,
JWM van der Meer2, B-J Kullberg2, MG Netea2, T-D Kanneganti1
1Department of Immunology, St Jude Children’s Research Hospital,
Memphis, TN, USA; 2Department of Medicine, Radboud University
Nijmegen Medical Centre, and Nijmegen Institute for Infection,
Inflammation and Immunity (N4I), Nijmegen, the Netherlands
Critical Care 2009, 13(Suppl 4):P31 (doi: 10.1186/cc8087) but a number of deficiencies persisted. Implementation of sepsis
guidelines remains a major challenge in clinical practice. Succinct
guidelines were helpful in this setting but need additional
educational and feedback support to improve standards of practice. Introduction IL-1β plays an important role in antifungal defense. The inflammasome is thought to be required for caspase-1
activation and processing of the inactive precursor pro-IL-1β into
its active form. Contradictory data have been reported regarding
the role of the inflammasome in Candida sepsis. In order to
address these discrepancies, we investigated host defense against
disseminated candidiasis in knockout mice defective in the various
components of the inflammasome. P30 Available online http://ccforum.com/supplements/13/S4 Experiments using the NADPH inhibitor diphenyl-
eneiodonium, or in monocytes isolated from CGD patients who
have defective capacity to form ROS, demonstrated that ROS did
not mediate inflammasome activation and C. albicans induced
IL-1β production. and histological assessment revealed preferential growth of
hyphae in the pyelum of the caspase-1–/– mice. In contrast, ASC–/–
mice did not have higher fungal loads, but they showed a
significant stronger inflammatory reaction in the kidneys. On days 3
and 7 of infection, the ASC–/– mice splenocytes that were
restimulated with Candida specifically showed a higher TNF
production. NLRP3–/– and P2X7–/– did not display an increased
susceptibility to disseminated candidiasis, as shown by normal
survival and fungal loads in the organs. Local IL-1β production was
lower in caspase-1–/– mice, but not in the ASC–/–, NLRP3–/– or
P2X7–/– animals. Experiments using the NADPH inhibitor diphenyl-
eneiodonium, or in monocytes isolated from CGD patients who
have defective capacity to form ROS, demonstrated that ROS did
not mediate inflammasome activation and C. albicans induced
IL-1β production. Methods Retrospective data collection of all patients with severe
sepsis or septic shock who were admitted to the ITU over 3 months
(December 2008, January and February 2009). All patients who
developed sepsis before admission to the ITU/HDU were included. Results Thirty-three patients were admitted to the ITU at West
Middlesex Hospital with either severe sepsis or septic shock. Median age was 72 years. The overall mortality rate was 50%. Patients with septic shock had a mortality rate of 52%. The results
of the initial resuscitation of the patients are summarized in Table 1. In septic shock patients, only 35% had ITU intervention within 6
hours (had CVP insertion and/or started on vasopressor and/or
inotropic support). Central venous oxygen saturation or mixed
venous oxygen saturation was not measured for these patients. Conclusions Early recognition and the initial resuscitation of
sepsis at this District General Hospital were assessed for the first
time. Patients with severe sepsis or septic shock were not
resuscitated appropriately and the SSC guidelines were not
implemented, resulting in a high mortality rate. The results showed
that there is a delay in recognizing sepsis at early stages resulting
in inadequate management of patients. In septic shock patients,
this resulted in delayed CVP measurement and administration of
vasopressors and/or inotropic support. Table 1 (abstract P32) S15
Table 1 (abstract P32)
Breakdown of tasks of the initial resuscitation bundle achieved within 6 hours
% of patients
Number of patients
where SSC
achieved/total number
recommendation
Initial resuscitation tasks (within 6 hours)
of patients
was followed
Serum lactate measured
25/33
76
Obtaining blood cultures prior to antibiotic administration
6/33
18
Broad-spectrum antibiotics within 3 hours from time of presentation for Emergency Department admissions
6/9
67
1 hour for non-Emergency Department ICU admissions
5/24
21
In patients with septic shock or serum lactate >4 mmol/l (36 mg/dl)
Fluid challenges
7/23
30
Vasopressors
17/23
74
CVP >8 mmHg in nonmechanically ventilated patients (12 to 15 in mechanically ventilated patients)
2/23
9 Breakdown of tasks of the initial resuscitation bundle achieved within 6 hours P33 Z Aboud, T Peters
ICU Department, West Middlesex Hospital, London, UK
Critical Care 2009, 13(Suppl 4):P32 (doi: 10.1186/cc8088) The selective V1a receptor agonist FE 202158 does not
cause von Willebrand factor release in sheep unlike
arginine vasopressin Introduction Mortality associated with severe sepsis remains high
at 30 to 50% and rises to 50 to 60% when shock is present. The
Surviving Sepsis Campaign (SSC) recommends two bundles for
severe sepsis management to achieve 25% reduction in mortality;
the Initial Resuscitation Bundle (within the first 6 hours) and the
Management Bundle (within 24 hours). West Middlesex University
Hospital set up a severe sepsis management protocol based on the
SSC initial resuscitation and management bundles. It is a 350-bed
hospital with an emergency department. Five hundred patients
(medical and surgical) are admitted to the critical care unit per year. Objective To assess the early recognition of sepsis and the
application of the initial resuscitation bundle according to SSC
guidelines at West Middlesex University Hospital. S Rehberg1, P Enkhbaatar1, R Laporte2, J Rehberg1, E La2,
K Wisniewski2, LD Traber1, CD Schteingart2, PJM Riviere2,
DL Traber1 1Investigational Intensive Care Unit, Department of Anesthesiology,
The University of Texas Medical Branch, Galveston, TX, USA;
2Ferring Research Institute, Inc., San Diego, CA, USA
Critical Care 2009, 13(Suppl 4):P33 (doi: 10.1186/cc8089) 1Investigational Intensive Care Unit, Department of Anesthesiology,
The University of Texas Medical Branch, Galveston, TX, USA;
2Ferring Research Institute, Inc., San Diego, CA, USA
Critical Care 2009, 13(Suppl 4):P33 (doi: 10.1186/cc8089) Introduction The mixed V1a/V2 receptor agonist arginine vaso-
pressin (AVP) is recommended by the guidelines of the Surviving
Sepsis Campaign as an adjunct vasopressor in norepinephrine-
resistant septic shock. However, AVP may be procoagulant Available online http://ccforum.com/supplements/13/S4 Methods Retrospective data collection of all patients with severe
sepsis or septic shock who were admitted to the ITU over 3 months
(December 2008, January and February 2009). All patients who
developed sepsis before admission to the ITU/HDU were included. Results Thirty-three patients were admitted to the ITU at West
Middlesex Hospital with either severe sepsis or septic shock. Median age was 72 years. The overall mortality rate was 50%. Patients with septic shock had a mortality rate of 52%. The results
of the initial resuscitation of the patients are summarized in Table 1. In septic shock patients, only 35% had ITU intervention within 6
hours (had CVP insertion and/or started on vasopressor and/or
inotropic support). Central venous oxygen saturation or mixed
venous oxygen saturation was not measured for these patients. Conclusions Early recognition and the initial resuscitation of
sepsis at this District General Hospital were assessed for the first
time. Patients with severe sepsis or septic shock were not
resuscitated appropriately and the SSC guidelines were not
implemented, resulting in a high mortality rate. The results showed
that there is a delay in recognizing sepsis at early stages resulting
in inadequate management of patients. In septic shock patients,
this resulted in delayed CVP measurement and administration of
vasopressors and/or inotropic support. Therefore, we have
suggested an educational programme running throughout the year
to educate medical and nursing teams about the early recognition
and management of sepsis, with emphasis on the strict
implementation of all tasks of sepsis protocol according to SSC
guidelines to reduce the mortality rate by 25%. We also suggest
setting up critical care beds on each ward that will be supported
by ITU outreach for CVP insertion and level 1 monitoring. and histological assessment revealed preferential growth of
hyphae in the pyelum of the caspase-1–/– mice. In contrast, ASC–/–
mice did not have higher fungal loads, but they showed a
significant stronger inflammatory reaction in the kidneys. On days 3
and 7 of infection, the ASC–/– mice splenocytes that were
restimulated with Candida specifically showed a higher TNF
production. NLRP3–/– and P2X7–/– did not display an increased
susceptibility to disseminated candidiasis, as shown by normal
survival and fungal loads in the organs. Local IL-1β production was
lower in caspase-1–/– mice, but not in the ASC–/–, NLRP3–/– or
P2X7–/– animals. Available online http://ccforum.com/supplements/13/S4 Therefore, we have
suggested an educational programme running throughout the year
to educate medical and nursing teams about the early recognition
and management of sepsis, with emphasis on the strict
implementation of all tasks of sepsis protocol according to SSC
guidelines to reduce the mortality rate by 25%. We also suggest
setting up critical care beds on each ward that will be supported
by ITU outreach for CVP insertion and level 1 monitoring. Conclusions Caspase-1-dependent processing of IL-1β is an
important step in antifungal host defense during Candida sepsis. However, this process is not dependent on the inflammasome
components NLRP3, the ATP receptor P2X7, or ROS. These data
confirm previous studies in human monocytes showing that IL-1β
processing during Candida infection did not require pathogen-
mediated inflammasome activation, due to the constitutive activation
of caspase-1. ASC also plays an important role in Candida sepsis,
but unexpectedly seems to have a different function, specifically by
regulating TNF production and local inflammation in the organs. P32 Early recognition and management of sepsis at West
Middlesex University Hospital Differential effects of IL-17 pathway in disseminated
candidiasis and zymosan-induced multiple organ failure Caspase-1–/– mice had a 100-fold increase
in fungal loads in the kidneys of the deficient animals (P <0.05) Introduction The role of the IL-17 pathway in fungal sepsis
remains controversial. Several studies suggested that IL-17 is
crucial for the protection against Candida sepsis, while other S14 Available online http://ccforum.com/supplements/13/S4 Serum lactate measured S15 Critical Care November 2009 Vol 13 Suppl 4
Sepsis 2009 Critical Care November 2009 Vol 13 Suppl 4
Sepsis 2009 through V2 receptor-mediated effects (for example, von Willebrand
factor (vWF) release). Methods Ewes were operatively prepared and randomized after a
7-day recovery period into control, MRSA, and Ps. aeruginosa
groups (n = 6). Injury consisted of instillation of (2 to 5) x 1011 CFU
live MRSA or Ps. aeruginosa into the airway, and the sheep were
sacrificed after 24 hours. In addition, groups of C57Bl/6J wild-type
mice and iNOS knockout mice (n = 6) were nasally inoculated with
(2 to 5) x 108 CFU live MRSA or Ps. aeruginosa and were
sacrificed after 8 hours. through V2 receptor-mediated effects (for example, von Willebrand
factor (vWF) release). Objective We hypothesized that the selective V1a receptor agonist
FE 202158, which lacks the activity at the V2 receptor, might not
have the procoagulant effects of AVP. This hypothesis was tested
by measuring vWF antigen (vWF:Ag) activity in plasma of healthy
sheep during administration of either FE 202158, AVP, the
selective V2 receptor agonist desmopressin, or vehicle. Methods After measurements of vWF:Ag activity and hemoglobin
concentration in blood over a 1-hour baseline period, 24 female
sheep were randomly assigned to receive either an intravenous
bolus of the selective V2 receptor agonist desmopressin (1 nmol/kg)
or a continuous intravenous infusion of AVP (3 pmol/kg/min), the
selective V1a receptor agonist FE 202158 (10 pmol/kg/min) or
vehicle (0.9% NaCl, n = 6 each). The infusion rates were represen-
tative of the requirements for the treatment of sepsis-induced
vasodilatory hypotension in sheep. vWF:Ag activity and hemo-
globin concentration were measured 60, 90 and 120 minutes after
initiation of treatment. Because of the V2 receptor-mediated fluid
retention, vWF:Ag activity was corrected for plasma volume
changes by calculating the ratio of vWF:Ag activity/hemoglobin
concentration (vWF:Ag/Hb). Data are expressed as a percentage
of the mean baseline value and presented as mean ± SEM. Results Ps. aeruginosa-treated sheep had a significantly higher
ADMA (1.79 ± 0.14 vs. 1.16 ± 0.24 μM, P <0.05), lower plasma
NOx (6.83 ± 0.17 vs. 9.63 ± 0.64 μM, P <0.05), and higher
arginase activity (1.55 ± 0.16 vs. 1.07 ± 0.11 μM urea/μg protein,
P <0.05) compared with MRSA-treated sheep. These changes
were associated with more pronounced lung injury in Ps. aerugi-
nosa sepsis compared with MRSA sepsis (PaO2/FiO2: 205 ± 72
vs. P34 Methods We prospectively studied 14 consecutive septic shock
patients with a pulmonary artery catheter in place. For 3 days after
admission, hemodynamic variables, and plasma levels of lactate,
thrombin–antithrombin complexes (TAT), tissue plasminogen
activator (tPA), plasminogen activator inhibitor (PAI) and plasmin–
α2-antiplasmin complexes and TNFα, IL-6 and complement
activation product C3a were measured 6-hourly. Serum lactate measured 319 ± 82, P <0.05). Ps. aeruginosa-treated mice had a signifi-
cantly higher arginase activity (0.60 ± 0.12 vs. 0.16 ± 0.0038 μM
urea/μg protein, P <0.05) and higher protein oxidation with
carbonyl groups (3,223 ± 440.7 vs. 1,124 ± 140.1, P <0.05)
compared with MRSA-treated mice. iNOS knockout mice treated
with MRSA had significantly lower arginase activity than wild-type
mice (0.21 ± 0.01 vs. 0.36 ± 0.04 μM urea/μg protein, P <0.05). Conclusions Suppressed NO production in Ps. aeruginosa sepsis
is caused by the increased expression of ADMA. Increased
arginase activity is most probably caused by augmented oxidative
stress, which results in a more severe lung injury. Results Whereas there were no significant changes in
vWF:Ag/Hb in vehicle-treated animals over time, desmopressin
and AVP caused an immediate increase in vWF:Ag/Hb after
60 minutes (129 ± 6% and 121 ± 2% of baseline (100%), respec-
tively; P <0.01 each). At each time point during the 120-minute
study period, vWF:Ag/Hb was significantly higher in desmo-
pressin-treated and AVP-treated animals than in vehicle-treated
animals (P <0.001 each). In contrast, there was no significant
difference between FE 202158-treated and vehicle-treated
animals (P = 0.225). Notably, vWF:Ag/Hb in the FE 202158 group
(maximum 108 ± 2% at 120 minutes) was significantly lower than
the AVP group (maximum 123 ± 2% at 60 minutes; P ≤0.005) and
the desmopressin group (maximum 138 ± 6% at 120 minutes;
P <0.001) at every time point. Disseminated intravascular coagulation during human
septic shock: relation with lactate levels KJ Hartemink1,2, CE Hack3, ABJ Groeneveld1
1Department of Intensive Care and the Institute for Cardiovascular
Research, 2Department of Surgery, and 3Department of Clinical
Chemistry, VU University Medical Center, Amsterdam, the
Netherlands
Critical Care 2009, 13(Suppl 4):P35 (doi: 10.1186/cc8091) Conclusions Unlike AVP, the selective V1a receptor agonist FE
202158 did not increase vWF:Ag/Hb ratios in plasma compared
with vehicle-treated animals. Therefore, a selective V1a receptor
agonist such as FE 202158 might be superior to AVP or other
mixed V1a/V2 receptor agonists under conditions that produce
activation of the coagulation system, such as severe sepsis and
septic shock. Introduction The exact pathogenic role of disseminated intra-
vascular coagulation (DIC) during septic shock is incompletely
understood. Introduction The exact pathogenic role of disseminated intra-
vascular coagulation (DIC) during septic shock is incompletely
understood. Objective We studied the relation between sensitive and specific
markers for DIC and lactate levels in the course of time, to evaluate
whether DIC could contribute to microvascular obstruction and
tissue hypoxygenation. R Barchetta, C Alessandrini, C Di Corato, F Candidi, F Turani,
M Falco
Department of Anaesthesia and Intensive Care, European Hospital,
Rome, Italy
Critical Care 2009, 13(Suppl 4):P36 (doi: 10.1186/cc8092) MV Schmidt1, P Paulus2, A-M Kuhn1, V Meilladec-Jullig1,
K Zacharowski2, B Bruene1, A von Knethen1
1Institute of Biochemistry I, Faculty of Medicine, Goethe-University
Frankfurt, Germany; 2Department of Anaesthesia, Intensive Care
Medicine & Pain Therapy, University Hospital Frankfurt, Germany
Critical Care 2009, 13(Suppl 4):P37 (doi: 10.1186/cc8093) Introduction Procalcitonin (PCT) and IL-6 are markers used in the
evaluation of systemic inflammation (SIRS) and septic states. The
purpose of this study is to analyse changes in plasma concen-
trations of PCT and IL-6 in patients undergoing cardiac surgery on-
pump and assess its reliability in the early detection of post-
operative infectious complications. In all patients the variation of
brain natriuretic peptide (BNP) was also evaluated in order to
stratify the clinical condition of patients. Introduction Immune paralysis with massive T-cell apoptosis is a
central pathogenic event during sepsis and correlates with septic
patient mortality. Previous observations implied a crucial role of
peroxisome proliferator-activated receptor gamma (PPARγ) during
T-cell apoptosis. Methods To elucidate mechanisms of PPARγ-induced T-cell
depletion, we used an endotoxin model as well as the caecal
ligation and puncture sepsis model to imitate septic conditions in
wild-type versus conditional PPARγ knockout (KO) mice. Methods We measured serum levels of PCT, IL-6 and BNP in
adult patients undergoing myocardial revascularization and/or valve
surgery performed in extracorporeal circulation. The measurements
were performed on the day before the intervention (T0), at the end
of surgery (T1) and then until the third and fourth postoperative day
(T2 to T4). We also recorded the onset of cardiac, respiratory,
neurological, renal and septic complications. The diagnosis of
sepsis was confirmed retrospectively on the basis of clinical,
radiological and microbiological data. All data are expressed as
mean and standard deviation. The Kruskal–Wallis test was used to
assess changes over time of variables. P <0.05 was considered
statistically significant. Results PPARγ KO mice showed a marked survival advantage
compared with control mice. Their T cells were substantially
protected against sepsis-induced death and showed a significantly
higher expression of the pro-survival factor IL-2. Since PPARγ is
described to repress nuclear factor of activated T cells (NFAT)
transactivation and concomitant IL-2 expression, we propose
inhibition of NFAT as the underlying mechanism allowing T-cell
apoptosis. Corroborating our hypothesis, we observed up-
regulation of the pro-apoptotic protein BIM and downregulation of
the anti-apoptotic protein Bcl-2 in control mice, which are down-
stream effector proteins of IL-2 receptor signaling. T-cell-specific peroxisome proliferator-activated receptor
gamma depletion inhibits T-cell apoptosis and improves
survival of septic mice via an IL-2-dependent mechanism T-cell-specific peroxisome proliferator-activated receptor
gamma depletion inhibits T-cell apoptosis and improves
survival of septic mice via an IL-2-dependent mechanism Table 1 (abstract P36) Table 1 (abstract P36)
Results of temporal changes and significance
T0
T1
T2
T3
T4
PCT (ng/ml)
Nonseptic
0.04
0.04
0.58
0.34
0.34
Septic
0.04
0.15
2.63
1.87
0.74
P
NS
<0.001
<0.001
<0.001
<0.01
IL-6 (pg/ml)
Nonseptic
12
160
129
78
75
Septic
18
184
145
261
92
P
NS
NS
NS
<0.01
NS
BNP
Nonseptic
159
154
347
428
492
Septic
373
627
731
756
798
P
<0.01
< 0.01
<0.01
<0.05
<0.05
Conclusions In patients who develop septic complications,
changes in PCT occur earlier than changes in IL-6. Furthermore,
BNP performs in the same fashion as PCT and correlates better
than IL-6 with the clinical data of the infection status. In conclusion,
monitoring PCT seems to be useful in early diagnosis of septic
complications in patients undergoing cardiac surgery and more
sensitive on the variations in IL-6. The combined study of variations
in PCT and BNP could improve the diagnostic accuracy in these
patients. R Barchetta, C Alessandrini, C Di Corato, F Candidi, F Turani,
M Falco
Department of Anaesthesia and Intensive Care, European Hospital,
Rome, Italy
Critical Care 2009, 13(Suppl 4):P36 (doi: 10.1186/cc8092) Application of a
neutralizing anti-IL-2 antibody reversed the pro-survival effect of
PPARγ-deficient T cells and confirmed IL-2-dependent apoptosis
during sepsis. Results There have been enrolled 60 patients undergoing cardiac
surgery in extracorporeal circulation. Among these, nine patients
developed septic complications. The results of temporal changes
and the significance are presented in Table 1. Table 1 (abstract P36) Conclusions Apparently antagonizing PPARγ in T cells might
improve their survival during sepsis, which concomitantly enhances
defence mechanisms and possibly provokes an increased survival
of septic patients. Increased asymmetrical dimethyl-arginine suppresses
nitric oxide production in Pseudomonas aeruginosa sepsis L Sousse, C Jonkam, D Traber, S Rehberg, L Traber,
D Herndon, P Enkhbaatar
Department of Experimental Pathology and Department of
Anesthesiology, University of Texas Medical Branch, Galveston,
TX, USA
Critical Care 2009, 13(Suppl 4):P34 (doi: 10.1186/cc8090) Results Of the 14 patients, eight died in the ICU. Patients had a
hyperdynamic circulation with tachycardia, mild hypotension and
increased cardiac index. The course of TAT, tPA and particularly of
PAI predicted the course of lactate levels, independently of
hemodynamic and inflammatory factors. Lactate and PAI elevations
persisted in nonsurvivors versus survivors. Introduction More than 750,000 patients in the United States
develop sepsis annually. Previously, we have shown that plasma
nitric oxide (NO) levels were approximately sevenfold higher and
that arginase activity is significantly lower in methicillin-resistant
Staphylococcus aureus (MRSA) sepsis than in Pseudomonas
aeruginosa sepsis. In the present study, we hypothesize that
increased asymmetrical dimethyl-arginine (ADMA), an endogenous
inhibitor of NO synthase, is responsible for the suppressed NO
production in Ps. aeruginosa sepsis. Introduction More than 750,000 patients in the United States
develop sepsis annually. Previously, we have shown that plasma
nitric oxide (NO) levels were approximately sevenfold higher and
that arginase activity is significantly lower in methicillin-resistant
Staphylococcus aureus (MRSA) sepsis than in Pseudomonas
aeruginosa sepsis. In the present study, we hypothesize that
increased asymmetrical dimethyl-arginine (ADMA), an endogenous
inhibitor of NO synthase, is responsible for the suppressed NO
production in Ps. aeruginosa sepsis. Conclusions Our observations show that, in the course of human
septic shock, activation of coagulation and, particularly, inhibition
of activated fibrinolysis are independently associated with
hyperlactatemia. This suggests a contribution of DIC resulting from
a coagulation/fibrinolysis imbalance to microvascular obstruction,
tissue hypoxygenation and thereby to ultimate demise. S16 Available online http://ccforum.com/supplements/13/S4 P36 P38 Induction of severe Staphylococcus aureus sepsis in pigs Monitoring of procalcitonin, IL-6 and brain natriuretic
peptide for sepsis diagnosis in cardiac surgery R Barchetta, C Alessandrini, C Di Corato, F Candidi, F Turani,
M Falco
Department of Anaesthesia and Intensive Care, European Hospital,
Rome, Italy
Critical Care 2009, 13(Suppl 4):P36 (doi: 10.1186/cc8092) Induction of severe Staphylococcus aureus sepsis in pigs r-Roscovitine (70 mg/kg) or vehicle was administered
24 hours later in combination with antibiotic therapy (ceftriaxon;
20 mg/kg). Mice were sacrificed after 48 hours. In a second
experiment, mice were infected and treated at 24 and 72 hours
and sacrificed 96 hours post infection. Methods Twelve pigs in four groups were inoculated intravenously
once or twice with 1 x 108 S. aureus/kg body weight and eutha-
nized consecutively from 6 to 48 hours after inoculation. Mock-
inoculated pigs served as controls. Body temperature was
measured and blood samples were taken at regular intervals for
bacteriology, haematology, clinical chemistry, and acute phase
reactant determinations. Full necropsy was done and tissue
samples were collected for bacteriology and histology. Apoptosis
was measured in the spleen. Results r-Roscovitine treatment significantly reduced phosphory-
lated CDK substrate and increased cleaved caspase 3 levels in
isolated neutrophils. During LTA-induced lung inflammation,
r-roscovitine treatment significantly reduced the amount of PMNs
in the BAL fluid and cytokines in lung homogenates. After 48 hours
of bacterial pneumonia, r-roscovitine-treated animals displayed
enhanced pulmonary bacterial outgrowth. Cytokine production and
lung damage scores were higher in the r-roscovitine-treated group
as compared with vehicle. Interestingly, when studying the animals
at 96 hours post infection, r-roscovitine treatment resulted in lower
bacterial outgrowth and chemokine levels in the lung. Results Onset of clinical disease (fever and lethargy) was seen at
7 to 8 hours after inoculation. Blood bacterial counts remained low
throughout the study. SIRS characterized by fever, leukocytosis,
increased levels of CRP, IL-6, IL-1β, TNFα, and decreased level of
serum iron was detected after 12 hours. Both CRP and IL-6 levels
peaked at 36 hours. Platelet numbers declined slightly and were
lower than in the controls at 48 hours. Thromboelastography
showed increased hypercoagulability over time. Levels of serum
aspartate aminotransferase and bilirubin were elevated at 24 and
36 hours. Blood urea nitrogen levels had increased at 36 hours;
however, no difference was seen in serum creatinine levels. Disseminated microabscesses were found in the lung at 6 hours,
but had disappeared at 48 hours. In the bones, the presence of
microabscesses progressed until 48 hours. Other histopatho-
logical signs related to inoculation were limited to a renal
microabscess at 12 hours, splenic microabscesses at 24 hours
and centrilobular hepatic necrosis with thrombosis in one animal at
48 hours. In the liver and kidneys, various degrees of fibrinous
exudation were found. Honey as an immunomodulator during sepsis in animal
model Honey as an immunomodulator during sepsis in animal
model Induction of severe Staphylococcus aureus sepsis in pigs TM Iburg1, PS Leifsson1, M Kjelgaard-Hansen2, P Heegaard3,
B Wiinberg2, B Aalbaek1, AE Olsson1, MGS Hansen1,
LB Thomsen1, HE Jensen1, JS Agerholm1, OL Nielsen1
1Department of Veterinary Disease Biology, and 2Department of
Small Animal Clinical Sciences, Faculty of Life Sciences, University
of Copenhagen, Denmark; 3Department of Veterinary Diagnostics
and Research, National Veterinary Institute, Technical University of
Denmark, Copenhagen, Denmark
Critical Care 2009, 13(Suppl 4):P38 (doi: 10.1186/cc8094) Introduction Organ dysfunction is an integrated part of severe
sepsis, and severe sepsis is one of the major causes of death in
ICUs. Lately Gram-positive bacteria accounted for more than one-
half of the overall sepsis cases reported in the USA, with
Staphylococcus aureus being the most commonly isolated
bacterium. Effective treatment of sepsis is still not optimal and
good animal models are needed for research in pathogenesis and
treatment. S. aureus infections are also common in pigs and are
isolated from approximately 40% of embolic lesions found in
slaughter-pigs. Conclusions In patients who develop septic complications,
changes in PCT occur earlier than changes in IL-6. Furthermore,
BNP performs in the same fashion as PCT and correlates better
than IL-6 with the clinical data of the infection status. In conclusion,
monitoring PCT seems to be useful in early diagnosis of septic
complications in patients undergoing cardiac surgery and more
sensitive on the variations in IL-6. The combined study of variations
in PCT and BNP could improve the diagnostic accuracy in these
patients. S17 g
p g
Objective To establish a porcine model of severe sepsis. Critical Care November 2009 Vol 13 Suppl 4
Sepsis 2009 Critical Care November 2009 Vol 13 Suppl 4
Sepsis 2009 70 mg/kg r-roscovitine or vehicle was injected intraperitoneally. Twenty-four hours later bronchoalveolar lavage (BAL) was
performed and differential cell counts were determined. Bacterial
pneumonia was induced by inoculation of 5 x 104 CFU S. pneumoniae. r-Roscovitine (70 mg/kg) or vehicle was administered
24 hours later in combination with antibiotic therapy (ceftriaxon;
20 mg/kg). Mice were sacrificed after 48 hours. In a second
experiment, mice were infected and treated at 24 and 72 hours
and sacrificed 96 hours post infection. 70 mg/kg r-roscovitine or vehicle was injected intraperitoneally. Twenty-four hours later bronchoalveolar lavage (BAL) was
performed and differential cell counts were determined. Bacterial
pneumonia was induced by inoculation of 5 x 104 CFU S. pneumoniae. Induction of severe Staphylococcus aureus sepsis in pigs The number of apoptotic cells in the splenic
white pulp was increased at 48 hours. Conclusions With this study, we reproduced earlier findings that
r-roscovitine treatment reduces CDK activity and induces
apoptosis in neutrophils; we demonstrated that r-roscovitine
diminishes inflammatory responses in sterile inflammation; and we
found that r-roscovitine treatment in bacterial pneumonia is
detrimental early in infection but beneficial at later time points. We
believe that the negative effect of r-roscovitine reflects the
importance of neutrophil antibacterial defense early in infection. Yet, during resolution of infection, apoptosis of neutrophils induced
by r-roscovitine could present a way of damage control as
opposed to unwanted side effects of neutrophil function. Conclusions All infected pigs developed sepsis with metastatic
abscesses and at 48 hours severe sepsis was present with signs
of dysfunction of the liver and the coagulation system. The splenic
apoptotic response indicates reduced function and immuno-
suppression. P39 M Kassim1, M Mansor1, M Achoui2, OS Yan1, S Devi3,
KM Yusoff4
1Department of Anesthesiology, 2Department of Pharmacology,
3Department of Medical Microbiology, and 4Department of
Molecular Medicine, Faculty of Medicine, University of Malaya,
Kuala Lumpur, Malaysia
Critical Care 2009, 13(Suppl 4):P40 (doi: 10.1186/cc8096) M Kassim1, M Mansor1, M Achoui2, OS Yan1, S Devi3,
KM Yusoff4 Cyclin-dependent kinase inhibitor r-roscovitine reduces
lipoteichoic acid lung inflammation and improves the
resolution of antibiotic-treated Streptococcus pneumoniae
pneumonia 1Department of Anesthesiology, 2Department of Pharmacology,
3Department of Medical Microbiology, and 4Department of
Molecular Medicine, Faculty of Medicine, University of Malaya,
Kuala Lumpur, Malaysia
Critical Care 2009, 13(Suppl 4):P40 (doi: 10.1186/cc8096) P41 Are phenylcarboxylic acids really markers in severe
sepsis? Are phenylcarboxylic acids really markers in severe
sepsis? Objective A multidisciplinary public health campaign was
developed to educate a local urban community about recognizing
the signs and symptoms of sepsis, preventing infection, and
seeking treatment early. Targeting education efforts at the
community level engages consumers to become involved in the
care of their health. NV Beloborodova, AS Khodakova, AJ Olenin
Bakoulev Scientific Center of Cardiovascular Surgery, Moscow,
Russian Federation
Critical Care 2009, 13(Suppl 4):P41 (doi: 10.1186/cc8097) Introduction Laboratory diagnostics of sepsis need to be
improved. There is no evidence in the literature whether microbial
metabolites could be used as sepsis markers. As a result of large-
scale screening of microbial compounds we showed that levels of
some phenylcarboxylic acids (PCAs) were increased in blood of
septic patients. The content of p-hydroxyphenyllactic acid (HPLA),
phenyllactic acid (PLA) and p-hydroxyphenylacetic acid (HPAA)
was significantly higher in blood of patients with severe sepsis
compared with control groups. The aim of the present study is to
evaluate the sensitivity and specificity of PLA, HPLA and HPAA as
markers for severe sepsis diagnostics. Methods A multimedia approach including print, television
broadcast, hospital website on the Internet, news article and
advertisement in the newspaper were widely distributed to
maximize the ability to reach citizens throughout the area. A live
seminar presented by two physicians and a clinical nurse specialist
was strategically used to enhance learning, and participants
completed an evaluation upon completion of the seminar. Results Ninety-six percent of the participants who attended the
live seminar rated the program as excellent. The participants
shared that the topic was very educational, informative, and felt
that their questions were answered. Because of the education
media campaign that targeted 320,000 households and the
hospital-wide implementation of the sepsis bundles, the mortality
for severe sepsis decreased from 40% at baseline to 32.8%,
which was a 18% relative risk reduction (Figure 1). Methods In total, 264 blood samples from 200 adults were
included to research. All persons were divided into groups with
infectious complications after cardiosurgery (35 severe sepsis, 35
local infection complication) and others (33 non-infection compli-
cation after surgery, 30 smooth recovery after surgery, 42 before
surgery and 25 healthy volunteers). Clinical characteristics and
procalcitonin (PCT), a well established biomarker of sepsis, were
assessed in all patients. Impact of community-based education on sepsis Impact of community-based education on sepsis K Choy, CA Agcaoili, K Halimi
Washington Hospital Healthcare System, Fremont, CA, USA
Critical Care 2009, 13(Suppl 4):P42 (doi: 10.1186/cc8098) Introduction Sepsis is an uncontrolled infection that can develop
very quickly throughout the body. Sepsis can strike anyone at any
age and people with pre-existing medical conditions may be at
greater risk. Patients with sepsis often present to the emergency
department from home. Community education programs focus on
raising public awareness of sepsis, its signs and symptoms, and
can positively impact outcomes. Introduction Sepsis is an uncontrolled infection that can develop
very quickly throughout the body. Sepsis can strike anyone at any
age and people with pre-existing medical conditions may be at
greater risk. Patients with sepsis often present to the emergency
department from home. Community education programs focus on
raising public awareness of sepsis, its signs and symptoms, and
can positively impact outcomes. pneumonia Obtained data indicate that quantitative measurement of PLA,
HPLA and HPAA in blood could be used for sepsis diagnostics in
clinical practice and also as a predictor of outcome in high-risk
surgery. Conclusions The following levels of PCAs are appropriate for
diagnostic of sepsis: HPAA 8 x 10–6 M (sensitivity 64.3%, speci-
ficity 88.9%), HPLA 3 x 10–6 M (sensitivity 75%, specificity
66.4%), PLA 1 x 10–6 M (sensitivity 75%, specificity 71.6%). Obtained data indicate that quantitative measurement of PLA,
HPLA and HPAA in blood could be used for sepsis diagnostics in
clinical practice and also as a predictor of outcome in high-risk
surgery. pneumonia AJ Hoogendijk1,2, JJTH Roelofs3, MHP van Lieshout1,2,
DC Blok1,2, T van der Poll1,2, CW Wieland2,4
1Center for Infection and Immunity Amsterdam, 2Center for
Experimental and Molecular Medicine, 3Department of Pathology,
and 4Laboratory of Experimental Intensive Care and
Anesthesiology, Academic Medical Center, Amsterdam, the
Netherlands
Critical Care 2009, 13(Suppl 4):P39 (doi: 10.1186/cc8095) Introduction Malaysian honey (Gelam) has antibacterial activity and
it also has a high antioxidant capacity and free radical scavenger
activities. Honey extracts showed potent activity against TNFα in
L929 cell and NO in RAW 264.7 macrophage as well as inhibitory
effects on the prostaglandin E2 and nitric oxide (NO) in inflammatory
tissues of rat. Sepsis is mediated in part by bacterial endotoxin,
which stimulates macrophages/monocytes to sequentially release
early (for example, TNF, IL-1) cytokines and inducible enzymes such
as inducible nitric oxide (iNOS) synthase and heme oxygenase 1
(HO-1) and late such as high-mobility group box 1 (HMGB1). Introduction Streptoccocus pneumoniae
pneumonia remains
associated with high morbidity and mortality. Antibiotic treatment
frequently is insufficient in limiting lung damage due to inflam-
mation. Therefore, additional treatment strategies are needed. The
drug r-roscovitine, a cyclin-dependent kinase (CDK) inhibitor, was
demonstrated to reduce inflammation in several models of
inflammation. Objective To investigate the role of honey as an immunomodulator
in sepsis induced by LPS in rats. Methods Four groups (n = 6) of rats were used. The treatment
group received honey with LPS, the positive control group were
given LPS (5 mg/kg), the negative control group were given saline
only, while the fourth group were only given honey; all doses were
1 ml by intravenous route. Blood samples were collected 4 hours
later and all rats were sacrificed after 24 hours. TNFα, IL-1β, IL-6,
IL-10, NO, HO-1 and HMGB1 were quantified using ELISA. The
effect of honey on coagulation (PT and APPT) in whole blood ex
vivo from healthy volunteers (n = 10) was measured. Objective We studied the potential of r-roscovitine to modulate
host defense during sterile inflammation and bacterial infection of
the lung. Methods Isolated neutrophils were treated with 20 μM r-roscovitine
and CDK and caspase 3 activity were determined by western blot
analysis. Sterile lung inflammation was induced by intranasal
administration of 100 μg lipoteichoic acid (LTA), a prominent cell
wall component of Gram-positive bacteria. Simultaneously S18 Available online http://ccforum.com/supplements/13/S4 Results After 4 hours of treatment, the cytokines, NO and HO-1
were measured in all groups. pneumonia Honey showed evidence of immuno-
modulatory effects with reduced cytokines (TNFα (P < 0.001),
IL-1β (P < 0.001), IL-10 (P < 0.001)) and NO (P < 0.037) in the
treatment group, while the change in IL-6 was not significant
between all groups, HO-1 (P < 0.001) was increased in the
treatment group, but only slightly increased in the honey group. After 24 hours of treatment, HMGB1 (P <0.025) and IL-1β
(P <0.001) were reduced in the treatment group as well. HO-1
(P <0.013) continuously increased in all groups. Curiously, honey
alone induced TNFα (P <0.001) and IL-1β (P <0.03) at 4 hours,
and HO-1 (P <0.028) at 24 hours compared with saline. Honey
prolonged the time of PT and APPT in a dose-dependent manner. Results After 4 hours of treatment, the cytokines, NO and HO-1
were measured in all groups. Honey showed evidence of immuno-
modulatory effects with reduced cytokines (TNFα (P < 0.001),
IL-1β (P < 0.001), IL-10 (P < 0.001)) and NO (P < 0.037) in the
treatment group, while the change in IL-6 was not significant
between all groups, HO-1 (P < 0.001) was increased in the
treatment group, but only slightly increased in the honey group. After 24 hours of treatment, HMGB1 (P <0.025) and IL-1β
(P <0.001) were reduced in the treatment group as well. HO-1
(P <0.013) continuously increased in all groups. Curiously, honey
alone induced TNFα (P <0.001) and IL-1β (P <0.03) at 4 hours,
and HO-1 (P <0.028) at 24 hours compared with saline. Honey
prolonged the time of PT and APPT in a dose-dependent manner. Conclusions Honey behaves as immunomodulator by acting in
two ways, by inducing HO-1, TNFα, and IL-1β and at the same
time inhibiting cytokines, NO and HMGB1 that is induced by LPS. However, the exact mechanism remain unclear, but our suggestion
is that since honey induces HO-1, TNFα and IL-1β this may cause
changes or inhibition in the signaling of cytokines and NF-κB. Honey could therefore be used as a pharmacological tool in sepsis
in the future. 34.7) x 10–6 M vs. 2.8 (2.2 to 5.0) x 10–6 M correspondingly), but
not for HPAA. Conclusions The following levels of PCAs are appropriate for
diagnostic of sepsis: HPAA 8 x 10–6 M (sensitivity 64.3%, speci-
ficity 88.9%), HPLA 3 x 10–6 M (sensitivity 75%, specificity
66.4%), PLA 1 x 10–6 M (sensitivity 75%, specificity 71.6%). P41 Severe sepsis was diagnosed according
to consensus criteria, also a level of PCT ≥2 ng/ml was an
additional criterion. Blood concentrations of PCAs were deter-
mined by gas chromatography–mass spectrometry. IL-33 protects mice from sepsis by inhibiting TLR4
signaling aureus was attempted using the following techniques directly
from the positive bottles: (1) morphology and organization of the
cocci on Gram stain (10 minutes), (2) rapid tube coagulase
(2 hours), (3) direct DNAse test using toluedine blue agar (4
hours), (4) species-specific multiplex PCR (4 hours), (5) direct
inoculation of selective commercial chromogenic media (18 hours),
and
(6)
direct
inoculation
of
mannitol-supplemented
Mueller–Hinton agar (MMHA) combined with disc diffusion
susceptibility testing (18 hours). Identifications were compared
with the standard laboratory identification at 36 hours. Objective To investigate the role of the IL-33/ST2 pathway in
experimental sepsis. Methods and results We show that IL-33 treatment (1 μg/mouse,
intravenously) markedly reduced mortality in WT mice (50%,
P <0.01) undergoing experimental sepsis induced by the cecal
ligation and puncture (CLP) model. We did not detect any
differences between ST2 KO and WT mice in peritoneal bacterial
load and mortality rate after CLP, suggesting that the endogenous
IL-33 does not participate in the pathophysiology sepsis. However,
while the exogenous injection of IL-33 markedly reduced the CLP-
induced mortality in WT mice, IL-33 failed to do so in ST2–/– mice,
indicating the critical role of ST2 on the protective effect of IL-33. Notably, we found that IL-33-treated mice developed significantly
increased neutrophil infiltration in the peritoneal cavity (fourfold) and
more efficient bacterial clearance than untreated mice (n = 10) after
CLP. Moreover, IL-33 treatment leads to marked reduction (30 to
70%) of systemic proinflammatory cytokines (TNFα, IL-6 and
CXCL2) but not a shift toward a Th2 immune response (IL-4 and
IL-13). The chemokine receptor CXCR2 plays a central role in the
recruitment of neutrophils into the site of infection. Flow cytometry
analysis showed that direct activation of TLR4 in neutrophils
downregulates the expression of CXCR2 and, consequently,
impaired CXCL2-driven and CLP-driven neutrophil migration, in
vitro and in vivo respectively. Notably, IL-33 prevented the
downregulation of CXCR2 on circulating neutrophils during CLP in
vivo or LPS-treated neutrophils in vitro. Finally, we demonstrated
that IL-33 reversed the TLR4-induced reduction of CXCR2 via the
inhibition of LPS-induced G-protein-coupled receptor kinase-2
(GRK2) expression, a potent negative regulator of CXCR2. Conclusions Altogether we provide here a novel mechanism of Results Definitive laboratory identification revealed S. epidermidis
87% (n = 98), S. aureus 11% (methicillin-sensitive S. aureus
(MSSA) n = 9, methicillin-resistant S. aureus (MRSA) n = 3),
Micrococcus spp. 2% (n = 2); one culture contained a mixture of
S. IL-33 protects mice from sepsis by inhibiting TLR4
signaling Introduction The ability to rapidly identify and determine the
antimicrobial susceptibility of bacterial pathogens is an undisputed
requirement for strategies aimed at improving the management of
sepsis. Staphylococci are amongst the most common organisms
isolated from blood cultures but it is difficult to rapidly distinguish
between those representing contamination of the blood culture
with harmless skin commensals (Staphylococcus epidermidis) and
those that contain pathogenic species (Staphylococcus aureus). IL-33 protects mice from sepsis by inhibiting TLR4
signaling JC Alves-Filho1,2, F Sonego1, FO Souto1, A Freitas1,
WA Verri Jr1, D Xu2, FQ Cunha1, FY Liew2
1Department of Pharmacology, School of Medicine of Ribeirao
Preto, University of São Paulo, Brazil; 2Division of Immunology,
Infection and Inflammation, Glasgow Biomedical Research Centre,
University of Glasgow, UK
Critical Care 2009, 13(Suppl 4):P43 (doi: 10.1186/cc8099) p
g
p
(
p y
)
Objective A number of phenotypic and genotypic tests with a vast
range of complexity, speed and cost have been proposed to help
distinguish these organisms. We evaluated the sensitivity, specificity
and speed of a range of these tests compared with the standard
laboratory identification protocol which takes up to 48 hours. Introduction Sepsis is an acute systemic inflammation following
infection, with a high mortality rate and limited therapeutic options. IL-33 is a recently identified member of the IL-1 family that binds to
ST2 receptor, which is preferentially expressed on Th2 and mast
cells. Accordingly, the IL-33/ST2 pathway is closely associated with
the activation and production of type-II cytokines (IL-4, IL-5 and IL-
13). However, ST2 has been implicated in inhibiting macrophage-
dependent inflammation in response to LPS by negatively regulating
Toll-like receptor-4 (TLR4) activation. Although TLRs have been
implicated as an important element of host defense against
infections, evidence indicates that these receptors may also play a
detrimental role in the pathophysiology of sepsis. y
p
p
Methods Positive blood culture sets (BACT/Alert 3D) (n = 113) in
which Gram-positive cocci in clusters were seen on the initial film
were included. Further identification as S. epidermidis versus
S. IL-33 protects mice from sepsis by inhibiting TLR4
signaling aureus and S. epidermidis. The sensitivity and specificity of
each of the direct techniques was calculated as follows: (1)
microscopy 53% and 98%, (2) rapid coagulase 92% and 100%,
(3) direct DNAse 69% and 98%, (4) multiplex PCR 88% (no
amplification in 12 samples) and 99%, (5) chromogenic S. aureus
media 90% and 100%, and (6) MMHA 100% and 78%. Conclusions All the methods had specificity >95%, except for
MMHA, although this has the added advantage of providing
susceptibility results. Chromogenic agar had the highest sensitivity
and specificity, although it provided a result only 12 hours quicker
than the standard protocol. Rapid tube coagulase was highly
specific and one of the most rapid tests. This may be the most
useful, inexpensive technique providing preliminary results to guide
empirical therapy, especially in resource-poor settings. P44 Conclusions This community-based education program on sepsis
demonstrated that education programs offered to the community
will improve overall outcomes and promote quality care of sepsis
patients. In the pursuit of evaluating the effectiveness of the
program, there will be ongoing monitoring of its impact. Future
education programs will continue to sustain improvements. Comparison of microscopic, phenotypic and molecular
techniques for the rapid identification and susceptibility
testing of Staphylococci from positive blood culture bottles Comparison of microscopic, phenotypic and molecular
techniques for the rapid identification and susceptibility
testing of Staphylococci from positive blood culture bottles A Shah, NC Gordon, L Pheel, DW Wareham
St Bartholomew’s and The London NHS Trust, London, UK
Critical Care 2009, 13(Suppl 4):P44 (doi: 10.1186/cc8100) A Shah, NC Gordon, L Pheel, DW Wareham Figure 1 (abstract P42) Figure 1 (abstract P42) Figure 1 (abstract P42) Figure 1 (abstract P42)
Severe sepsis mortality. Results The levels of PCA in two control groups (healthy people
and patients before surgery) were HPAA 0.4 to 0.8 x 10–6 M,
HPLA 1.2 to 1.5 x 10–6 M, PLA 0.3 to 0.4 x 10–6 M and were not
significantly different. Otherwise the levels of HPAA, HPLA and
PLA as 11.6 (3.3 to 33.6) x 10–6 M, 7.5 (3.0 to 14.4) x 10–6 M and
1.8 (1.1 to 4.9) x 10–6 M in all severe sepsis patients were
significantly increased versus control groups and versus all other
groups (P <0.0001). In addition the levels of PCAs from surviving
and nonsurviving severe sepsis patients were compared with each
other. Nonsurviving sepsis patients had a significantly higher content
of PLA (3.6 (1.5 to 6.4) x 10–6 M vs. 1.2 (0.8 to 1.6) x 10–6 M for
survivors); the same trend was observed for HPLA (12.5 (5.6 to Severe sepsis mortality. S19 Critical Care November 2009 Vol 13 Suppl 4
Sepsis 2009 Acknowledgements Financial support from Wellcome Trust, MRC
and FAPESP. Pre-treatment with simvastatin prevents LPS-induced
hyporesponsiveness of porcine isolated coronary artery S Al-Shalmani1, S Chinniah2, R Mahajan2, V Wilson1
1School of Biomedical Sciences, and 2Academic Division of
Anaesthesia and Intensive Care, University of Nottingham Medical
School, Nottingham, UK
Critical Care 2009, 13(Suppl 4):P46 (doi: 10.1186/cc8102) Introduction Several retrospective studies suggest that prior use
of statins can reduce hospital mortality in patients diagnosed with
either bacteraemia or sepsis. Simvastatin has been shown to
modify the proinflammatory effect of lipopolysaccharide (LPS) on
neutrophils and endothelial cells. However, it is not clear whether
these effects are also manifest on vascular smooth muscle, which
becomes hyporesponsive to vasoconstrictor agents due to the
induction of nitric oxide synthase. We have investigated the effect
of pre-treatment with simvastatin on LPS-induced changes in
contractions of porcine isolated coronary artery (PCA). Introduction Several retrospective studies suggest that prior use
of statins can reduce hospital mortality in patients diagnosed with
either bacteraemia or sepsis. Simvastatin has been shown to
modify the proinflammatory effect of lipopolysaccharide (LPS) on
neutrophils and endothelial cells. However, it is not clear whether
these effects are also manifest on vascular smooth muscle, which
becomes hyporesponsive to vasoconstrictor agents due to the
induction of nitric oxide synthase. We have investigated the effect
of pre-treatment with simvastatin on LPS-induced changes in
contractions of porcine isolated coronary artery (PCA). Methods Segments (5 mm) of the PCA were dissected from
hearts and incubated in Krebs–Henseleit (K–H) solution at 37°C in
the presence of an antibiotic mixture (60 μg/ml benzylpenicillin and
20 μg/ml streptomycin sulphate), with or without 1 μg/ml LPS,
3 μM simvastatin or a combination of the two (simvastatin added
60 minutes before LPS). After 16 to 18 hours, segments were
prepared for isometric tension recording in K–H solution. The
segments were then exposed to cumulatively increasing concen-
trations of KCl and then U46619. In some experiments, some
segments were exposed to 10 μM 1400W, a selective inhibitor of
inducible nitric oxide synthase prior to the addition of the agonists. Responses are shown as gram weight or calculated as the
concentration causing 50% of the maximum effect (–log EC50). Differences between mean values were assessed by ANOVA
(post-hoc Dunnett test). Pre-treatment with simvastatin prevents LPS-induced
hyporesponsiveness of porcine isolated coronary artery R
lt
KCl
d
U46619
d
i
d
d Methods Segments of the PCA were dissected from hearts and
incubated (2 x 5 mm) in DMEM at 37°C in the presence of an
antibiotic mixture (60 μg/ml benzylpenicillin, and 20 μg/ml strepto-
mycin sulphate), with or without 1 μg/ml LPS, 3 μM simvastatin or
a combination of the two (simvastatin added 60 minutes before
LPS). The medium also contained 1 mM L-arginine. After 24 hours,
segments were removed and weighed (mg wet weight). The
nitrite/nitrate content (nmol) of the bathing medium was deter-
mined by spectrophotometry using the Griess reaction. In a
separate experiment, segments were prepared for immunohisto-
chemical determination of the presence of iNOS and CD31. Differences between mean values were assessed by ANOVA
(post-hoc Dunnett test). Methods Segments (5 mm) of the PCA were dissected from
hearts and incubated in Krebs–Henseleit (K–H) solution at 37°C in
the presence of an antibiotic mixture (60 μg/ml benzylpenicillin and
20 μg/ml streptomycin sulphate), with or without 1 μg/ml LPS,
3 μM simvastatin or a combination of the two (simvastatin added
60 minutes before LPS). After 16 to 18 hours, segments were
prepared for isometric tension recording in K–H solution. The
segments were then exposed to cumulatively increasing concen-
trations of KCl and then U46619. In some experiments, some
segments were exposed to 10 μM 1400W, a selective inhibitor of
inducible nitric oxide synthase prior to the addition of the agonists. Responses are shown as gram weight or calculated as the
concentration causing 50% of the maximum effect (–log EC50). Differences between mean values were assessed by ANOVA
(post-hoc Dunnett test). Results Under control conditions the coronary artery segments
produced 25.9 ± 3.7 nmol/mg wet weight nitrite/nitrate (n = 8)
over 24 hours. Exposure to 1 μg/ml LPS caused a sevenfold
increase in nitrite/nitrate production (199.0 ± 40.6 nmol/mg wet
weight, n = 8). Although 3 μM simvastatin did not affect basal
nitrite/nitrate production, it inhibited the response to LPS
(35.6 ± 7.4 nmol/mg wet weight nitrite/nitrate) by 94.7 ± 5.7%
(n = 8). Immunohistochemical assessment of four arteries revealed
the presence of CD31 on endothelial cells under control
conditions. Exposure to 1 μg/ml LPS was associated with an
increase in endothelial CD31 and the appearance of iNOS in the
adventitia. P46 P46 either bacteraemia or sepsis. In an accompanying abstract we
demonstrated that pre-treatment with simvastatin prevents LPS-
induced hyporesponsiveness of the porcine isolated coronary
artery (PCA); an observation consistent with the clinical data. Although this effect of simvastatin is qualitatively similar to that of a
known inhibitor of inducible nitric oxide synthase (1400W), it
unlikely that direct inhibition of the enzyme is implicated. We have
investigated whether the beneficial effect of simvastatin on LPS-
induced changes in the PCA involves alteration in the induction of
nitric oxide synthase (iNOS). P46
Pre-treatment with simvastatin prevents LPS-induced
hyporesponsiveness of porcine isolated coronary artery
S Al-Shalmani1, S Chinniah2, R Mahajan2, V Wilson1
1School of Biomedical Sciences, and 2Academic Division of
Anaesthesia and Intensive Care, University of Nottingham Medical
School, Nottingham, UK
Critical Care 2009, 13(Suppl 4):P46 (doi: 10.1186/cc8102) Evidence that simvastatin prevents induction of nitric oxide
synthase by LPS in the porcine isolated coronary artery S Al-Shalmani1, S Chinnah2, R Mahajan2, V Wilson1
1School of Biomedical Sciences, and 2Academic Division of
Anaesthesia and Intensive Care, University of Nottingham Medical
School, Nottingham, UK
Critical Care 2009, 13(Suppl 4):P45 (doi: 10.1186/cc8101) Conclusions Altogether we provide here a novel mechanism of
action of IL-33 and establish a potential therapeutic role of this
new cytokine in sepsis. Introduction Several retrospective studies suggest that prior use
of statins can reduce hospital mortality in patients diagnosed with Acknowledgements Financial support from Wellcome Trust, MRC
and FAPESP. S20 Available online http://ccforum.com/supplements/13/S4 Pre-treatment with simvastatin prevents LPS-induced
hyporesponsiveness of porcine isolated coronary artery Co-incubation of segments with 3 μM simvastatin and
LPS produced a profile similar to that of control segments (CD31-
positive, iNOS-negative, n = 4). Results
KCl and U46619 caused concentration-dependent
contraction of the PCA. Table 1 shows that treatment with 1 μg/ml
LPS overnight (and subsequent removal) significantly reduced the
maximum response to KCl and U46619 in the PCA by
32.0 ± 4.5% (n = 12) and 28.9 ± 12.3% (n = 12), without
changing the potency of either agent. These effects of LPS on
vasoconstrictor responses were not observed when 10 μM
1400W was added to the organ bath, after removal of the
endotoxin (Table 1). LPS also failed to impair constrictor responses
if 3 μM simvastatin was present during the incubation period and
was subsequently removed (Table 1). Conclusions Simvastatin suppressed the induction of nitric oxide
caused by LPS and the associated increase in nitrite/nitrate
production. This finding helps to explain our observation that
simvastatin prevented LPS-induced hyporesponsiveness of the
coronary artery, and is also consistent with clinical studies
suggesting that prior use of statins may afford protection against
bacterial sepsis. Table 1 (abstract P46) Effects of statins on postoperative sepsis, systemic
inflammatory response syndrome and mortality after
colorectal surgery Multiple organ dysfunction syndrome: the scapegoat? Assessment of organ dysfunction between surviving and
dying mice in the acute phase of polymicrobial sepsis A Khan1, D Yeung1, B Wyatt1, T Rafai1, J Byant1, A Coates1,
E Fitzgerald2, A Acheson2, V Wilson1
1School of Biomedical Sciences, and 2Division of Surgery,
University of Nottingham Medical School, Nottingham, UK
Critical Care 2009, 13(Suppl 4):P47 (doi: 10.1186/cc8103) Introduction Colorectal surgery carries significant risks of
postoperative morbidity and mortality. One of the major hazards is
an increased risk of sepsis; an important component of which is
systemic inflammatory response syndrome (SIRS). Several recent
studies suggest that the noncholesterol-related, pleiotropic effects
of statins may limit the development of sepsis and associated
inflammation. This study investigates the impact of prior statin
therapy on the incidence and outcome of postoperative sepsis and
SIRS in colorectal surgery patients. Introduction Multiple organ dysfunction syndrome (MODS)
frequently complicates sepsis contributing to poor outcome. Yet
the evolution of MODS in the early septic mortality (ESM) is
unclear. To delineate the ESM–MODS relationship, we compared
the development and magnitude of organ injury between dying and
surviving mice in the acute phase (days 1 to 5) of polymicrobial
sepsis. Methods Female OF-1 mice were subjected to cecal ligation and
puncture (CLP). In the first mouse subset, 20 μl blood was
collected daily for 5 days or until death (mice followed for 28 days). To define the pre-lethal changes in circulating parameters, mice
were retrospectively divided into two groups based on outcome in
the acute sepsis: DEAD (all died within 5 days, n = 39) and
survivors (SUR; alive at day 28, n = 40). In the second subset,
mice were sacrificed within 24 hours of projected death (based on
the body T <28°C, 100% specificity, n = 7) and matched with
SUR (body T >35°C, 100% sensitivity) from the same post-CLP
day and controls. Methods A retrospective cohort analysis of 577 patients who
underwent curative surgery for colorectal cancer was conducted to
evaluate postoperative morbidity and mortality (within 30 days of
surgery). The primary endpoints were: 30-day in-hospital mortality,
admission to intensive care (ICU), and a positive diagnosis of SIRS
or sepsis. Results Prior to admission, 21.7% of patients were taking either
simvastatin, atorvastatin, fluvastatin, pravastatin or rosuvastatin. Patients on statins were significantly older than those not on
statins (statin – 74.7 years (SD = 6.5) vs. Effects of statins on postoperative sepsis, systemic
inflammatory response syndrome and mortality after
colorectal surgery nonstatin – 69.2 years
(SD = 13.4), P = 0.022), more likely to have pre-existing co-
morbidities and in receipt of antidiabetic agents and other
cardiovascular drugs. Table 1 shows there was no difference in
mortality rate between the two groups. Furthermore, the incidence
of nosocomial infection and sepsis did not differ between the statin
and nonstatin groups. Despite being more likely to be admitted to
the ICU, the statin group was significantly less likely to develop
either SIRS (in or out of the ICU) or postoperative wound infection
or be admitted to the ICU for infective/inflammatory sequelae. y
Results In the first subset, significant difference was observed
between SUR and DIED in the circulating urea, ALT, LDH and
glucose during the 1-day to 5-day time course but the magnitude
of these changes varied among post-CLP days. Therefore, we
used the day of death as a reference point clustering all pre-lethal
parameter values as the 72, 48 and 24 hours prior-to-death (on
any 1 to 5 post-CLP day) time points for comparison with the SUR
values. A significant separation between SUR and DIED occurred
generally at 24 hours prior to death: pre-lethal urea increased to 78
(vs. 40 mg/dl in SUR), ALT to 173 (vs. 106 U/l) and LDH to 798
(vs. 445 U/l), while pre-lethal glucose declined to 41 (vs. 74 mg/dl). In the second subset (only 24 hours prior-to-death time
point), acute deaths were not preceded by a significant rise in
creatinine (DIED 6.8 vs. 4.3 μM/l in SUR) and troponin I (239 vs. 119 pg/ml). Similarly, respiratory function of mitochondria in the
liver and kidney was not impaired in either DIED and SUR
compared with controls. Injury scores in the liver, kidney, heart and
lung showed no apparent morphological disparity between
moribund, surviving or control mice. Incidence of cell apoptosis
(TUNEL) in organs was not increased in either of the groups. Conclusions The increase of selected organ function/metabolic
markers was manifested at least 24 hours prior to death. Despite
statistical significance, the relatively small magnitude of these
changes questions organ failure as a direct cause of death in the
early phase of CLP sepsis. Table 1 (abstract P47) Table 1 (abstract P46) S21
(
)
Effect of LPS, 1400W and simvastatin on maximum response and potency of vasoconstrictor agent on the PCA
KCl
U46619
Incubation conditions
Maximum (g wt)
–log EC50
Maximum (g wt)
–log EC50
Control (n = 12)
10.23 ± 0.71
1.56 ± 0.04
10.39 ± 0.71
7.52 ± 0.08
LPS
6.83 ± 0.48*
1.60 ± 0.04
6.77 ± 0.80**
7.64 ± 0.06
LPS then 1400W
9.35 ± 1.21
1.64 ± 0.06
9.41 ± 0.46
7.54 ± 0.04
Control (n = 18)
12.13 ± 0.58
1.55 ± 0.01
13.46 ± 0.51
7.83 ± 0.01
LPS
8.41 ± 0.58**
1.51 ± 0.02
9.90 ± 0.47**
7.75 ± 0.06
Simvastatin and LPS
11.82 ± 0.34
1.53 ± 0.03
13.92 ± 0.38
7.73 ± 0.04
Data presented as mean ± SEM (n = 12 to 18). *P <0.05, **P <0.01, denotes a statistically significant difference from control. W and simvastatin on maximum response and potency of vasoconstrictor agent on the PCA Effect of LPS, 1400W and simvastatin on maximum response and potency of vasoconstrictor agent on the PC S21 Critical Care November 2009 Vol 13 Suppl 4
Sepsis 2009 Critical Care November 2009 Vol 13 Suppl 4
Sepsis 2009 Conclusions Prolonged exposure to LPS caused hyporesponsive-
ness of the PCA by a mechanism that appears to involve the
induction of nitric oxide synthase. Since pre-treatment of the PCA
with simvastatin reduced LPS-induced changes in vasoconstrictor
responses, it unlikely that the effect of the statin involves direct
inhibition of NOS (compare 1400W). These findings are consis-
tent with clinical studies suggesting that prior use of statins may
afford protection against bacterial sepsis. Conclusions Statin patients were older than nonstatin patients,
and had a greater burden of co-morbidities, yet the mortality rate
did not differ between the two groups. The possibility that prior use
of statins may influence inflammatory or infective events associated
with this surgical procedure is supported by the significantly lower
incidence of wound infection, SIRS and sepsis in statin-treated
patients, illustrating the potential for statins to confer protection
against these insults in the most critically ill patients. Conclusions Statin patients were older than nonstatin patients,
and had a greater burden of co-morbidities, yet the mortality rate
did not differ between the two groups. Table 1 (abstract P46) The possibility that prior use
of statins may influence inflammatory or infective events associated
with this surgical procedure is supported by the significantly lower
incidence of wound infection, SIRS and sepsis in statin-treated
patients, illustrating the potential for statins to confer protection
against these insults in the most critically ill patients. P48 Multiple organ dysfunction syndrome: the scapegoat? Assessment of organ dysfunction between surviving and
dying mice in the acute phase of polymicrobial sepsis
M Osuchowski1, K Weixelbaumer1, P Raeven1, D Remick2,
K Reise1, A Kozlov1, M van Griensven1, H Redl 1, S Bahrami1
1Ludwig Boltzmann Institute for Experimental and Clinical
Traumatology in the Trauma Research Center of AUVA, Vienna,
Austria; 2University of Boston, School of Medicine, Boston, MA,
USA
Critical Care 2009, 13(Suppl 4):P48 (doi: 10.1186/cc8104) Effects of statins on postoperative sepsis, systemic
inflammatory response syndrome and mortality after
colorectal surgery Available online http://ccforum.com/supplements/13/S4 The model
incorporates a virtual clinician, an automated system to examine
simulated patients’ status at clinically relevant intervals and
administer standard of care interventions as necessary, thereby
altering the dynamics of the disease state. The model reproduces
many characteristics of systemic response to an infection,
including the time course of cytokines, coagulation factors, clinical
markers, early and late organ failure, and early versus late deaths. Conclusions The ability of our model to reproduce a large variety
of patients with a relatively small number of parameter changes
illustrates the robustness of the underlying biological processes
being modeled. The model may help identify real signals in
immensely variable and noisy multidimensional sepsis patient data,
and distinguish real patient responses from clinical study-site-
related variability. This model is currently undergoing further
validation. Future capabilities include assessment of risk and
benefit of new drugs for sepsis or new treatment strategies (for
example, early goal-directed therapy) in different patient cohorts. Available online http://ccforum.com/supplements/13/S4 Available online http://ccforum.com/supplements/13/S4 P49 demographics and co-morbidities (for example, COPD, cardio-
vascular disease), a handful of parameters were changed to fit the
model to median time course data from 14 subgroups. These
parameters included pathogen virulence, effectiveness of anti-
biotics, baseline status of patients at study inclusion, and
occurrence of secondary infection. By overlaying variations in the
above parameters about the median model, the model can
encompass the entire spectrum of patients observed in the
GenIMS study. The virtual clinician administers individualized
treatment for each simulated patient. The model simulations
provide clear evidence of changes in disease progression as a
function of differences in treatment. Mathematical modeling of community-acquired pneumonia
patients Mathematical modeling of community-acquired pneumonia
patients J Sarkar1, DD Marathe1, AM Inglis1, KW Hurst1, JA Kellum2,
DC Angus2, Y Vodovotz3, S Chang1
1Immunetrics Inc., Pittsburgh, PA, USA; 2Department of Critical
Care Medicine, University of Pittsburgh, PA, USA; 3Department of
Surgery, University of Pittsburgh, PA, USA
Critical Care 2009, 13(Suppl 4):P49 (doi: 10.1186/cc8105) Introduction Sepsis is defined by the systemic response to an
infection, governed by dynamic interactions between the tissues,
immune cells and inflammatory mediators. We used the
Immunetrics platform to build a large-scale mathematical model
that encompasses these biological components. The model
incorporates a virtual clinician, an automated system to examine
simulated patients’ status at clinically relevant intervals and
administer standard of care interventions as necessary, thereby
altering the dynamics of the disease state. The model reproduces
many characteristics of systemic response to an infection,
including the time course of cytokines, coagulation factors, clinical
markers, early and late organ failure, and early versus late deaths. Methods The ordinary differential equation-based model was used
to simulate the progression of sepsis over a 30-day hospital stay. This model was fit to published human endotoxemia data as well as
data from severely septic community-acquired pneumonia (CAP)
patients from the GenIMS study. The model was fit to 15
biomarkers and clinical markers, including mean arterial pressure,
PaO2, creatinine, TNFα, IL-6, and PAI-1. Introduction Sepsis is defined by the systemic response to an
infection, governed by dynamic interactions between the tissues,
immune cells and inflammatory mediators. We used the
Immunetrics platform to build a large-scale mathematical model
that encompasses these biological components. P50 Heart rate variability in the early resuscitation of septic
shock RC Arnold1, DJ Lundy1, L Glaspey1, G Green2, AJE Seely2
1Cooper University Hospital, UMDNJ-Robert Wood Johnson
Medical Center, Camden, NJ, USA; 2The Ottawa Hospital and
Research Institute, Ottawa, ON, Canada
Critical Care 2009, 13(Suppl 4):P50 (doi: 10.1186/cc8106) 2
Results Figure 1 illustrates simulated output as compared with the
median time course data for surviving CAP patients without co-
morbidities and for CAP patients without co-morbidities who died
between 4 and 8 days after admission. After incorporating
changes in physiological and immune function due to patient Introduction The assessment of heart rate variability (HRV) has
provided valuable insight in sepsis. Impaired HRV has been shown Table 1 (abstract P47) S22
Outcomes for statin and nonstatin patients following colorectal
surgery
Parameter
Statin
Nonstatin
Sig. Overall mortality
9/125 (7.2%)
27/452 (6.4%)
0.77
Nosocomial infections
42/125 (33.6%)
132/452 (29.2%)
0.40
Sepsis
10/75 (13.3%)
42/203 (20.7%)
0.22
SIRS
16/125 (12.8%)
148/452 (32.7%)
<0.001
Wound infection
11/125 (8.8%)
66/452 (14.6%)
0.04
Admitted to the ICU
34/125 (27.2%)
63/452 (13.9%)
<0.001
Sig,. significance. Outcomes for statin and nonstatin patients following colorectal
surgery S22 Available online http://ccforum.com/supplements/13/S4 Figure 1 (abstract P49) Time domain factors: SDNN = standard deviation of the
normal R–R interval; RMSSD = root mean square of the difference of
successive R–R intervals. Frequency domain factors of HRV spectrum:
LF = low frequency (0.04 to 0.15 Hz); HF = high frequency (0.18 to
0.4 Hz). Introduction Critically ill patients often have abnormally low
plasma thyroxine (T4) concentrations even in the absence of
thyroidal illnesses, with the lowest T4 values being observed in
patients (particularly older individuals) with sepsis. Staphylococcal
infection of the lung is the most common presentation of sepsis in
the medical ICU setting, and is associated with a much higher
mortality in older individuals than in younger. In studies where the
lungs of rats are challenged with staphylococcal components
lipoteichoic acid and peptidoglycan, we have noted negative corre-
lations between plasma MIF and T4. Objective To examine the dynamics of MIF and T4 in a clinically
relevant, large animal model of sepsis. Methods Adult female sheep (30 to 40 kg) with age approximately
equivalent to humans of 55 years were surgically prepared for
chronic study. After 5 days recovery, approximately 2.5 x 1011 CFU
methicillin-resistant Staphylococcus aureus (ATCC 4300, a clinical
isolate) were instilled into the lung via a bronchoscope. The
animals were ventilated, and hemodynamics were monitored
continuously for 24 hours post instillation. Blood and lymph
samples were collected at baseline and at 3, 6, 12, 18, and
24 hours. MIF was measured by semiquantitative western blot and
free-T4 by ELISA. to be diagnostic of sepsis, heralding its onset, and prognostic of its
impact, correlating with the development of sepsis-induced organ
failure and death. Objective To analyze the ability of HRV to act as a prognostic aid
in the early resuscitation of septic shock, and to study the effect
that early fluid resuscitation will have on the direction and
magnitude of subsequent measurements of HRV in patients with
septic shock. Methods Subjects were prospectively identified within the
emergency department of an urban-based tertiary-care medical
center during their initial evaluation and treatment for septic shock,
defined as a systemic infection with a systolic blood pressure
<90 mmHg after intravenous fluid or a serum lactate >4.0 mmol/l. Continuous cardiac telemetry was obtained for the assessment of
HRV using a standardized set of multiple parameters including
variables of frequency and time domain analyses. Fluid
administration was recorded during the initial resuscitation. Figure 1 (abstract P49) Figure 1 (abstract P49)
Comparison of time course data from model simulation (solid line) and median of patients (filled circles) from (a) surviving CAP patients without co-
morbidities and (b) CAP patients without co-morbidities dying between 4 and 8 days after admission. Comparison of time course data from model simulation (solid line) and median of patients (filled circles) from (a) surviving CAP patients without co-
morbidities and (b) CAP patients without co-morbidities dying between 4 and 8 days after admission. S23 Critical Care November 2009 Vol 13 Suppl 4
Sepsis 2009 Figure 1 (abstract P50)
Heart rate variability parameters measured during the initial
resuscitation of patients with septic shock. Nonsurvivors showed
significantly impaired variability as measured by LF and the HF/LF ratio. Data presented as logarithmic transformation to control for scale and
allow visual comparison. Differences between survivors and
nonsurvivors were calculated using raw averages without logarithmic
conversion. Time domain factors: SDNN = standard deviation of the
normal R–R interval; RMSSD = root mean square of the difference of
successive R–R intervals. Frequency domain factors of HRV spectrum:
LF = low frequency (0.04 to 0.15 Hz); HF = high frequency (0.18 to
0.4 Hz). Figure 1 (abstract P50) Figure 1 (abstract P50) independent of organ severity measurement. While the use of fluid
administration in the early resuscitation of shock did not correlate
with changes in HRV measured at 2 hours, this relationship may
exist when measured at longer time points allowing for the
physiologic response to manifest in the HRV trend. The
assessment of HRV in patients with infection can identify those at
high risk for clinical deterioration and can potentially serve as an
endpoint of resuscitation in patients with septic shock. P51
Changes in MIF and thyroxine, in a clinically relevant large
animal model of sepsis
E Miller1, S Rehberg2, H Linge1, P Enkhbaatar2, L Traber2,
D Traber2, Y Al-Abed1
1The Feinstein Institute for Medical Research, Manhasset, NY,
USA; 2The University of Texas Medical Branch, Galveston, TX,
USA
Critical Care 2009, 13(Suppl 4):P51 (doi: 10.1186/cc8107) Heart rate variability parameters measured during the initial
resuscitation of patients with septic shock. Nonsurvivors showed
significantly impaired variability as measured by LF and the HF/LF ratio. Data presented as logarithmic transformation to control for scale and
allow visual comparison. Differences between survivors and
nonsurvivors were calculated using raw averages without logarithmic
conversion. Available online http://ccforum.com/supplements/13/S4 We examined several classes of
endogenous small molecules and their metabolites and observed
that MIFnl1 binds to, and inhibits, the hydrophobic cavity of MIF in
a dose-dependent manner with an IC50 of 15.8 μM. Importantly,
MIFnl1 was a more potent inhibitor of MIF than ISO-1 (IC50 =
25 μM), the gold standard synthetic inhibitor of MIF. In addition, in
plasma from patients with sepsis, we found an inverse correlation
between the increased level of MIF and the decreased
concentration of MIFnl1. Therefore, we hypothesized that
supplementation of this ligand during sepsis should compensate
for its dramatic reduction and improve survival in our peritonitis
model of sepsis in C57/Bl6 mice. Administration of MIFnl1
improved the 7-day survival rate to 60% compared with 20%
observed for the vehicle-treated mice. Conclusions Our data identify for the first time the presence of a
natural, ligand antagonist of MIF in plasma. This suggests that,
during severe sepsis, increased production and release of MIF
leads to an imbalance of the MIF:MIFnl1 regulatory mechanism
resulting in the development of an overwhelming systemic
inflammatory response leading to cardiovascular collapse and
death. A better understanding of the kinetics of MIF/ligand Introduction Macrophage migration inhibitory factor (MIF) is a pro-
inflammatory cytokine that plays a critical role in the pathogenesis
of sepsis. Plasma MIF concentrations are significantly higher in
nonsurvivors than survivors of severe sepsis, and administration of
antibodies that neutralize MIF activity improves survival in an
experimental model of sepsis. Three-dimensional X-ray crystallo-
graphy shows MIF has a homotrimeric conformation and we have
determined that the hydrophobic cavity formed between two
adjacent subunits of the homotrimer is required for the pro-
inflammatory activity of the molecule. We have designed several
small molecules that fit into the site critical for the proinflammatory
action of MIF, and confirmed the interaction by the crystal structure
of the MIF complex. Binding of MIF in this way inhibits its
proinflammatory activity, improves the clinical outcome in sepsis,
and recapitulates immunotherapy and gene deletion. However, no
natural soluble ligand of MIF has been reported previously. Methods Lipoteichoic acid (LTA) and peptidoglycan (PGN) are
major inflammatory components of the staphylococcal cell wall
known to induce shock. Male Fischer 344 rats either Young
(6 months) or Old (>18 months), approximately equivalent to
humans of 18 and 60 years respectively, were anesthetized and
LTA and PGN (1.5/5 mg/kg in 200 μl) were administered intra-
tracheally. P54 Conclusions Our data identify for the first time the presence of a
natural, ligand antagonist of MIF in plasma. This suggests that,
during severe sepsis, increased production and release of MIF
leads to an imbalance of the MIF:MIFnl1 regulatory mechanism
resulting in the development of an overwhelming systemic
inflammatory response leading to cardiovascular collapse and
death. A better understanding of the kinetics of MIF/ligand
regulation in patients with sepsis may lead to improved outcome in
this devastating disease. Neutrophil recruitment by macrophage migration inhibition
factor and CXCL1 to the lung following staphylococcal
stimulation is significantly elevated in advanced age Available online http://ccforum.com/supplements/13/S4 Six hours later, blood was collected from the heart, and
post mortem, the lungs were lavaged for analysis of the epithelial
lining fluid. Young, untreated cage control animals (Control) were
also assessed. Results The plasma MIF concentration significantly increased with
LTA/PGN challenge (Control, 24.9 ± 0.6 ng/ml; Young, 38.8 ±
5.9 ng/ml; Old, 64.2 ± 12.1 ng/ml) and the mean concentration in
the old animals was significantly higher than in the young;
P = 0.004. Conversely, plasma free-T4 significantly decreased with
LTA/PGN challenge (Control, 2.3 ± 0.9 ng/dl; Young, 1.7 ± 0.7 ng/dl;
Old, 1.0 ± 0.4 ng/dl) and the mean concentration in the old
animals was significantly lower than in the young; P <0.05. Significant age-specific differences were also noted in the
response with respect to neutrophils and IL-6 within the lavage
fluid. Hypothesis MIFnl1 binds to the hydrophobic cavity of MIF. Increased concentrations of MIF in sepsis deplete plasma MIFnl1
and lead to a critical MIF:MIFnl1 imbalance. Results We have discovered a natural ligand, designated MIFnl1,
that binds the proinflammatory site of MIF with high affinity, and
effectively modulates its activity. We examined several classes of
endogenous small molecules and their metabolites and observed
that MIFnl1 binds to, and inhibits, the hydrophobic cavity of MIF in
a dose-dependent manner with an IC50 of 15.8 μM. Importantly,
MIFnl1 was a more potent inhibitor of MIF than ISO-1 (IC50 =
25 μM), the gold standard synthetic inhibitor of MIF. In addition, in
plasma from patients with sepsis, we found an inverse correlation
between the increased level of MIF and the decreased
concentration of MIFnl1. Therefore, we hypothesized that
supplementation of this ligand during sepsis should compensate
for its dramatic reduction and improve survival in our peritonitis
model of sepsis in C57/Bl6 mice. Administration of MIFnl1
improved the 7-day survival rate to 60% compared with 20%
observed for the vehicle-treated mice. Conclusions Typically, critically ill patients have abnormally low
plasma T4 concentrations even in the absence of thyroidal
illnesses (the so-called euthyroid sick phenomenon) with the
lowest values being observed in septic and/or elderly individuals,
suggesting a strong negative correlation between prognosis and
T4 concentration. The data suggest that an increased early
inflammatory response in older compared with young animals
results in an exaggerated imbalance between MIF and T4, possibly
leading to development of an uncontrolled systemic response. Neutrophil recruitment by macrophage migration inhibition
factor and CXCL1 to the lung following staphylococcal
stimulation is significantly elevated in advanced age HM Linge, K Takahashi, E Miller
Cardiopulmonary Research Center for Heart and Lung Research,
The Feinstein Institute for Medical Research, Manhasset, NY, USA
Critical Care 2009, 13(Suppl 4):P54 (doi: 10.1186/cc8124) Introduction Mortality from sepsis is greater in the elderly than in
the young although incidence only increases slightly. Pulmonary
infections caused by Staphylococcus aureus, which progress into
sepsis, are a major cause of death in elderly patients. Adiponectin,
multifaceted adipokine with anti-inflammatory properties, is
secreted primarily from adipose tissue. It is increasingly
acknowledged that the tissue microenvironment changes with old
age. With increasing age, ectopic fat accumulates, increasing the
possibility of elevated levels of adipose-derived mediators in the
older individual. Figure 1 (abstract P49) A
composite endpoint of increasing organ failure and in-hospital
mortality was measured. Results The plasma concentration of MIF increased from
24.9 ± 4.5 ng/ml at baseline to 30.1 ± 2.5 ng/ml in the first 6 hours
post instillation of the bacteria. During this time period, free-T4
concentration decreased from 1.6 ± 0.5 ng/dl to 0.4 ± 0.3 ng/dl. The concentration of MIF in the pulmonary lymph was approxi-
mately 10% of the plasma level, and showed no time-dependent
decrease, although the lymph flow increased approximately
fourfold over the course of the study. Conclusions We have found that intratracheal instillation of
staphylococcal cell wall components in rats results in an imbalance
of plasma MIF and free-T4, and that pulmonary-derived MIF in
sepsis induces cardiocirculatory depression. Here we show that, in
a clinically relevant, large animal model of sepsis induced by
pulmonary infection with methicillin-resistant S. aureus, increased
plasma concentration of MIF and decreased free-T4 occurred in a
similar manner to our rat studies, and that the total amount of MIF
in the lymph also increased. Since strong negative correlations
exist between prognosis and levels of T4 or MIF in patients with
sepsis, our findings underscore interest in possible interactions
between the MIF and T4 molecules that may be critical for the
outcome of the disease. Results Prospective analysis of 15 patients with septic shock was
made. The in-hospital mortality rate was 67% (10/15). Non-
survivors had a significantly impaired HRV compared with survivors
when measured through multiple parameters (P < 0.01), as seen in
Figure 1. There was no difference between fluid administration
between survivors and nonsurvivors (4,475 vs. 5,220 ml, P = NS). There was no relationship seen between intravenous fluid
administration and the change in 2-hour HRV seen in the early
resuscitation of septic shock. Conclusions In the early resuscitation of septic shock, HRV
assessment
can
differentiate
survivors
from
nonsurvivors, S24 Available online http://ccforum.com/supplements/13/S4 Available online http://ccforum.com/supplements/13/S4 P52 older individuals. While most animal models of sepsis have studied
polymicrobial or Gram-negative sepsis from an abdominal origin in
young animals, the most common presentation in the medical ICU
is in older individuals (median age 64 years), with Gram-positive
bacteria (predominantly Staphylococcus aureus) infection in the
lung. Our recent studies reveal a critical role for macrophage
migration inhibitory factor (MIF) in the pathogenesis of sepsis, and
thyroxine (T4), two molecules with major implications in sepsis. Therefore, we reasoned that the age-related severity of sepsis may
be due to an exaggerated imbalance between MIF and its inhibitor. Objective To examine age-dependent differences in inflammatory
responses following pulmonary staphylococcal challenge. Discovery of a natural antagonist of macrophage migration
inhibitory factor Discovery of a natural antagonist of macrophage migration
inhibitory factor Discovery of a natural antagonist of macrophage migration
inhibitory factor Y Al-Abed, C Metz, KF Cheng, B Aljabari, H Linge, M Ochani,
X Lin, V Pavlov, T Coleman, K Tracey, EJ Miller
The Feinstein Institute for Medical Research, Manhasset, NY, USA
Critical Care 2009, 13(Suppl 4):P52 (doi: 10.1186/cc8108) Introduction Macrophage migration inhibitory factor (MIF) is a pro-
inflammatory cytokine that plays a critical role in the pathogenesis
of sepsis. Plasma MIF concentrations are significantly higher in
nonsurvivors than survivors of severe sepsis, and administration of
antibodies that neutralize MIF activity improves survival in an
experimental model of sepsis. Three-dimensional X-ray crystallo-
graphy shows MIF has a homotrimeric conformation and we have
determined that the hydrophobic cavity formed between two
adjacent subunits of the homotrimer is required for the pro-
inflammatory activity of the molecule. We have designed several
small molecules that fit into the site critical for the proinflammatory
action of MIF, and confirmed the interaction by the crystal structure
of the MIF complex. Binding of MIF in this way inhibits its
proinflammatory activity, improves the clinical outcome in sepsis,
and recapitulates immunotherapy and gene deletion. However, no
natural soluble ligand of MIF has been reported previously. Hypothesis MIFnl1 binds to the hydrophobic cavity of MIF. Increased concentrations of MIF in sepsis deplete plasma MIFnl1
and lead to a critical MIF:MIFnl1 imbalance. Results We have discovered a natural ligand, designated MIFnl1,
that binds the proinflammatory site of MIF with high affinity, and
effectively modulates its activity. Age-associated increased inflammatory response to
pulmonary bacterial challenge Age-associated increased inflammatory response to
pulmonary bacterial challenge HM Linge, K Lin, Y Al-Abed, EJ Miller
Cardiopulmonary Research, Center for Heart and Lung Research,
The Feinstein Institute for Medical Research, Manhasset, NY, USA
Critical Care 2009, 13(Suppl 4):P53 (doi: 10.1186/cc8109) Introduction Sepsis, the systemic inflammation following infectious
insult, is a major cause of morbidity and mortality particularly in Objective To investigate age-dependent changes in the intra-
pulmonary response to staphylococcal challenge. We hypothesized S25 Critical Care November 2009 Vol 13 Suppl 4
Sepsis 2009 Critical Care November 2009 Vol 13 Suppl 4
Sepsis 2009 Critical Care November 2009 Vol 13 Suppl 4
Sepsis 2009 Corporation, Sagamihara, Japan). Auto anti-HMGB1 antibodies
were detected in serum by a homemade ELISA test. Corporation, Sagamihara, Japan). Auto anti-HMGB1 antibodies
were detected in serum by a homemade ELISA test. Corporation, Sagamihara, Japan). Auto anti-HMGB1 antibodies
were detected in serum by a homemade ELISA test. that older animals have higher levels of adiponectin due to adipose
tissue accumulation and that these levels will neutralize the
proinflammatory consequences of staphylococcal stimulation of
the lung. Results Forty-two septic shock patients were included. Median
age was 70 (59 to 78) years, SAPS 2 was 68 (51 to 83), and the
mortality rate was 29%. HMGB1 was undetectable in the plasma
of a control population. In contrast, high levels of HMGB1 were
found in all septic patients (median = 5.71 ng/ml at D7). Thirteen
patients (38%) presented a significant production of auto anti-
HMGB1 IgG antibodies during the course of sepsis. The age, sex
ratio, median HMGB1 level and mortality rate were similar in
patients producing (PPAb) and not producing antibodies (PNPAb). However, as compared with the PNPAb patients, the PPAb group
had significant higher APACHE II (P = 0.027) and SOFA scores
(P = 0.02) at the onset of shock and had a more important but
nonsignificant decrease of the cardiovascular SOFA score
between day 1 and day 7 (P = 0.063). Methods Young (6 months) and old (>18 months) male Sprague–
Dawley rats were challenged intratracheally with S. aureus cell wall
components lipoteichoic acid (LTA, 0.15 mg) and peptidoglycan
(PGN, 0.5 mg) or saline alone. After 24 hours, plasma was
collected and lungs lavaged post mortem. Concentrations of total
protein, chemokines and MIF were assessed. P55 Naturally acquired anti-high mobility group box 1
antibodies during septic shock Age-associated increased inflammatory response to
pulmonary bacterial challenge In vitro studies
examined the accumulation of adiponectin in the culture medium of
the adipocyte cell line 3T3-L1 following direct challenge with
LTA/PGN (3 μg/ml; 100 μg/ml) for 6 hours. Results The older age group, compared with the young group
receiving the same stimulus, showed significantly elevated alveolar
levels of MIF and CXCL1 (KC), both involved in neutrophil
recruitment. Neutrophils (controls: 0.029 ± 0.033, young: 13.9 ±
7.8, old: 29.3 ± 10.5, x 106) and the total number of cells
(controls: 0.72 ± 0.26, young: 16.4 ± 9.7, old: 32.9 ± 11.2, x 106)
within the alveolar space were significantly and age-dependently
increased, following pulmonary insult. Plasma adiponectin did not
change significantly. However, instillation of LTA/PGN significantly
elevated the levels of adiponectin found within the alveolar space
(controls: 3.9 ± 2.4, young: 22.5 ± 8.8, old: 29 ± 10.8 ng/ml,
P <0.01). Interestingly,
following
LTA/PGN
challenge
of
adipocytes, there was a significant decrease in adiponectin
concentration in the culture medium (control: 20.3 ± 0.78,
LTA/PGN: 18.23 ± 1.14 pg/ml, P = 0.016). Conclusions Naturally acquired anti-HMGB1 antibodies are
produced during septic shock. The presence of antibodies is
associated with higher severity scores and might be associated
with an improvement of the haemodynamic dysfunction. The
neutralizing capacity of these autoantibodies and their physio-
logical role remain to be investigated. Naturally acquired anti-high mobility group box 1
antibodies during septic shock B Sauneuf1, D Grimaldi1,2,3, C Rousseau1, J-D Chiche1,2,3,
C Desgranges1, J-P Mira1,2,3
1Department of Cellular Biology, Host–Pathogen Interactions,
Cochin Institute, University Paris Descartes, INSERM U567, CNRS
(UMR 8104), Paris, France; 2Medical Intensive Care Unit, Cochin
Hospital, Paris, France; 3University Paris Descartes, Paris, France
Critical Care 2009, 13(Suppl 4):P55 (doi: 10.1186/cc8125) Objective To assess capacity of S100A9 mRNA in whole blood
from SS patients to predict survival and the occurrence of HAI. Objective To assess capacity of S100A9 mRNA in whole blood
from SS patients to predict survival and the occurrence of HAI. Methods The authors conducted a cohort study. This study was
conducted in two ICUs in Lyon University Hospital. The study
included 166 SS patients and 44 healthy volunteers. PAXgene
blood samples were obtained regularly in the course of the
syndrome for S100A9 gene expression analysis using qRT-PCR. Results The overall mortality was 38% and the mean SAPS II on
shock onset was 52. Thirty-seven patients (23%) experienced at
least one HAI after septic shock. We found that S100A9 mRNA
levels at days 1 to 3 after the onset of shock were significantly
higher in SS patients compared with healthy volunteers (median:
1,460 vs. 16,620; P <0.0001) but not significantly different in
nonsurviving versus surviving patients (median: 18,070 vs. 16,310;
P = 0.1278). In contrast, systemic S100A9 mRNA levels
measured at days 7 to 10 were significantly higher in the group of Introduction High mobility group box 1 (HMGB1) is a pleiotropic
cytokine, implicated in the pathophysiology of sepsis. This alarmin,
usually located in the nucleus, is released after tissue injury and
activates various innate immunity receptors, leading to sustained
inflammatory response. Inhibition of HMGB1 by anti-HMGB1
antibodies has been reported to decrease mortality in experimental
models of sepsis. In the present work, we analyse whether
HMGB1 secretion during septic shock leads to the production of
naturally acquired anti-HMGB1 antibodies during septic shock. Methods All patients with septic shock criteria and no
immunosuppression were included during a 6-month period of
time. After informed consent, blood samples (200 μl) were drawn
on the day of shock and at D3, D7, and D14. Plasma HMGB1
levels were measured, using a commercial ELISA kit (Sino-Test Introduction High mobility group box 1 (HMGB1) is a pleiotropic
cytokine, implicated in the pathophysiology of sepsis. P56 Delayed increased S100A9 mRNA predicts
hospital-acquired infection after septic shock Delayed increased S100A9 mRNA predicts
hospital-acquired infection after septic shock M Fontaine1, A Pachot2, A Larue2, B Mougin2, C Landelle1,
C Allombert2, F Venet3, M-A Cazalis2, G Monneret3, A Lepape1
1Intensive Care Units, Hospices Civils de Lyon, CH Lyon-Sud,
Lyon, France; 2Joint Unit Hospices Civils de Lyon ‘bioMerieux,
Hôpital E. Herriot, Lyon, France; 3Immunology Laboratory,
Hospices Civils de Lyon, Hôpital E. Herriot, Lyon, France
Critical Care 2009, 13(Suppl 4):P56 (doi: 10.1186/cc8126) Conclusions The proinflammatory but not anti-inflammatory
components of the immune response (assessed by neutrophil
recruitment and adiponectin concentrations, respectively) differed
significantly between the age groups. Since adiponectin
decreased on direct stimulation of adipocytes, the increased
adiponectin within the lungs may reflect increased lung perme-
ability, and/or production by other cells within the lung. These
findings are important in understanding the response to pulmonary
infections in the older patient and may lead to the identification of
novel targets for age-dependent therapeutic strategies. Introduction Septic shock (SS) remains a serious disease with
high mortality and increased risk of hospital-acquired infection
(HAI). Access to biomarkers assessing prognosis of these
outcomes is of utmost importance in order to select patients for
future therapeutic strategies. Alarmins are normal cell constituents
that can be released into the extracellular milieu during states of
cellular stress and subsequently activate the innate immune
system. Several alarmins of the S100 family are released by
phagocytes in response to cell stress, recognized by RAGE and/or
TLR4 on monocytes and therefore highlighted as relevant mediators
in sepsis pathophysiology. Among them, S100A8 and S100A9
exist mainly as a heterodimer called calprotectin. However, several
studies suggest independent functioning of S100A9, making it an
interesting candidate biomarker in septic syndromes. Conclusions Our results showed that S100A9 mRNA is
overexpressed in blood from SS. We showed that its persistent
overexpression over time is associated with the occurrence of
secondary HAI. This biomarker may be of major interest in identi-
fying patients at increased HAI risk who could benefit from either
targeted therapy aimed at restoring immune functions (in case of
associated immunosuppression) or reinforced antibiotherapy and
measures against cross-transmission. patients that were going to develop HAI compared with patients
that were not (median: 10,140 vs. 7,160; P = 0.009). Multivariate
analysis showed that the S100A9 mRNA level at days 7 to 10 after
the onset of septic shock significantly increases the probability of
HAI with odd ratios of 1.12 per unit (P = 0.0054). Available online http://ccforum.com/supplements/13/S4 Naturally acquired anti-high mobility group box 1
antibodies during septic shock This alarmin,
usually located in the nucleus, is released after tissue injury and
activates various innate immunity receptors, leading to sustained
inflammatory response. Inhibition of HMGB1 by anti-HMGB1
antibodies has been reported to decrease mortality in experimental
models of sepsis. In the present work, we analyse whether
HMGB1 secretion during septic shock leads to the production of
naturally acquired anti-HMGB1 antibodies during septic shock. Methods All patients with septic shock criteria and no
immunosuppression were included during a 6-month period of
time. After informed consent, blood samples (200 μl) were drawn
on the day of shock and at D3, D7, and D14. Plasma HMGB1
levels were measured, using a commercial ELISA kit (Sino-Test S26 Available online http://ccforum.com/supplements/13/S4 Available online http://ccforum.com/supplements/13/S4 S27
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https://openalex.org/W3217631785
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http://eprints.lse.ac.uk/115191/1/93_1266_1_PB.pdf
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English
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Colombia, the Drug Wars and the Politics of Drug Policy Displacement – from La Violencia to UNGASS 2016
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Journal of illicit economies and development
| 2,021
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cc-by
| 13,030
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Collins, J and Alarcón, KT. 2021. Colombia, the Drug Wars and the
Politics of Drug Policy Displacement – from La Violencia to UNGASS
2016. Journal of Illicit Economies and Development, 3(2), pp. 190–205.
DOI: https://doi.org/10.31389/jied.93 Collins, J and Alarcón, KT. 2021. Colombia, the Drug Wars and the
Politics of Drug Policy Displacement – from La Violencia to UNGASS
2016. Journal of Illicit Economies and Development, 3(2), pp. 190–205. DOI: https://doi.org/10.31389/jied.93 Collins, J and Alarcón, KT. 2021. Colombia, the Drug Wars and the
Politics of Drug Policy Displacement – from La Violencia to UNGASS
2016. Journal of Illicit Economies and Development, 3(2), pp. 190–205. DOI: https://doi.org/10.31389/jied.93 RESEARCH
Colombia, the Drug Wars and the Politics of Drug
Policy Displacement – from La Violencia to UNGASS
2016 John Collins1 and Karen Torres Alarcón2
1 Global Initiative against Transnational Organised Crime, AT
2 London School of Economics (LSE), UK Corresponding author: John Collins (john.collins@globalinitiative.net) This article breaks new conceptual ground by questioning orthodox interpretations of nation
state agency in the global drug wars. Specifically, it challenges the David vs. Goliath conception
of Colombia as a passive, client state simply abiding to the United States’ hegemonic war on
drugs. It provides the first published analysis of Colombia’s leadership during the UN General
Assembly Special Session on Drugs (UNGASS) in 2016. It argues that the UN served as a useful
forum for Colombia’s displacement of state building dilemmas, including drug control, and that
Bogota utilised the UN as a proxy negotiating mechanism with the US and other international
donors. Keywords: Colombia; War on Drugs; United States of America; FARC; Plan Colombia; Prohibi
tion; Cocaine; Coca; Marijuana; Cannabis; United Nations; UNODC; UNGASS; Drug Trafficking;
Drug Policy Introduction 191 Collins and Alarcón: Colombia, the Drug Wars and the Politics of
Drug Policy Displacement – from La Violencia to UNGASS 2016 This paper links broader revisionist drug histories with ‘mutual negotiation’ and ‘Colombian-agency’ based
approaches to PC and the UNGASS in 2016. In this rendering, Bogota appears far less a client state of grander
US regional designs, and far more an active protagonist both in terms of its own destiny and in terms of the
international drug policy consensus. The government of President Juan Manuel Santos undoubtedly played
a key role in challenging prohibitionist drug policy precepts in the run up to UNGASS 2016, arguing that
prohibitionist drug policies had failed to achieve their aims and were imposing unacceptable burdens on
producer and transit states. However, to fully understand the drives to do so, one must look beyond tradi
tional narratives of David versus Goliath and instead view his government’s approach as a manifestation of
complex domestic, regional, and international political imperatives. Only then, it argues, can a more com
prehensive account of the politics of policy displacement within the war on drugs become apparent. The paper thereby utilises revisionist accounts of Colombian-US relations, particularly those focused on
PC. It links these bilateral accounts with increased revisionism in both drug policy historiography, one which
highlights greater role and agency to individual countries and localities, as well as with revisionist accounts
of the evolution of the UN drug treaty system. It continues in this trend of challenging orthodox interpreta
tions which ascribe sole agency to the hegemon, the US, and portraying national and local actors as lacking
agency or control of their drug agenda. It in many ways reverses this narrative, highlighting the frequent
role that drugs played within Colombian diplomatic initiatives and state building exercises, from the 1970s,
through Plan Colombia up to the 2012–2016 drug policy reform era. The paper suggests a further research agenda, one which unpacks the internal power relations that the
war on drugs informs and the frequent utility the latter provides to national elites’ agendas. Moreover, it
challenges the general portrayal of UNGASS 2016 as an earnest effort by ‘client’ Latin American states to
radically challenge the drug policy status quo. It instead highlights a continuation of a complex process of
international bargaining and negotiation around funding, influence and policy choices, underpinned by a
continuity in domestic coalition politics and state formation. Introduction This is not to say that many Colombian and other Latin American political elites did not hope for policy
improvements via the UNGASS process. It instead highlights the UN as a forum where Colombia could
safely displace the policy dilemmas of drugs and thereby both challenge a status quo that was increasingly
out of step with reform debates, while leveraging the possibility of some additional funding and political
legitimacy from new policy linkages such as development, gender, and human rights. This also served to
remove the issue, to some degree, from fraught domestic politics and enabled a discussion on policy reform
that would likely be infeasible in a domestic context. Further it enabled Colombia and other Latin American
states to triangulate their complex bilateral drug diplomacy with the US via the UN and thereby sidestep
difficult issues in an era of peace processes, policy fragmentation, popular delegitimization of the war on
drugs, and cannabis legalization in the US and Latin America. Introduction This paper analyses nation state agency in the global drug wars. Specifically, it looks at the case of Colombia,
long seen as ground zero in the United States (US)-led war on drug production and trafficking in the Ameri
cas. Plan Colombia (PC) remains a centre point of this bilateral relationship and is one of the most discussed
and debated US policy interventions in the region. The popular and academic conception of PC as a US
imposition is widespread (Pastor 2001; Stokes 2016, 2005). However, this paper challenges this conventional
wisdom both on the supposed passiveness of Colombian elites and society in accepting US impositions. Further it challenges the idea of the war on drugs being a solely US hegemonic construct rather than a multi
state negotiated process, underpinned by numerous social, political, and economic power determinants that
eschew simple analysis based on power asymmetries between states. p
y
p
y
This paper is based on an extensive literature review analysis and is intended to chart a new analytic frame
work in understanding the relationship between member states and the international drug control system
more broadly. It suggests a further research agenda based on primary source research and interviews with
policy makers. Attempting to undertake both here would have resulted in a paper that is too thin on both
points. The paper’s significant contribution to the field is a linking of the siloed research on (a) Plan Colombia
and (b) the UN drug control system, particularly the latter’s more recent history. It uses these key case stud
ies as they represent arguably the most significant bilateral and multilateral drug policy engagements for
Colombia and ones of major national and international significance for the US as well as governments under
the UN drug control system more broadly. Plan Colombia represents one of the most extensive and insti
tutionalised bilateral counter-narcotics efforts in global drug control history as well as an epoch shaping
strategic framework in Colombia’s drug wars and its state strengthening efforts. Meanwhile, Colombia’s
engagement with the UN drug control system under President Santos, from 2012 through the UN General
Assembly Special Session (UNGASS) on Drugs in 2016 represented a pivotal moment for international drug
policy reform discussions, and one in which Colombia played a key strategic leadership and normative role. Rethinking Drug Control and Policy Displacement g
g
y
p
The Lancet Commission on Drug Policy discussed the many ways in which drug policies have too often
worked to counter health, security and human rights (Csete et al. 2016). The high financial and human bur
den of drug war policies and their disproportionate displacement onto poor and marginalized communities
is a point widely accepted within the existing literature (Collins 2014a). The question is how these policies
are displaced, imported, exported, and/or developed. The notion of drug control as a policy displacement from the global north, specifically the US, remains
widespread in the policy literature. For the purposes of this article, we consider the concept of policy dis
placement as equating to the viewpoint outlined by Mejia and Restrepo in 2014: From the perspective of producer and transit countries, prohibitionist drug policies can be under
stood as a transfer of the costs of the ‘drugs problem’ faced by consumer countries to producer and
transit countries. On the one hand, under complete legalisation, consumer countries would end up
bearing most of the costs associated with drug consumption (Mejia & Restrepo 2014: 27). In other words, the costs of the policy of prohibition are widely borne by producer and transit countries,
while the apparent public health benefits of reduced consumption are received by consumer countries. This
is a point that scholars sympathetic to prohibition, utilizing a cost-benefit analysis from consumer states,
agree with. Caulkins accepts that it does not apply to source or transshipment countries. If there were a country whose people would
have no interest in using a drug, and that country were beset by violence, corruption and other ills 192 Collins and Alarcón: Colombia, the Drug Wars and the Politics of
Drug Policy Displacement – from La Violencia to UNGASS 2016 from hosting production or international trafficking, then that country might benefit from legalisa
tion, even if final market countries would not (Caulkins 2014: 25). Meanwhile, proponents of what Collins has termed the ‘orthodox school’ of drug regime analysis (2021), por
tray a uniform and analytically parsimonious story on the displacement of these costs. The line of thought
within the orthodox school suggests that the war on drugs, and all interventions resulting therefrom, are
the product of US hegemonic ambitions to instil its moralising and interventionist drug policies at the inter
national level. Rethinking Drug Control and Policy Displacement It continues, arguing that the US legally enshrined prohibition via the League of Nations and
subsequently the United Nations and pursued aggressive imperialist policies to enshrine these in subservi
ent nations, either by UN legislative fiat or intervention by military-economic means (Bewley-Taylor 1999). y
g
y
y
(
y
y
)
PC is a neat fit within this orthodox narrative, suggesting a military intervention funded and guided
by the US with minimal legitimacy within Colombia. Further, President Santos’ 2012–2016 leadership of
global drug policy reform discussions have often assumed a David versus Goliath narrative, one of a small
supplicant state challenging the hegemonic status quo. Neither of these conceptions is correct. Both miss
the deep complexity of politics and power dynamics within global, US and Colombian drug policies; the
US-Colombian relationship; PC and the UNGASS process. p
p
This is not, of course, to negate the role or responsibility of the US in propagating a broader policy of
repressive drug policies internationally and how that interacted with national political imperatives. But
interact it did. As is often highlighted in many other areas of drug policy, the war on drugs has served as a
mechanism for other socio-economic goals, such as enshrining unequal power relations, racial divides and/
or maintaining socioeconomic inequalities. These same factors applied to local political elites and constella
tions. The US in many ways exerted a form of regional policy hegemony, but as the Colombia example dem
onstrates, it was often only at the invitation of, and thereby influence of, local political elites and alliances. The same is true for Colombia’s leadership role in international drug policy over the 2012–2016 period. Without a recognition of the policy shifts and national-international dynamic underlying it, the changing
international drug policy landscape over this period remains inexplicable. Far from a David versus Goliath
story of reformist states versus the US-dominated drug regime (Jelsma & Bewley-Taylor 2016; Oakford 2016),
UNGASS 2016 represented a continuation of the negotiated power processes of Colombian internal and
regional politics and Colombian-US relations. The latter point extends beyond US interventions in Colombia,
as the US was undergoing its own legal fragmentation on cannabis, impacting national-international treaty
law and state-federal law. From La Violencia to Plan Colombia It remains unclear why Colombia so radically monopolised the cocaine mar
ket at the time (Thoumi 2003, 1995). Gaviria and Mejia posit that coca base from Bolivia and Peru, which they processed into cocaine and exported to the United States and
Europe (Suárez & Santos 2018). It remains unclear why Colombia so radically monopolised the cocaine mar
ket at the time (Thoumi 2003, 1995). Gaviria and Mejia posit that The dominance of Colombia might have been due to fortuitous events, historical accidents that
were then exploited and maintained for economic reasons, and the experience and specialization
that resulted might have created a competitive advantage (Gaviria & Mejia 2017: 3). The scale of the emerging illicit market began to further disrupt Colombian political economy. The New York
Times wrote as early as 1978 that ‘the drug traffickers have arisen not only as a new economic class, but also
as a powerful political force’ (Quoted In: Gaviria & Mejia 2017). Bogota adopted a repressive strategy early
on, issuing decree 1206 and creating the National Narcotics Council (CNE) in 1973 (Zorro-Sánchez 2017). Some suggest that conditionality attached to US development assistance drove this institutional develop
ments (Guáqueta 2005). While the timing coincided with the earliest counter-narcotics policies, the relative
unimportance of Colombia in the early Nixonian war on drugs suggests as much a home-grown decision as
an imposition of the US or the UN drug control system. Indeed, rather than fixating on drugs, in many ways
the US at the time was focused on maintaining close clientelist ties with the Colombian military through
training cooperation to serve as a bulwark against communism in the region (Richani 2020). With the growing dominance of drug cartels, violent crime soon ‘reached epidemic proportions’ and
tripled between the 1970s and 1990s, increasing tenfold in some regions, while leaving others broadly
untouched (Gaviria 2000: 2). Homicides almost doubled from 57 per 100,0000 in 1985, to 95 in 1993. In the
province of Antioquia, where Medellín is the capital, the estimated homicide rate reached 400 per 100,000
in the early 1990s (Rosen 2014). In this climate, cartels bid for political protection and sought to corrupt
government institutions through bribery, judicial intimidation, and murder, earning Colombia its infamous
title as a ‘narco-state’ (Mendez 2017). From La Violencia to Plan Colombia The seeds of Plan Colombia are generally traced to the emergence of the country’s ‘unholy trinity’: insurgent
organisations, vigilante paramilitary groups and the illegal drug industry (Manwaring 2002). The modern
configuration of these groups in turn can be traced to the era known simply as La Violencia – the Violence
(Franz 2016). La Violencia exploded in April 1948 with the assassination of Jorge Eliécer Gaitán, a social reformer presi
dential candidate. Nationwide clashes between liberals and conservatives deepened decades-old social divi
sions (Méndez 2012). While the conservative elites remained ostensibly aloof, their client groups killed an
estimated 250,000, largely in the rural areas. This only served to further solidify urban-rural divisions and
embed frontier justice systems (Méndez 2012). Eventually a relatively static political alignment emerged in
the 1960s, divided between ‘leftist guerrillas, rightist paramilitaries and blackmarketeering mafias’, again
only further mobilising discontent in rural areas (Méndez 2012: 92). The leftist guerrilla Revolutionary Armed Forces of Colombia (FARC) was founded in 1964. In its early
years, the group supported socialist ideology, independent peasant republics and specific policies such as
land reform, minimum agricultural prices, expanded agrarian credit, health care, and education for peasants
(Franz 2016). Following US-backed military campaigns against the FARC, the group underwent an ideologi
cal hardening emerging as a Marxist-Leninist guerrilla movement (Mendez 2017). The National Liberation
Army (ELN) was also founded in 1964 by young Marxists educated in Cuba (Rosen 2014). Meanwhile, right
wing paramilitaries, supported and financed by elite political groups, quickly began to form in the 1960s to
counter the momentum of Marxist movements (Franz 2016). The most prominent group would emerge as
the United Self-Defence Forces of Colombia (Autodefensas Unidas de Colombia, AUC), an umbrella paramili
tary organised in 1998 (Richani 2020). In parallel, the mid-1970s in Colombia saw the emergence and consolidation of power by the drug cartels. The resilience of the coffee and emerald mafias had long testified to Bogota’s weakness to impose legal
control in many regions (Méndez 2012). Finding a market gap, the Cali and Medellín cartels began to import 193 Collins and Alarcón: Colombia, the Drug Wars and the Politics of
Drug Policy Displacement – from La Violencia to UNGASS 2016 coca base from Bolivia and Peru, which they processed into cocaine and exported to the United States and
Europe (Suárez & Santos 2018). From La Violencia to Plan Colombia The most illustrative example of the control and penetration of drug
cartels in the country’s institutions was Pablo Escobar’s election as an alternate to the National Chamber of
Representatives in 1982 (Bushnell 1993). Eventually political leaders began to view drug trafficking groups as a preeminent threat to the country’s
institutions, prompting the agreement of a new extradition treaty between Colombia and the United States
(Gaviria & Mejia 2017). The threat of extradition sought to curb the violence caused by drug cartels, but
instead achieved the opposite effect: The feral, implacable violence engulfed the nation in assassinations of journalists, judges, politi
cians, police officers and thousands of innocents, beginning with the virtual annihilation of the
Colombian Supreme Court ten months after the first extraditions (Mendez 2017: 61). On November 6, 1985, members of the M-19 a leftist revolutionary guerrilla group took over the Palace of
Justice in Bogotá, assassinating 100 hostages, including half of the Supreme Court. Various key extradition
documents were destroyed that day, strengthening claims that the Medellín cartel financed part of the siege
(Méndez 2017). On November 6, 1985, members of the M-19 a leftist revolutionary guerrilla group took over the Palace of
Justice in Bogotá, assassinating 100 hostages, including half of the Supreme Court. Various key extradition
documents were destroyed that day, strengthening claims that the Medellín cartel financed part of the siege
(Méndez 2017). The following years were characterised both by the government’s ‘kingpin strategy’ and the reign of terror
of drug cartels (Rosen 2014). Targeting key members of drug cartels resulted in notorious backlashes such as
the assassination of Presidential candidate Luis Carlos Galán in 1989 or the bombing of Avianca Flight 203
(Rosen 2014). Responding to these circumstances, President César Gaviria requested technical assistance
from the United States, bringing about the capture and death of Pablo Escobar in December 1993 (Crandall
2008). With the partial dismantling of the Medellin cartel, the power vacuum left in the illegal drug business
was sought to be filled by FARC and soon AUC (Franz 2016). Colombia-US relations and the genesis of Plan Colombia
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with a novel approach to statecraft and international cooperation, an approach that assumed drug
production and traffic to be security threats that warranted bilateral military intervention in periph
eral areas where national sovereignty and US hemispheric hegemony were challenged (Britto 2020: 3). The conditions for this were not just an aggressive US export of its war on drugs on an unwilling neighbour. The overlap in agendas, interests and international cooperation were the by-product of forces within both
countries and their hemispheric and global strategies. To some degree the increased US focus on Latin
America was a natural outgrowth of its broader foreign policy and its more constrained geographic focus on
international drug policies. Increasingly marginalized from drug policy debates in the Middle East and Asia
in the 1960s and 1970s, and following its failed effort to strengthen the Single Convention, the US seemed
naturally to gravitate towards greater bilateral drug engagement in Latin America (Collins 2015). The second phase, during the 1980s, was characterised by a ‘slow expansion’ of the war on drugs in both
countries alongside the rapid expansion in cocaine production and trafficking. As insurgents’ and cartels’
power grew, so too did kidnappings, assassinations and a broad trend of insecurity (Gaviria & Mejia 2017). US
President Ronald Reagan responded by authorising foreign aid for Colombian law enforcement programmes
thereby expanding US participation in the design of Colombia’s drug policies (Guáqueta 2005). The third
phase, ‘deep institutionalization’ ran from 1989 through 1998 and was characterised by a juxtaposition of
strong institutional cooperation as well as periodic diplomatic tensions and hostility between the two coun
tries (Guáqueta 2005: 34; Suárez & Santos 2018). In 1989, US President George H.W. Bush authorised the
provision of $65 million in military supplies to the Colombian army and police and in 1990 an extraordinary
regional summit produced the Cartagena Declaration, announcing a new phase in the Colombian-US anti-
drug alliance (Guáqueta 2005). The election in 1994 of Ernesto Samper led to an overt diplomatic rupture, in what Crandall describes
as a period of ‘hypernarcotization’ of Colombian-US relations, as the pervasiveness of drug cartel money in
Colombian political institutions became evident (Crandall 2008: 45). In 1995, the US government revoked
President Samper’s visa and decertified Colombia due to insufficient drug cooperation (Rosen 2014). Colombia-US relations and the genesis of Plan Colombia
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Richani describes the prolonged Colombian conflict as ‘a war system’ resulting from ‘fragmented hegemony’
under which ‘violence becomes a conflict management mechanism…regulating multiple sets of contentious
relationships including intra-elite relationships’ (Richani 2020: 326). Within this war system, the US was an
important protagonist but only insofar as it could balance within these competing relationships and power
dynamics. The US was indeed a key support structure for parts of the Colombian state, but its relationship to
drugs and other issues were mediated by its situational role within Bogota’s fragmented hegemony. Alexan
dra Guáqueta argues that US-Colombia bilateral drug relations up until PC can be divided into four phases. 194 Collins and Alarcón: Colombia, the Drug Wars and the Politics of
Drug Policy Displacement – from La Violencia to UNGASS 2016 The first phase, in the 1970s, began with Nixon’s declaration of the war on drugs alongside the 1971 UN
drug convention (Guáqueta 2005). Financial support from the US Agency for International Development (USAID) included conditional
ity around building a drug policy infrastructure, which at the time was heavily focused on cannabis pro
duction in the Caribbean region (Guáqueta 2005). As the state drug control apparatus grew, the US Drug
Enforcement Administration (DEA) was there to embed, train, and conduct investigations (Nadelmann
1993). It was unclear at the time but at the beginning of the 1970s Colombia was just embarking on its two
decades transition ‘from a coffee republic to a narcotics nations’ (Britto 2020: 1). Britto attributes the curious rise of the Colombian cannabis industry to ‘a series of state interventions
that the Colombian government carried out’ with the support of the US and private business interests ‘in
pursuit of agrarian development and nation-state formation’ (Britto 2020: 2). The early attempts to ‘mod
ernise’ and ‘develop’ these outlier regions came not from US external pressure but internal anthropological,
and in many ways fundamentally liberal, initiatives to encourage progress. The United Nations Educational,
Scientific, and Cultural Organization (UNESCO) along with a collection of philanthropic groups and the
US federal government, sought to create a mixed growth economy along the coast, which would move the
economic centre of gravity of the region away from Venezuela and towards Bogota. The initiative failed, but
associated projects laid key tangible and intangible infrastructure that was to partly-enable the development
of the cannabis export industry (Britto 2020). Colombia-US relations and the genesis of Plan Colombia
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coincided with a more hawkish US drug policy where both parties pushed a more assertive approach to
foreign governments, one which the US State Department enthusiastically embraced. In response President
Samper sought to reassure US and domestic audiences of his drug war sincerity. However, as one author
comments, the reality was continuity of institutional cooperation and Samper ‘was not forcing prohibition
onto the state bureaucracy because it was already institutionalized’ (Guáqueta 2005: 44). The fourth phase centred on the genesis of PC (Guáqueta 2005). Colombia was one of the most unstable
and dangerous countries in the region, where a three-front war between FARC, paramilitary groups and drug
cartels had critically undermined the government’s monopoly of force and exacerbated capital flight (Suárez &
Santos 2018). This unravelling had also fundamentally changed Colombian-US relations. As Guáqueta writes, 195 Collins and Alarcón: Colombia, the Drug Wars and the Politics of
Drug Policy Displacement – from La Violencia to UNGASS 2016 The old war against drugs had nothing to do with the Colombian conflict. In contrast, the new war
was concerned with ending the 40-year-old Colombian conflict in which drugs pose a major secu
rity threat because they provide money and local social support to illegal armed groups (Guáqueta
2005: 46). The old war against drugs had nothing to do with the Colombian conflict. In contrast, the new war
was concerned with ending the 40-year-old Colombian conflict in which drugs pose a major secu
rity threat because they provide money and local social support to illegal armed groups (Guáqueta
2005: 46). The blueprint for PC originated during the presidential campaign of Andrés Pastrana. A conservative run
ning a ‘Candidacy for Peace’ platform he called for a ‘new Marshall Plan’ along with peace talks with insur
gent groups to lead Colombia back from the brink of collapse (Mendez 2017: 87). When elected in 1998,
Pastrana unveiled a broader drug policy that included enforcement-oriented supply centric measures as
well as alternative development measures. Seeking international funding Pastrana looked to the US (Suárez
& Santos 2018). Alternative development is ostensibly a model of drug policy intervention which seeks to
address the root causes of illicit drug production by offering support and incentives for farmers to transition
beyond their reliance on drug crops (See for example: Brombacher & Westerbarkei 2019). Colombian Agency and Plan Colombia
As early as 2005 Alexandra Guáqueta wrote that [m]ost academic and policy-oriented writing on illegal drugs in Colombia and the United States
invariably focuses on diplomatic disagreements between the two countries caused by clashing pol
icy interests and an imbalance of power (Guáqueta 2005: 27). She continues, Colombia-US relations and the genesis of Plan Colombia
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In 2000, after strong lobbying by the Colombian government, the Republican-dominated congress allo
cated $1.3 billion to Plan Colombia with the support of President Bill Clinton (Suárez & Santos 2018). In
theory, the aid package was to support judicial reform, foster alternative economic development, reduce
drug supply, assist the Colombian police, and expand counter-narcotics operations (Oehme 2010). In
practice, the majority of PC’s funding was destined to the Colombian military and in many ways the pro
gramme supported a counter-insurgency effort against FARC (Guáqueta 2005). A report by the Government
Accountability Office of the United States outlines the objectives and budget distribution of PC, emphasis
ing with equal importance counter-narcotics and counter-insurgency efforts. The first of three general objec
tives was ‘to reduce the flow of illicit narcotics and improve security’ (GAO 2008: 11). Specific targets were
illicit crop eradication, interdiction activities and reclaiming control of FARC-dominated territory ‘through a
variety of means, including the growth and professionalism of the Colombian military and National Police,
systematic military engagement with insurgent groups, and an expanded police presence throughout the
country’ (GAO 2008: 1). Given the interwoven nature of these objectives, it is impossible to distinguish how
much of the $7.5 billion that was destined to PC by 2005 was intended for counterinsurgency or counter-
narcotics efforts, or if one was prioritised over the other. She continues, The picture offered, that of a coerced U.S. imposition on a reticent and powerless Colombia is inac
curate (Guáqueta 2005: 27). As in the broader discussion of Colombian-US relations, the question of agency in PC has featured in a
number of academic debates and disciplines. Stances can be categorised into three groups: proponents of
Colombian passiveness, proponents of mutual negotiation, and proponents of Colombian agency. Propo
nents of Colombian passiveness predominate US foreign policy analysis, human rights, and security studies
(Buxton 2006; Callaway 2008; Franke & Reed 2005; Holloway 2012; Rochlin 2011; Stokes 2007; William L. Marcy 2010). These lenses view PC as unilaterally imposed, based on American geopolitical and economic
concerns. These echo closely the ‘orthodox’ interpretive lens for international drug control discussed else
where in this article whereby nation states were simply railroaded, duped, or blindly led into a system of
global prohibition at the behest of the US (Collins 2020). In a discourse analysis on American constructions of Latin American identities, Holloway highlights
how conceptions of Latin American inferiority (derived via perceptions of particular qualities about Latin
Americans, such as volatility, infantilism, and barbarism) underpin Colombian-US relations (Holloway 2012). Plan Colombia thus exemplified a ‘natural hemispheric leadership’ by the US, which was constructed from
the perception of Latin American countries as ‘incapable of self-control and effective governance (...) [and]
as a breeding ground for regional instability’ (Holloway 2012: iii). In what Holloway posits as the dominant Collins and Alarcón: Colombia, the Drug Wars and the Politics of
Drug Policy Displacement – from La Violencia to UNGASS 2016 196 academic discourse on PC, where Colombia is defined through a composite set of identities such as ‘drug
producer/distributor, impotent, unstable, corrupt, indifferent, backward, subordinate’, the US locates itself
as the victim of an external threat, an unchecked trade, in which it had no involvement (Holloway 2012:
199). The US is thus defined as ‘the drug victim, powerful, controlled, honorable, guardian’ (Holloway 2012:
199). According to Holloway, this framework explains the US’ approval of the multibillion-dollar package to
improve the Colombian military. However, the author emphasises how this framing of PC discourse does not
entirely write off Colombian agency. In a context of weak institutions and poor governance, Colombia’s only
option to fight the drug war against insurgent groups was to resort to the intervention of a better-equipped
ally, such as the US (Holloway 2012). She continues, y
(
y
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Presenting another angle of the Colombian passiveness argument, Stokes characterises PC as a continu
ation of Cold War counter-insurgency efforts hidden under the facade of humanitarian aid and drug con
trol (Stokes 2007). Framing it as a programme designed to counter insurgency groups which undermine
American interests in South America, the author emphasises the disproportionate focus on suppressing
FARC insurgents and not paramilitaries, despite their heavy involvement in drug trafficking. The empty
humanitarian claims of PC are also evident in the rampant human rights violations committed by the
Colombian military, the lack of oversight controls, and the presence of a ‘presidential waiver’ (Stokes 2007). Stokes views the ostensible goals of PC, for example social development, as ‘propaganda devices employed
to construct the US as a moral agent in world politics while hiding motives that have more to do with geo-
strategic reasoning’ (Stokes 2007: 72). In particular this refers to the US’ economic interests of stable oil
production, which was threatened by the FARC and ELN bombings of oil pipelines. p
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Similarly, Oehme posits that Washington’s intervention in Colombian politics derived from a simple cost-
benefit analysis (Oehme 2010). PC enabled the US to tackle insurgency and drug trafficking, two problems
which were very costly to US interests. Therefore, despite Pastrana’s desire to focus on the social develop
ment potential of PC, ‘the United States used diplomatic and economic leverage to ensure drug interdiction
and massive aerial eradication, conducted by both Colombian military and police units, were the focus of
the plan’ (Oehme 2010: 225). Buxton echoes this point from a drug control perspective by emphasising the
dominance of the US’ model of criminalisation and supply eradication in international drug control frame
works, which, she argues, have been devised to ultimately aid American interests (Buxton 2006). ‘Mutual negotiation’ authors suggest that Colombians had some limited degree of agency in the develop
ment of PC. Crandall and Tickner argue that PC was a result of pragmatic negotiations and aligned interests
between Bogotá and Washington, who ‘shared the common goals of promoting political stability, economic
growth, and most important, anticommunism’ (Crandall 2008: 3; A. Tickner 2007). Guáqueta Argues that
ultimately the Colombian-US drug relationship was one of continuity and overarching overlap of interests
(Guáqueta, 2005). She continues, As he writes, ‘astute observers knew the
Colombian state was headed for failure, and it was clear that the political elite were powerless by themselves
to halt it’ (Mendez 2017: 181). The Colombian political elite ‘had far more to gain from the success, and far
more to lose from the failure of their efforts to interest the US in intervening, than did the US itself’ (Méndez
2012: 13). More specifically, he argues that the Colombian government’s agency was apparent through its
‘Janus-facedness’ in framing PC according to the audience: a revamped military funded by American aid (Mendez 2017). As he writes, ‘astute observers knew the
Colombian state was headed for failure, and it was clear that the political elite were powerless by themselves
to halt it’ (Mendez 2017: 181). The Colombian political elite ‘had far more to gain from the success, and far
more to lose from the failure of their efforts to interest the US in intervening, than did the US itself’ (Méndez
2012: 13). More specifically, he argues that the Colombian government’s agency was apparent through its
‘Janus-facedness’ in framing PC according to the audience: The Plan was represented to the US Congress as a supply interdiction effort in the drugs war; it was
represented to the Colombia people as an internal peace and development plan; to the Europeans
a process of accommodating romantic revolutionaries. Amongst themselves, the Colombian elites
knew it was a state-building and rehabilitation project. The Pastrana administration was endeavour
ing to win the support of stakeholders everywhere whilst avoiding the fallout from their adversarial
interests (Mendez 2017: 184). The author suggests that the dominant rhetoric of Colombian passiveness and American imposition is the
result of scholars only seeing the ‘face’ that was presented to the US – that of a country in desperate need
of foreign intervention to sustain the war on drugs (Mendez 2017). Sandra Borda continues this narrative
with a focus on Colombian government efforts to ‘pivot’ towards key international issues as a way to solidify
the relevance of the Colombian conflict to donor governments. In this sense, the narrative Borda portrays is
very much a drug war constructed in Bogota and sold in various forums in a manner tailored to perceived
audiences’ interests (Borda 2016). This is something substantiated by Colombia’s engagement with the UN
drug control system. The International Drug Control System g
y
The origins of multilateral drug control system are generally dated to the Shanghai Opium Commission of
1909 and the Hague Opium Convention of 1912 (McAllister 2012). The system developed over numerous
subsequent treaties building on the aspirational foundations enunciated in the 1912 treaty, namely limiting
global drug consumption to ‘medical and scientific purposes’. Its current legal architecture is built on three
international drug conventions, the 1961 Single Convention on Narcotic Drugs (as amended by the 1972
Protocol), the 1971 Convention on Psychotropic Substances of 1971 and the 1988 United Nations Convention
against Illicit Traffic in Narcotic Drugs and Psychotropic Substances (UNODC 2008). The international drug control system is often examined as an archetype robust regime. Colgan et al. write, ‘robust regimes’ emerge where there are low conflicts of interest and high concentrations of power
(Colgan et al. 2012). Orthodox ‘prohibition regime’ theories are predicated on such a conception. Namely,
the US represented the hegemon, providing stability, leverage and the diplomatic initiatives necessary for
the production of a coherent Global Drug Prohibition Regime (GDPR) (Bewley-Taylor 2012, 1999). Under
this conception, smaller or weaker states emerge as passive rule-takers, beholden to US power and its ideo
logical pursuit of an interventionist war on drugs. This approach fits closely with orthodox narrative about
Colombia’s client state role within the US war on drugs. Historiographical developments, such as the ‘new drug history’ approach to Latin America (Gootenberg
& Campos 2015) shed a more complicating light. First, US drug hegemony represented a relatively delayed
structural development (Collins 2020). The US was not even a member of the League of Nations; European
states were more activist participants than earlier US-focused historiography gave credit to (Mills 2014);
modern drug prohibitions did not originate in the US, but in Asia (Windle 2013); decolonization agendas
often buttressed and drove prohibitions rather than passively inculcating them (Collins 2017a); the Cold
War and the realities of bipolarity acted as a fundamental constraint on US drug hegemony and thereby the
UN’s ability to adhere solely to US interests (Collins 2015; Kinder 1981). The analytic parsimony of the US-led
global drug prohibition regime narrative does not, as such, hold up to empirical evaluation. She continues, Oehme also suggests that, despite the different strategic approaches between Washington
and Bogotá, there was ‘a clear consensus about the overarching security imperative’ (Oehme 2010: 225). The Colombian agency authors, suggest that PC was conceived in Bogotá and pursued a Colombian stra
tegic agenda (DeShazo et al. 2009; Marcella 2001; Mendez 2017; Suárez & Santos 2018). DeShazo et al. highlight how PC was a ‘broad-based approach to security and development’ in light of the government’s
sovereignty gap due to FARC and ELN dominance in crucial territories (DeShazo et al. 2009: VII). In this
view, the shift from Pastrana’s social development and counter-narcotics agenda to a counter-insurgency
campaign in later years was more reflective of the differing political priorities between Pastrana and his suc
cessor Álvaro Uribe Velez, rather than primarily the US’ interests for the region (DeShazo et al. 2009). In a
different vein, Suárez argues that PC’s shift to a counter-insurgency agenda was a direct result of Pastrana’s
decision to involve the international community to avoid state failure. Indeed, by inviting international aid,
Pastrana inevitably surrendered to the pressures of intermestic issues (such as the failed peace negotiations
with FARC) in the development of PC (Suárez & Santos 2018). This approach thereby decentres the hegemon
in the negotiation process, seeks to uncover and reiterate the agency of the smaller power in exerting influ
ence over the superpower and in some cases overcoming the reluctance of the hegemon ‘to be drawn too far
into its internal affairs of its victim’ (Méndez 2012). Arlene Tickner and Álvaro Méndez, argue that Plan Colombia represented a continuation of a broader
international phenomenon of ‘empire by invitation’ (Lundestad 1999; Méndez 2012). In the case of Plan
Colombia, both Tickner and Mendez label it ‘intervention by invitation’ (Mendez 2017; A. B. Tickner 2007). Méndez builds on Tickner’s earlier exposition that President Pastrana proactively invited US intervention for
the purposes of state rebuilding after a confluence of drug, security and economic emergencies, as well as
‘chronic debility with deep historical roots’ (A. B. Tickner 2007; Mendez 2017: 182). In this view, Pastrana’s
agenda was characterised by a desire to leverage the FARC and ELN towards peace negotiations through 197 Collins and Alarcón: Colombia, the Drug Wars and the Politics of
Drug Policy Displacement – from La Violencia to UNGASS 2016 a revamped military funded by American aid (Mendez 2017). Colombia and International Drug Control This called for a special conference to discuss a global plan
of action and reiterated the call for drug trafficking to be treated as a crime against humanity. This time
Argentina, Bolivia, Brazil, Ecuador, Peru, and Venezuela signed, with Colombia absent (United Nations 1998). The UN General Assembly accepted the Quito Declaration as demonstrating the need for a new conven
tion. Latin American suggestions to expand the jurisdiction of the International Court of Justice (ICJ) and
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t d This was followed in quick succession by the New York Declaration against Drug Trafficking and the Illicit
Use of Drugs, submitted to the UN in 1985. This called for a special conference to discuss a global plan
of action and reiterated the call for drug trafficking to be treated as a crime against humanity. This time
Argentina, Bolivia, Brazil, Ecuador, Peru, and Venezuela signed, with Colombia absent (United Nations 1998). The UN General Assembly accepted the Quito Declaration as demonstrating the need for a new conven
tion. Latin American suggestions to expand the jurisdiction of the International Court of Justice (ICJ) and
to treat drug trafficking as a crime against humanity were massaged out of the proposals by less affected
and non-interventionist states. As with the UNGASS 2016 process (see below), Latin American governments
floated open ended and more radical departures from status quo policies with the ultimate outcome of pro
ducing a brokered and more limited consensus shift. Within a year the emerging Convention proposal had
received ‘worldwide’ support from governments as well as international, regional, and non-governmental
organizations (United Nations 1998). g
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Governments had broadly and implicitly expanded a principle of shared responsibility on the issue of drug
trafficking through an enumeration of 14 elements to be included in the convention. These included a focus
on asset seizure, extradition, control of precursor chemicals, intelligence sharing, and all activities related to
trafficking, including ‘crop substitution’ (United Nations 1998: 7). Within a decade, Latin American govern
ments had gone from secondary actors in the multilateral drug story to key proponents of transnational
legal developments. European governments, ostensibly seeking common ground, supported this idea of
a shared responsibility of consumers and producers to mitigate the effects of policy displacement. Colombia and International Drug Control Colombia and International Drug Control
Colombia’s involvement with the UN and drug control has often, if not generally, been driven by an effort ‘to
advance its internal political agenda’ (Borda 2016: 103). Colombia’s engagement in UN negotiations of the
1961 and 1971 Conventions were relatively limited, aside from overtly acting as a good citizen of global insti
tutions. This began to change in the 1970s and accelerated in the 1980s with the rapid expansion of, and
policy interest in, the illicit drug trade in all parts of the world. Expert groups within the UN Commission on Collins and Alarcón: Colombia, the Drug Wars and the Politics of
Drug Policy Displacement – from La Violencia to UNGASS 2016 198 Narcotic Drugs (CND) began advocating new treaty measures. Law enforcement officials in the Asia Pacific
region advocated ‘streamlining mutual judicial and other assistance…to facilitate the tracing, freezing and
forfeiture of the proceeds of drug crimes at the international level’ (United Nations 1998: 2). Extradition sup
port became a key focus for the UN drug secretariat as did drug precursor control, intelligence sharing, and
air and sea interdiction efforts (United Nations 1998). These issues, widely exceptionalised in Colombian-US
bilateral relations at the time, were but one thread of a broader multilateral momentum on these issues. The US is also widely ascribed as the propagator of the archetypically prohibitionist 1988 Convention. In fact, Latin American governments provided the key impetus. Struggling to muster national and mul
tinational responses to the growing power of illicit drug actors, Latin American governments sought to
in some way displace the burden to, and via, the United Nations. Latin American governments submitted
the Quito Declaration against Traffic in Narcotic Drugs to the UN General Assembly in 1984. Signed by
Bolivia, Colombia, Ecuador, Nicaragua, Peru, and Venezuela, it called for, among other measures, drug traf
ficking to be treated as a crime against humanity (United Nations 1998). Facing domestic opposition and a
weak state apparatus, displacing the problem to the UN appeared a logical choice for Colombia and other
governments. Adherence to international obligations was an easy bypass for complex domestic legislative
processes, while strength in numbers would raise enforcement capabilities across the region, ostensibly
preventing safe havens. p
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This was followed in quick succession by the New York Declaration against Drug Trafficking and the Illicit
Use of Drugs, submitted to the UN in 1985. Colombia and International Drug Control Europe
remained reluctant to support repressive eradication policies and often sought other proactive frameworks
grounded in development interventions (Brombacher & Westerbarkei 2019; Laurent 2017). The resulting
blurred strategic relationship between states focusing on drug enforcement goals and those targeting devel
opment outcomes has led many to critique development approaches to drugs as lacking a true development
orientation, and being instead bound to drug control, supply reduction goals (Buxton 2015). The early efforts at Colombian Alternative Development (AD) saw a shift of eradication strategy away
from a sole focus on fumigation and some attempt to incorporate crop substitution as a ‘complementary
strategy’. This was in line with President Betancur’s National Rehabilitation Plan (PNR) (Zorro-Sánchez 2017:
68). The UN undertook the first efforts, ultimately focusing in Cauca and Nariño under the administration of
President Virgilio Barco (1986–1990). These were generally isolated and aimed at halting the spread of cul
tivation. They produced ‘no sustainable results’ (Zorro-Sánchez 2017: 69). The 1988 Convention had opened
new avenues of substitution funding which encouraged the Colombian state to become more directly
involved. The administration of César Gaviria (1990–1994) maintained a mixed, ‘contradictory’, approach,
expanding on crop spraying, while scaling-up substitution programmes with UN support (Zorro-Sánchez
2017). The years 1993–2002 saw the government further expand and mainstream these interventions. International institutions, such as the International Development Bank (IDB) and nongovernmental devel
opment organizations increasingly sought to engage. USAID also significantly increased support for sub
stitution programs (Zorro-Sánchez 2017). New programmes, driven by community mobilisation in the 199 Collins and Alarcón: Colombia, the Drug Wars and the Politics of
Drug Policy Displacement – from La Violencia to UNGASS 2016 mid-1990s in opposition to fumigation of small-scale producers, led to a firm policy shift towards alterna
tive development as a core policy approach rather than one subservient to fumigation (Zorro-Sánchez 2017). Although external funding helped create institutional structures and programmes that would be internal
ised by the Colombian state and civil society, it was the delicate negotiation of various domestic coalitions
and political movements which ultimately drove Colombian policy towards a greater development orienta
tion. With the adoption of Plan Colombia in 1999 under President Andrés Pastrana, AD became one of its
ten strategies, but many would argue was subservient to eradication efforts. Colombia and International Drug Control The US was a co-financier of
these efforts, which some authors view as enabling the US to ensure a strong focus on fumigation alongside
development efforts (Zorro-Sánchez 2017). ‘Shared responsibility’ formally became part of the international drug control strategy at the 1998 UNGASS
(UNGA 1998). Further the 1998 UNGASS Action Plan had specifically included AD, in what some com
mentators have referred to as a ‘crucial moment’ for donor countries to engage with drug control beyond
purely policing-oriented approaches (Brombacher & Westerbarkei 2019: 90). The reality of course was that
Colombia was a country ahead of the international curve and the 1998 UNGASS merely served to elaborate
what was already an evolving reality on the ground. Nevertheless, ‘the principle of shared responsibility
turned out to be the life insurance for AD in the decades to come’ (Brombacher & Westerbarkei 2019: 90). The administration of Álvaro Uribe again maintained some continuity, particularly based on the mix of
economic alternatives and eradication, albeit under changed or evolved domestic institutional structures
(Zorro-Sánchez 2017). (
)
Through the early 2000s Colombia maintained this framework as a good citizens of the drug control
system, while promoting interests around AD and enforcing anti-production and trafficking laws (Borda
2016). The initiation of the US ‘war on terror’ led to a deprioritisation of the war on drugs, but the Uribe
administration proved adroit at linking its domestic drug agenda to terror, for example the President in a
speech to the UN in 2002: There is concern about weapons of mass destruction in the United Nations forum, and we share this
anguish. Please, we must understand that drugs have the capability for mass destruction equivalent
to that of the most feared chemical weapon (Quoted in: Borda 2016: 108). The administration also expanded its active linkage between drugs, human rights and the environment, but
to a far less effective degree. As Borda writes, ‘[t]he central idea is that illegal drug money interposes itself
between the state and its objective of guaranteeing the security and human rights of its citizens’ (Borda
2016: 114). This ultimately warped the mainstream vision of human rights, including state complicity in
human rights abuses and ‘the penetration of the same [drug] monies into the Colombian political class and
the very apparatus of the state’ (Barrett 2012; Borda 2016: 114; Lines 2017). Colombia and International Drug Control It would take the administration
of President Santos before Colombia found a tone on drugs and human rights that would resonate with
international human rights groups, largely framing human rights problems as systemic to the war on drugs
itself (Collins 2017b). Further, sitting world leaders, including in Colombia and Mexico adopted a more reformist tone. In 2012
President Santos of Colombia wrote in a London School of Economics report, He continued, The Colombian Government strongly believes that the time has come to take a fresh look and we
invite world leaders, scientists and experts to start an open, serious and honest debate about this
war. The time has come to think outside the box. Our invitation is to dutifully study new formulas
and approaches screened through an academic, scientific and non-politicised lens, because this war
has proven to be extremely challenging and oftentimes, highly frustrating. This is a global problem
that demands a global solution, and therefore a new international consensus is needed (Santos
2012: 2). Ultimately his scope for action was limited by his complex domestic coalition as well as the delicacies of the
peace process. These were only complicated by regional dynamics and growing anti-drug war sentiments. All of these factors augured for a removal of the drug issue from domestic politics in so far as possible, while
also suggesting the need for some significant changes in Colombian and international drug policy. Displac
ing the dilemma to international fora to mitigate, and potentially to solve, seemed to represent perhaps the
best option for the administration and one which it embraced. These constraints set the tone for the Colombian and Latin American reformist governments leading
into the UNGASS 2016 meeting. They offered a challenge to the status quo within a consensus institutional
setting in the hope of finding an evolved or potentially alternative consensus. In parallel, Latin American
governments tended to follow a broad continuity in domestic policies. The Colombian peace process saw
a much greater focus placed on peacebuilding and development in drug crop settings (Meger & Sachseder
2020), but this tended to follow in the broader domestic institutional lineage of drugs and development pol
icies. Meanwhile, the US government tacitly enabled this dialogue, often against the more hawkish elements
within its own administration. In 2012 Vice-President Joe Biden declared the debate on drug war orthodoxy
in the region ‘totally legitimate’, paving the way for a more substantial regional dialogue (Williams & Cattan
2012). )
The Organization of American State (OAS) embraced this opening, producing the report Scenarios for the
Drug Problem in the Americas, 2013–2025 (Organization of American States, 2012). Alongside this, Uruguay,
Colorado and Washington State moved ahead with cannabis legalization. Again, the Obama administrations’
drug war scepticism won out, producing an effectively accommodationist approach at the Federal level, led
by the Department of Justice. The Fragmentation of International Control, 2008–2016 The Fragmentation of International Control, 2008–2016
One author dates the onset of global drug control fragmentation to 2008 (Collins 2017c). US President
Barack Obama, both a vocal sceptic of the war on drugs and attempting to repair regional relations following
the perceived unilateralism of the George W. Bush administration, adopted a greater multilateral tone. This
coincided with domestic fiscal crises which forced local governments to reassess drug war policies as well as
to begin frank assessments of cannabis legalization as a revenue measure (Collins 2017c). Latin American leaders also began to speak out. The Latin American Commission on Drugs and Democracy,
comprising a high-profile list of former presidents and leaders, launched a report challenging the war
on drugs and advocating an open discussion on drug policy (Latin American Commission on Drugs and
Democracy 2009). This was followed by a 2011 Global Commission on Drug Policy report which called for
‘not just alternatives to incarceration and greater emphasis on public health…but also decriminalization
and experiments in legal regulation’ (The Global Commission on Drug Policy 2011). By 2014, the Global
Commission was explicitly advocating a more robust focus on the legal regulation of drugs (The Global
Commission on Drug Policy 2014). Further, sitting world leaders, including in Colombia and Mexico adopted a more reformist tone. In 2012
President Santos of Colombia wrote in a London School of Economics report, 200 Collins and Alarcón: Colombia, the Drug Wars and the Politics of
Drug Policy Displacement – from La Violencia to UNGASS 2016 Colombia has experienced progress in this fight, and historic results have been achieved. We are no
longer the world’s top cocaine producer…However, we are now witnessing, with profound concern,
how this situation is drifting to neighbouring countries, along with all the pain, violence and cor
ruption it entails, which we have endured in our country for too many years (Santos 2012: 2). He continued, This forced the US State Department to seek an uncomfortable accommoda
tion with the UN drug conventions, suggesting treaty flexibility and the reality that ‘some countries will
have very strict drug approaches; other countries will legalize entire categories of drugs’ (Brownfield 2014). To some degree, the controversial US response, what one commentator termed the ‘Brownfield doc
trine’ (Collins 2014b) was not just about squaring US local regulatory changes with international law. It
had opened the door for Latin American governments to walk through, perhaps recognising that few, if
any, would. None did. Instead the pursuit of consensus continued via the UN and its expansive, but hardly
radical – in the sense of really challenging the tenets of the drug control system – UNGASS 2016 Outcome
Document (United Nations General Assembly, 2016). (
y
)
Civil society had staked heavy hopes that the UNGASS era would lead to a collapse of the consensus and
the onset of radical systemic drug policy reforms. Many were left reeling in the immediate aftermath (Jelsma
& Bewley-Taylor 2016). Some came to recognize the incremental victories it contained (Lines & Barrett 2016),
and this ultimately became a consensus view among civil society with time. One commentator suggested
that civil society had ignored political realities and ‘the assumption that the status-quo arose from a con
spiracy of silence rather than the intersection of very tangible interests, concerns and political alignments’
(Collins, 2017c, p. 11). Instead, Collins argues, ‘member states were always and inevitably set to subjugate
reformist rhetoric to consensus’ (Collins 2017c: 11). The 2016 Outcome Document moved beyond a three-pillar approach of drug control based on supply
reduction, demand reduction and international cooperation, towards a seven-chapter thematic focus. This
directly addressed areas of human rights, gender, and development. The concept of ‘development-oriented 201 Collins and Alarcón: Colombia, the Drug Wars and the Politics of
Drug Policy Displacement – from La Violencia to UNGASS 2016 drug control’, adopted in the 2009 Political Declaration and Plan of Action (Commission on Narcotic Drugs
2009), reached a new level with the UNGASS in 2016. Chapter VII assigns a key role to AD and also concep
tually expands the range of development interventions towards urban drug markets and drug trafficking
(Brombacher & Westerbarkei 2019; United Nations General Assembly 2016). He continued, For Colombia, the apotheosis
of UNGASS 2016 in drugs and development terms coincided perfectly with a peace process that required
large injections of domestic and international funding into areas affected by drug crop production. As UN
discussions moved further beyond the sole focus on policing and enforcement the areas of potential donor
overlap only increased. The real outcome of UNGASS 2016 for Colombia and other Latin American governments was not so much
about a radical change in policies or consensus, but in a shift in emphasis, priorities and routes for scaling
domestic, and to a lesser degree foreign, funding. For Colombia, the UNGASS process had, in many ways,
been an instrument in pursuit of a wider state building project, rather than pursuing an end of drug policy
reform in and of itself. Again, the narrative of Colombian agency looms large. Colombia was unquestionably
a leading and highly credible voice in the UNGASS process, supported by the particular normative vision of
its leader. This occurred at an exact moment when the US lost credibility among conservative states due to
cannabis legalisation and a softening of the drug war rhetoric at home, again driven in no small part by the
anti-drug war beliefs of President Obama and his Attorney General Eric Holder. Colombia’s reformist rhetoric almost undoubtedly derived from a strong drug war scepticism within
the Santos administration, originating with the President himself. However, the UNGASS process must be
viewed within the larger history of Colombian policy formation around drugs, security and development. Further, it must be seen as part of a broader effort to secure financial aid and international legitimacy for
the fledgling peace process. Lastly, it represented a useful venue to displace the difficulties and complexi
ties of negotiating a shift in drug policy emphasis and priorities away from tense and entrenched national
fora and interests and towards legitimating international bodies which could lead on problem-solving and
reformulating the approach from the outside. g
pp
Unable to maintain a broad drug policy reform coalition at home, the Colombian government displaced
the policy problem, first to the OAS, through drug policy scenario planning (OAS 2013), and then to the
UN. Conclusion This paper straddles between the Colombian agency and mutual negotiation framework for PC and drug
control more broadly. It suggests a far greater Colombian agency than has been traditionally assumed, while
recognising the clear and important role the US has played. That the pursuit of prohibition and a war on
drugs has imposed immense harm on Colombia is beyond question. That Colombia has served as ground
zero in this global fight is further accepted as consensus. This article challenges the conception of the war
on drugs as a one-way policy displacement on Colombia as a drug production country. It looks to the evi
dence of Plan Colombia and Colombia’s involvement with the UN drug control system, particularly around
UNGASS 2016, to highlight that Colombia was at least a participant is a negotiation of displacement, both
to international fora and from the US and UN drug control apparatus. Indeed, it highlights that Colombia
utilised drug control to secure desired outcomes from US and UN institutions. Finally, it suggests that the
OAS and UN served as useful fora for Colombia’s displacement of statebuilding dilemmas, including drug
control, and utilised these to serve as a proxy negotiating forum with the US and other international donors. Taken at face value, Colombia’s efforts to ‘challenge’ the global consensus on drug policy in the UNGASS
2016 period seems like a major departure with the past. Instead, this paper has argued that it fits well within
a continued evolution of Colombian drug policy within various national, bilateral, and international align
ments. Viewed in isolation Colombia’s newfound reticence to blindly pursue a war on drugs appeared a
radical departure. Viewed in terms of the history of bargaining and elite politics underpinning its approach
to Colombian-US relations, most exemplified by Plan Colombia, it seems less exceptional. Colombia was part
of a growing wave of disenchantment with war on drugs policies, both within the US and within producer
and transit countries. It was well positioned to utilize its credible position to both externally challenge the
status quo and simultaneously negotiate new power relations within the drug control system. This paper straddles between the Colombian agency and mutual negotiation framework for PC and drug
control more broadly. It suggests a far greater Colombian agency than has been traditionally assumed, while
recognising the clear and important role the US has played. He continued, Should these organizations be able to coalesce a new vision for drug policy and explain the technical
implementation of such a policy, Colombia would benefit from a repatriation of drug policy control and the
possibility of a more effective ‘post-war on drugs’ approach’. Should these organisations fail to elaborate a
new vision, the political costs of this failure would not directly accrue to the Colombian government, nor the
domestic political alliances underpinning it. Conclusion That the pursuit of prohibition and a war on
drugs has imposed immense harm on Colombia is beyond question. That Colombia has served as ground
zero in this global fight is further accepted as consensus. This article challenges the conception of the war
on drugs as a one-way policy displacement on Colombia as a drug production country. It looks to the evi
dence of Plan Colombia and Colombia’s involvement with the UN drug control system, particularly around
UNGASS 2016, to highlight that Colombia was at least a participant is a negotiation of displacement, both
to international fora and from the US and UN drug control apparatus. Indeed, it highlights that Colombia
utilised drug control to secure desired outcomes from US and UN institutions. Finally, it suggests that the
OAS and UN served as useful fora for Colombia’s displacement of statebuilding dilemmas, including drug
control, and utilised these to serve as a proxy negotiating forum with the US and other international donors. Taken at face value, Colombia’s efforts to ‘challenge’ the global consensus on drug policy in the UNGASS
2016 period seems like a major departure with the past. Instead, this paper has argued that it fits well within
a continued evolution of Colombian drug policy within various national, bilateral, and international align
ments. Viewed in isolation Colombia’s newfound reticence to blindly pursue a war on drugs appeared a
radical departure. Viewed in terms of the history of bargaining and elite politics underpinning its approach
to Colombian-US relations, most exemplified by Plan Colombia, it seems less exceptional. Colombia was part
of a growing wave of disenchantment with war on drugs policies, both within the US and within producer
and transit countries. It was well positioned to utilize its credible position to both externally challenge the
status quo and simultaneously negotiate new power relations within the drug control system. 202 Collins and Alarcón: Colombia, the Drug Wars and the Politics of
Drug Policy Displacement – from La Violencia to UNGASS 2016 As Méndez writes, ‘[t]he US government intervened in Colombia’s internal affairs on a much grander scale
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followed to ensure the author had no knowledge or involvement in the article’s peer review process. Conclusion PC
was borne of this vision of ‘soft’ US intervention on the Colombian political and military elites’ terms and
indeed the Colombian government succeeded in drawing the US into its internal affairs to a far greater
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DOI: https://doi.org/10.31389/jied.93 Submitted: 20 January 2021 Accepted: 01 November 2021 Published: 23 November 2021 Copyright: © 2021 The Author(s). This is an open-access article distributed under the terms of the Creative
Commons Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and
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Oridonin Sensitizes Cisplatin-Induced Apoptosis via AMPK/Akt/mTOR-Dependent Autophagosome Accumulation in A549 Cells
|
Frontiers in oncology
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Oridonin Sensitizes
Cisplatin-Induced Apoptosis via
AMPK/Akt/mTOR-Dependent
Autophagosome Accumulation in
A549 Cells
Huahong Yang 1, Yun Gao 1, Xiaoye Fan 2, Xingkai Liu 3, Liping Peng 1* and Xinxin Ci 2*
1 Department of Respiratory Medicine, The First Hospital of Jilin University, Changchun, China, 2 Institute of Translational
Medicine, The First Hospital of Jilin University, Changchun, China, 3 Department of Hepatopancreatobiliary Surgery, The First
Hospital of Jilin University, Changchun, China Huahong Yang 1, Yun Gao 1, Xiaoye Fan 2, Xingkai Liu 3, Liping Peng 1* and Xinxin Ci 2*
1 Department of Respiratory Medicine, The First Hospital of Jilin University, Changchun, China, 2 Institute of Translational
Medicine, The First Hospital of Jilin University, Changchun, China, 3 Department of Hepatopancreatobiliary Surgery, The First
Hospital of Jilin University, Changchun, China Edited by: Edited by:
Simona Rapposelli,
University of Pisa, Italy Reviewed by:
Daniela Cesselli,
University of Udine, Italy
Grazia Chiellini,
University of Pisa, Italy *Correspondence:
Liping Peng
plp640317@163.com
Xinxin Ci
cixinxin@jlu.edu.cn Specialty section:
This article was submitted to
Pharmacology of Anti-Cancer Drugs,
a section of the journal
Frontiers in Oncology Received: 10 May 2019
Accepted: 30 July 2019
Published: 14 August 2019 Keywords: oridonin, non-small cell lung cancer, cisplatin sensitivity, autophagy, apoptosis, AMPK/Akt/mTOR ORIGINAL RESEARCH
published: 14 August 2019
doi: 10.3389/fonc.2019.00769 ORIGINAL RESEARCH
published: 14 August 2019
doi: 10.3389/fonc.2019.00769 ORIGINAL RESEARCH
published: 14 August 2019
doi: 10.3389/fonc.2019.00769 ORIGINAL RESEARCH
published: 14 August 2019
doi: 10.3389/fonc.2019.00769 AMPK-mediated autophagy and Akt/mTOR pathways play important roles in current
cancer treatments. Oridonin (Ori), an ent-kaurane diterpenoid isolated from Isodon
rubescens, exerts extensive anti-tumor potential and controversial effects on autophagy. In this study, we investigated the effect of Ori on the autophagy, apoptosis, and
AMPK/Akt/mTOR pathways and determined whether Ori was related to the increased
cisplatin sensitivity observed in A549 cells. First, we found that Ori suppressed
Akt/mTOR, Bcl2 and autophagy flux with enhanced levels of Atg3, P62, and
LC3II, which was also shown as the accumulation of autophagosomes. AMPK and
pro-apoptotic proteins (caspase3, Bax, and PARP) were activated in Ori-treated
cells. With the pretreatment of compound c (AMPK inhibitor), the activation of
autophagosomes, apoptosis and the inhibition of Akt/mTOR pathways induced by Ori
were all reversed. The Ori-activated apoptosis-related markers mentioned previously
and the cell-killing effect were restrained by 3-MA (inhibitor of autophagosomes)
treatment. Therefore, we hypothesized that the Ori-induced pro-apoptotic effect was
mediated by AMPK/Akt/mTOR-dependent accumulation of impaired autophagosomes. Furthermore, Ori could increase the sensitivity of cisplatin through its increased
cell-killing, autophagy-suppressing and apoptosis-inducing activities. In addition to
sensitizing cisplatin, Ori also alleviated cisplatin-induced acute renal injury in vivo,
manifested as depleted BUN, CRE, kidney index, and weight loss compared to
the cisplatin group. In summary, apart from its protective effect on cisplatin-induced
nephrotoxicity, Ori enhanced cisplatin sensitivity via its pro-apoptotic activity mediated
by AMPK/Akt/mTOR-dependent autophagosome activation, which may be a potential
therapeutic target for non-small cell lung cancer. Edited by:
Simona Rapposelli,
University of Pisa, Italy
Reviewed by:
Daniela Cesselli,
University of Udine, Italy
Grazia Chiellini,
University of Pisa, Italy
*Correspondence:
Liping Peng
plp640317@163.com
Xinxin Ci
cixinxin@jlu.edu.cn INTRODUCTION human glioma cells (21). Unfolded protein response suppresses
cisplatin-induced apoptosis via autophagy regulation in human
hepatocellular carcinoma cells (22). Therefore, in our study, we
mainly studied the role of above pathways in cisplatin resistance
in NSCLC cells. With the highest morbidity and mortality, lung cancer is a
serious disease affecting public health (1). In terms of biological
characteristics, lung cancer is divided into small cell lung
cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC
accounts for 80–85% of all lung cancers with poor prognosis
(2). Cisplatin remains the first-line chemotherapy drug for
NSCLC, despite the occurrence of cisplatin resistance. Numerous
studies show that some cytotoxic agents can reverse the cisplatin
resistance of NSCLC patients, however, the survival rates are still
low (3–5). Therefore, there is an urgent need to develop new
effective adjuvant therapies against NSCLC. An ent-kaurane diterpenoid isolated from Isodon rubescens,
Oridonin (Ori) and its analogs exert anti-tumor potential in
cancer cells (23–25). Besides, the mechanisms involved are
mainly concentrated on autophagy and apoptosis (26, 27). However, the effect of Ori on autophagy in cisplatin-induced
lung cancer cells has not been thoroughly elucidated to date. It has been verified that Ori could induce the conversion
of LC3II/I in A549 cells, but the fusion of autophagosome-
lysosome and the expression of P62 (marker of autophagy
degradation) have not been investigated (27). So in our study,
we aimed to explore the role and correlation of autophagy,
apoptosis and AMPK/Akt/mTOR induced by Ori in cisplatin-
treated A549 cells, and provided a new therapeutic target against
carcinogenesis and cisplatin resistance in lung cancer. j
p
g
Autophagy and relevant pathways are closely related to the
emergence of cisplatin resistance (6). Autophagy, also known as
“self-eating,” is the process of transporting damaged, denatured
or aged proteins and organelles to lysosomes for digestion
and degradation. Autophagy consists of three stages: formation
of autophagosomes, fusion of autophagosome-lysosome and
degradation of autophagolysosomes. There is growing evidence
that autophagy takes part in many human diseases, such
as cancer, neurodegenerative disease, metabolic diseases and
immune-related disorders (7–9). From the studies of autophagy
and cancer, autophagy takes part in tumorigenesis, metastasis
and cisplatin resistance (10). Cisplatin resistance can be induced
by the activation of autophagy, and the autophagy inhibitor
chloroquine (CQ) can improve the sensitivity of endometrial
cancer cells to cisplatin (11, 12). Citation: Yang H, Gao Y, Fan X, Liu X, Peng L
and Ci X (2019) Oridonin Sensitizes
Cisplatin-Induced Apoptosis via
AMPK/Akt/mTOR-Dependent
Autophagosome Accumulation in
A549 Cells. Front. Oncol. 9:769. doi: 10.3389/fonc.2019.00769 August 2019 | Volume 9 | Article 769 Frontiers in Oncology | www.frontiersin.org 1 Oridonin Sensitizes Cisplatin in A549 Cells Yang et al. INTRODUCTION CQ blocks the fusion of
autophagosomes and lysosomes and causes the accumulation
of autophagosomes, which highlights the relationship between
autophagosomes and cisplatin resistance. Moreover, recent
studies show that the activation of autophagosomes inhibits
the proliferation of tumor cells and sensitizes cisplatin-induced
resistance in cervical cancer cells (13, 14). Therefore, finding a
compound that induces autophagosome accumulation may be a
new measure to reverse chemotherapy resistance. Frontiers in Oncology | www.frontiersin.org Cell Culture A549
(human
NSCLC
cell)
and
B2b
(human
bronchial
epithelium cell) were purchased from the China Cell Line Bank
(Beijing, China). Cells were maintained in DMEM supplemented
with 10% FBS, 1% antibiotic-antimycotic and incubated at 37◦C
in a 5% CO2 atmosphere. As a vital effector of autophagy regulation, apoptosis can be
initiated to counteract the proliferation, metastasis, and cisplatin
resistance of cancer cells, so that many activators of apoptosis
are used in cancer treatment (20). Studies have demonstrated
that 3-MA (autophagy inhibitor) increased cisplatin-induced
apoptosis by increasing endoplasmic reticulum stress in U251 Reagents Oridonin was obtained from Chengdu Pufeide Biotechnology
Company. All of the primary antibodies LC3, P62, Atg3, Bax,
Bcl2, caspase3, PARP, P-AMPK, AMPK, P-Akt, Akt, P-mTOR,
mTOR, and β-actin were from Cell Signaling Technology or
Abcam. FITC-annexin V and propidium iodide (PI) were from
BD or Invitrogen. Cell culture medium DMEM, antibiotic-
antimycotic and trypsin–EDTA were from Corning, MBI and
Biofil, respectively. Fetal bovine serum (FBS) and PBS were
obtained from BI. Autophagy detection kit was obtained from
Abcam. Cell Counting Kit-8 (CCK-8) was purchased from
Bimake. BUN and CRE detection kits were obtained from
Nanjing jiancheng Biotechnology Company. BCA protein assay
kit was provided from Thermo. Many signaling pathways are involved in the regulation
of autophagy, such as AMPK and Akt/mTOR (15, 16). The
mammalian target of rapamycin (mTOR) acts as a negative
regulator of autophagy and exists in two complexes (mTORC1
and mTORC2). Protein kinase B (Akt) and AMP-activated
protein kinase (AMPK) pathways can regulate mTORC1
positively and negatively (17). Previous studies have shown
that Akt inhibition significantly reduces the phosphorylation of
mTOR and enhances cell autophagy in human oral cancer CAR
cells (18). Additionally, AMPK inhibits mTORC1 through direct
phosphorylation of the tumor suppressor TSC2 on Ser 1387
or subunit Raptor on two conserved serines and subsequently
triggers autophagy flux (17). In addition, targeting AMPK and
Akt/mTOR signaling can overcome cisplatin resistance in ovarian
and oral cancer cells, respectively (18, 19). Animals C57BL/6 WT mice were obtained from Liaoning Changsheng
Technology Industrial, Co., Ltd. (Certificate SCXK2010-0001;
Liaoning, China). All mice were kept in SPF-grade animal room
and fed with sterile water and standard rodent chow. All animal
studies were reviewed and approved by the Animal Welfare and
Research Ethics Committee of Jilin University. Flow Cytometry (FCM) Analysis of
Apoptosis After 18 h of drug treatment (20 µM Ori and 20 µM cisplatin),
the cells were detached from the culture plates, washed with PBS
and suspended in 5 µl of Annexin V binding buffer for 20 min in
the dark. Then, the cells were stained with 2 µl of PI and analyzed
with a FACS. Cell Viability Assay A549 and B2b cells were seeded in 96-well plates (1.5 × 104
cells/well) and treated with different doses of Ori or cisplatin for
18 h. Then 10 µl CCK-8 was added to every well for 1 h and
measured at 450 nm according to the instructions. August 2019 | Volume 9 | Article 769 2 Oridonin Sensitizes Cisplatin in A549 Cells Yang et al. Oridonin Sensitizes Cisplat
Ori regulates autophagy, AMPK, Akt/mTOR signaling and apoptosis. (A) The structure of Oridonin (Ori). (B) A549 cells and B2b cells were t
es (10, 20, and 30 µM) of Ori for 18 h. Cell viability was calculated as the percentage relative to untreated cells which were considered as 10
cology | www frontiersin org
3
August 2019 | Volum FIGURE 1 | Ori regulates autophagy, AMPK, Akt/mTOR signaling and apoptosis. (A) The structure of Oridonin (Ori). (B) A549 cells and B2b cells were treated with
different doses (10, 20, and 30 µM) of Ori for 18 h. Cell viability was calculated as the percentage relative to untreated cells which were considered as 100%. (Continued) August 2019 | Volume 9 | Article 769 3 Frontiers in Oncology | www.frontiersin.org Yang et al. Oridonin Sensitizes Cisplatin in A549 Cells FIGURE 1 | (C–H) Cells were treated with different doses of Ori (5, 10, and 20 µM) for 18 h. Representative western blots and statistical results showed the protein
levels of P62, LC3, Atg3, P-AMPK, AMPK, P-Akt, Akt, P-mTOR, mTOR, caspase3, Bax, Bcl2, and PARP. (I,J) Cells were treated with Ori (20 µM) in the absence or
presence of CQ (50 µM) for 18 h. Acidic vesicular organelles (autophagosomes) were stained with acridine orange. Scale bars: 10 µM. (K–N) Cells were treated with
Ori (20 µM) in the absence or presence of CQ (50 µM) for 18 h. LC3 was measured with immunoblot and immunofluorescence. Scale bars: 20 µM. The results
showed the average of three independent experiments. *p < 0.05 and **p < 0.01 vs. the control group. ##p < 0.01 vs. the CQ group. FIGURE 1 | (C–H) Cells were treated with different doses of Ori (5, 10, and 20 µM) for 18 h. Representative western blots and statistical results showed the protein
levels of P62, LC3, Atg3, P-AMPK, AMPK, P-Akt, Akt, P-mTOR, mTOR, caspase3, Bax, Bcl2, and PARP. Western Blot Cells were homogenized in RIPA lysis buffer that contained
protease and phosphatase inhibitors. Protein concentrations
were determined using a BCA protein assay kit (Thermo
23227). According to the instructions, reagent A and B
were mixed together in a ratio of 50 to 1 and added
into every well for the measurement of OD. Then proteins
were quantified and separated by SDS-PAGE (10–12.5%) and
transferred to PVDF membrane. Then blocked with non-
fat milk for 1 h and incubated with primary antibodies
overnight. The next day, the membrane were washed and
followed with secondary antibodies for 1 h and detected
by ECL. Experimental Models of AKI and
Biochemical Assay The mice were fasted for 12 h and randomly divided into
4 groups: the control group without any treatment (Con, n
= 5); cisplatin-treated group (20 mg/kg, Cis, n = 5); Ori-
treated group without cisplatin (20 mg/kg, Ori, n = 5); Ori-
treated group with cisplatin treatment (Ori+Cis, n = 5). Cisplatin (20 mg/kg) was injected for 72 h to construct an
acute kidney injury model, and 20 mg/kg Ori was given for
3 consecutive days. Mice were sacrificed at 24 h after the last
Ori administration and body and kidney weights were recorded. Blood was collected to test BUN and CRE levels according to the
manufacturer’s instructions. Cell Viability Assay (I,J) Cells were treated with Ori (20 µM) in the absence or
presence of CQ (50 µM) for 18 h. Acidic vesicular organelles (autophagosomes) were stained with acridine orange. Scale bars: 10 µM. (K–N) Cells were treated with
Ori (20 µM) in the absence or presence of CQ (50 µM) for 18 h. LC3 was measured with immunoblot and immunofluorescence. Scale bars: 20 µM. The results
showed the average of three independent experiments. *p < 0.05 and **p < 0.01 vs. the control group. ##p < 0.01 vs. the CQ group. Immunofluorescence Staining Cells were seeded in glassy plates and treated with Ori (20 µM),
cisplatin (20 µM) or compound c (5 µM) for 18 h. After fixation
in 4% paraformaldehyde for 30 min, cells were permeabilized
with 0.2% Triton X-100 for 10 min and blocked with goat
serum. Then, cells were incubated with primary antibody (anti-
LC3) overnight and the corresponding secondary antibody. The nuclei were stained with DAPI for 20 min and images
were captured with a confocal microscope. LC3 puncta were
green and localized in the cytoplasm and nuclei were stained
blue. Then the intensity of LC3 puncta was examined by
Image J. RESULTS Ori Regulates Autophagy, AMPK,
Akt/mTOR Signaling, and Apoptosis
First, we treated cells with different doses of Ori (10, 20, and
30 µM), and found that Ori could significantly inhibit the
viability of A549 cells in a dose-dependent manner. Then the
same doses were used in B2b cells, and we ultimately chose
Ori (5, 10, and 20 µM) for the following vitro experiments
(Figure 1B). As autophagy and related pathways play a vital
role in cancer treatment, we investigated whether Ori had Statistical Analysis Data
presented
are
representatives
from
at
least
three
independent experiments. All results in this study were
expressed as the means ± SEM and analyzed using SPSS19.0
(IBM). Statistical analysis was employed the unpaired Student’s
t-test by using GraphPad 5 Software. Value of p < 0.05 was
considered significant. Quantification of Autophagosomes This step was performed using autophagy detection kit (Abcam
139484). Cells were seeded in 96-well plates and treated with
different reagents (20 µM Ori, 20 µM cisplatin and 5 µM
compound c) for 18 h. Starvation and CQ (50 µM) were positive
controls. Then, the cells were stained with dual detection reagent
and incubated for 30 min at 37◦C. After washed with assay
buffer, the cells were fixed with 4% formaldehyde. Finally,
images were captured with a fluorescence microscope and the
fluorescence intensity of autophagosomes (green) was examined
by Image J. Determination of Combination Index orange solution for 15 min and images were collected by
fluorescence microscope. The interaction between Ori and cisplatin was determined by
the combination index (CI) according to Chou-Talalay’s median-
effect plots and isobologram principles (28). Cells were treated
with different doses of each single drug or their combinations
at the set molar ratios (1:1). The equation for the isobologram
was shown as CI = (D)1/(Dx)1 + (D)2/(Dx)2. (Dx)1 and (Dx)2
indicated the individual doses of Ori and cisplatin required to
inhibit a given level of cell viability, and (D)1 and (D)2 were
the doses of them necessary to produce the same effect in
combination. The combination effects were indicated as follows:
CI = 1, additive effect; CI > 1, antagonism; CI < 1, synergism. Acridine Orange Staining g
g
A549 cells (1 × 106 per well) were treated with Ori (20 µM)
and CQ (50 µM) for 18 h and incubated with acridine August 2019 | Volume 9 | Article 769 Frontiers in Oncology | www.frontiersin.org 4 Oridonin Sensitizes Cisplatin in A549 Cells Yang et al. FIGURE 2 | Ori initiates autophagy, apoptosis and Akt/mTOR signaling by the regulation of AMPK. Cells were treated with Ori (20 µM) in the absence or presence of
compound c (5 µM) for 18 h. (A,B) Cells were stained with an autophagosome specific green reagent and examined by fluorescence microscope. The green
fluorescence intensity was analyzed by Image J. Scale bars: 10 µM. (C,D) Cells were stained with LC3 and DAPI and images were captured with confocal
microscope. The LC3 fluorescence intensity was analyzed by Image J. Scale bars: 20 µM. (E–J) Western blots and statistical results showed the protein levels of
P-AMPK, AMPK, P-Akt, Akt, P-mTOR, mTOR, P62, LC3, Atg3, caspase3, Bax, Bcl2, and PARP. The results showed the average of three independent experiments. **p < 0.01 vs. the control group. #p < 0.05 and ##p < 0.01 vs. the Ori group. FIGURE 2 | Ori initiates autophagy, apoptosis and Akt/mTOR signaling by the regulation of AMPK. Cells were treated with Ori (20 µM) in the absence or presence of
compound c (5 µM) for 18 h. (A,B) Cells were stained with an autophagosome specific green reagent and examined by fluorescence microscope. The green
fluorescence intensity was analyzed by Image J. Scale bars: 10 µM. (C,D) Cells were stained with LC3 and DAPI and images were captured with confocal
microscope. The LC3 fluorescence intensity was analyzed by Image J. Scale bars: 20 µM. (E–J) Western blots and statistical results showed the protein levels of
P-AMPK, AMPK, P-Akt, Akt, P-mTOR, mTOR, P62, LC3, Atg3, caspase3, Bax, Bcl2, and PARP. The results showed the average of three independent experiments. **p < 0.01 vs. the control group. #p < 0.05 and ##p < 0.01 vs. the Ori group. August 2019 | Volume 9 | Article 769 5 Frontiers in Oncology | www.frontiersin.org Yang et al. Oridonin Sensitizes Cisplatin in A549 Cells Ori Initiates Autophagy, Apoptosis, and
Akt/mTOR Signaling by the Regulation of
AMPK the corresponding regulatory effects in A549 cells. Autophagy-
related proteins (P62, LC3, and Atg3) were activated in a dose-
dependent manner (Figures 1C,F). We subsequently observed
that Ori (10 and 20 µM) evidently activated AMPK, caspase3,
Bax, and PARP, but decreased the protein levels of Akt, mTOR,
and Bcl2 (Figures 1D,E,G,H). From our results, 20 µM Ori
showed the most obvious regulator effects. These results indicate
that Ori can regulate autophagy, AMPK, Akt/mTOR signaling
and apoptosis in A549 cells. From the results that P62 and
LC3 were all overexpressed in Ori-treated cells, we added CQ
(50 µM) through acridine orange staining, LC3 staining and
Western blotting to explore whether Ori inhibited autophagy
flux. Results showed that Ori (20 µM) induced more acidic
impaired autophagosomes and LC3 expression in CQ-treated
cells (Figures 1I–N), which indicated that Ori, similar to CQ,
could block the maturation and degradation of autophagosomes. the corresponding regulatory effects in A549 cells. Autophagy-
related proteins (P62, LC3, and Atg3) were activated in a dose-
dependent manner (Figures 1C,F). We subsequently observed
that Ori (10 and 20 µM) evidently activated AMPK, caspase3,
Bax, and PARP, but decreased the protein levels of Akt, mTOR,
and Bcl2 (Figures 1D,E,G,H). From our results, 20 µM Ori
showed the most obvious regulator effects. These results indicate
that Ori can regulate autophagy, AMPK, Akt/mTOR signaling
and apoptosis in A549 cells. From the results that P62 and
LC3 were all overexpressed in Ori-treated cells, we added CQ
(50 µM) through acridine orange staining, LC3 staining and
Western blotting to explore whether Ori inhibited autophagy
flux. Results showed that Ori (20 µM) induced more acidic
impaired autophagosomes and LC3 expression in CQ-treated
cells (Figures 1I–N), which indicated that Ori, similar to CQ,
could block the maturation and degradation of autophagosomes. Ori Alleviates Cisplatin-Induced
Nephrotoxicity in vivo According to the above results, we found the protective effect
of Ori in cancer treatment and could enhance the sensitivity
of cisplatin. While clinically recognized that with the use of
chemotherapy drugs, nephrotoxicity induced by cisplatin also
appears. So we chose a dose of 20 mg/kg cisplatin intraperitoneal
injection for 3 days to induce acute kidney injury and Ori (20
mg/kg) injection simultaneously as a treatment (Figure 6A). We
examined some indicators of kidney function and found that Ori
could reduce cisplatin-induced high levels of BUN, CRE, kidney
index, and weight loss (Figures 6B–E). Ori Induces Apoptosis Through Autophagy
Initiation consistent with related protein expression levels (Figures 5C,D). In summary, these results indicate that Ori possesses anti-
tumor and cisplatin sensitizer potential by the activation of
apoptosis, which was mediated by AMPK/Akt/mTOR-dependent
autophagy initiation. Pro-apoptotic molecules are widely used to enhance the
regulation of autophagy in cancer treatment. So we added 3-
MA (autophagy inhibitor) to examine the relationship between
autophagy and apoptosis in Ori-treated cells. First, we examined
the effects of 3-MA (1 mM) and CQ (50 µM) on Ori-induced
cytotoxicity by CCK-8 assay (Figure 3A). The combination
of CQ and Ori exerted better cytotoxic effects in A549
cells, while 3-MA antagonized the anti-tumor effects of Ori. These results indicated that autophagosomes induced by Ori
possessed cell-killing effects in A549 cells. Then, Western
blotting was used to detect the proteins related to apoptosis
and autophagy (Figures 3B–E). Autophagy and apoptosis
activated by Ori (20 µM) were restrained by 3-MA. Above
all, Ori induced apoptosis by the AMPK/Akt/mTOR-dependent
impaired autophagosomes accumulation. DISCUSSION Ori, an ent-kaurane diterpenoid isolated from Isodon rubescens,
functions as an anti-tumor compound. It has been demonstrated
that Ori could regulate the levels of LC3II/I and some Atg
proteins in A549 cells. However, Ori-regulated the levels
of P62 in tumor growth and cisplatin resistance of lung
cancer have not been investigated. P62 is critical protein to
decide the process of autophagy flux. Generally, P62 will
be degraded and autophagy flux is complete and fluent. Once the autophagy flux is blocked and the degradation of
P62 will be also inhibited. Therefore, the levels of P62 and
autophagosomes are vital in cancer treatment (27). In our study,
Ori
induced
AMPK/Akt/mTOR-dependent
autophagosome
accumulation, which further activated apoptosis to inhibit
lung cancer cell growth and increase cisplatin sensitivity. Moreover,
Ori
alleviated
cisplatin-induced
renal
damage
indexes (BUN, CRE and body weight). Based on the results mentioned later (Figures 5A,B), we chose
the corresponding doses and tested whether Ori sensitized
cisplatin-induced cell death by the regulation of autophagy. Immunofluorescence and Western blotting were used to
display impaired autophagosome accumulation. LC3 puncta
and autophagosomes formation were also promoted in the
group of Ori (20 µM) plus cisplatin (20 µM) (Figures 4A–D). P62, LC3, Atg3, and AMPK/Akt/mTOR expressions were more
obvious in cotreatment with Ori and cisplatin (Figures 4E–H). Taken together, these results indicate that Ori increases cisplatin
sensitivity through the role of autophagy and AMPK/Akt/mTOR. Akt/mT
AMPK AMPK signaling acts as an upstream effector of autophagy
and participates in oncotherapy. Based on Ori’s protective
effect on the above pathways, we explored the interrelation
between them by the addition of compound c (AMPK inhibitor). The number of autophagosomes was counted and LC3 puncta
formation was analyzed by immunofluorescence. Ori-induced
autophagosomes and representative protein LC3 accumulation
were reduced by compound c (Figures 2A–D). Then Ori-
activated P62, LC3, Atg3, and apoptotic proteins (Bax, caspase3,
PARP) were reduced by compound c (5 µM), while the
low levels of Akt/mTOR and Bcl2 in Ori-treated cells were
elevated (Figures 2E–J). FIGURE 3 | Ori induces apoptosis through autophagy initiation. (A) Cells were exposed to different concentrations (10 and 20 µM) of Ori either alone or in the
presence of autophagy inhibitors (50 µM CQ and 1 mM 3-MA) for 18 h. Cell viability was calculated as the percentage relative to untreated cells which were
considered as 100%. (B–E) Cells were seeded in 6-well plates and treated with Ori (20 µM) in the absence or presence of 3-MA (1 mM) for 18 h. Western blots and
statistical results showed the protein levels of P62, LC3, Atg3, caspase3, Bax, Bcl2, and PARP. The results showed the average of three independent experiments. **p < 0.01 vs. the control group. #p < 0.05 and ##p < 0.01 vs. the Ori group. FIGURE 3 | Ori induces apoptosis through autophagy initiation. (A) Cells were exposed to different concentrations (10 and 20 µM) of Ori either alone or in the
presence of autophagy inhibitors (50 µM CQ and 1 mM 3-MA) for 18 h. Cell viability was calculated as the percentage relative to untreated cells which were
considered as 100%. (B–E) Cells were seeded in 6-well plates and treated with Ori (20 µM) in the absence or presence of 3-MA (1 mM) for 18 h. Western blots and
statistical results showed the protein levels of P62, LC3, Atg3, caspase3, Bax, Bcl2, and PARP. The results showed the average of three independent experiments. **p < 0.01 vs. the control group. #p < 0.05 and ##p < 0.01 vs. the Ori group. August 2019 | Volume 9 | Article 769 6 Frontiers in Oncology | www.frontiersin.org Oridonin Sensitizes Cisplatin in A549 Cells Yang et al. Frontiers in Oncology | www.frontiersin.org Ori Enhances Chemotherapeutic
Sensitivity Through the Activation of
Apoptosis Chemotherapy resistance has always been an important factor
in reducing the survival rate of cancer patients, so researchers
investigate sensitizers to reverse the resistance. On the basis of
anti-tumor potential of Ori, we further examined whether Ori
could enhance cisplatin sensitivity by CCK-8 assay. Cisplatin
(10, 20 µM) showed slight inhibition of tumor growth, while cell
viability was evidently suppressed after the addition of Ori (10,
20 µM) (Figure 5A). Addtionally, 30 µM cisplatin showed cell-
killing effects on B2b cells. Then, added these data into CalcuSyn
and calculated the combination index according to the formula. CI < 1 indicates synergism, while CI > 1 indicates antagonism. From our results, the combination of Ori (20 µM) and Cis
(20 µM) showed the best synergism and these two doses were
used in our experiments (Figure 5B). We further investigated the
role of apoptosis in cisplatin plus Ori group, and the effect of Ori
(20 µM) on apoptotic proteins (caspase3, Bax, Bcl2, and PARP)
was more remarkable in cisplatin-treated cells (Figures 5E,F). Then we detected apoptosis activity by FASC and the results were Under stress conditions, autophagy exerts a pro-survival
activity in carcinogenesis and drug resistance (29, 30). When
autophagy
occurs,
cytoplasmic
components
(organelles,
proteins) are wrapped by double-layer membrane vesicles
and form autophagosomes, which are then transferred to
lysosomes for degradation. This procedure aims to provide
nutrients and energy for cellular activity. In these reactions,
Atg genes and LC3 regulate the formation of autophagosome
double-layer membranes. Under the participation of Atg3,
LC3I is transformed into LC3II and tightly binds to the
surface of autophagic vesicles, taking part in the extension of
autophagosomes (31). As a vital tumor suppressor, P62 interacts
with mTORC1 and negatively regulates lysosome acidification
and autophagosome-lysosome fusion (32). Increasing evidence
indicates that cisplatin can induce autophagy, and suppression
of autophagy by 3-methyladenine(3-MA) strongly enhanced
cisplatin sensitivity (21). Our results showed that Ori evidently
inhibited cell growth and induced the over-expressions of August 2019 | Volume 9 | Article 769 7 Oridonin Sensitizes Cisplatin in A549 Cells Yang et al. FIGURE 4 | Ori enhances chemotherapeutic sensitivity through the modulation of autophagy and AMPK/Akt/mTOR pathways. A549 cells were exposed to Ori
(20 µM) in the absence or presence of cisplatin (20 µM) for 18 h. (A,B) Cells were stained with an autophagosome specific green reagent and examined by
fluorescence microscope. The green fluorescence intensity was analyzed by Image J. Frontiers in Oncology | www.frontiersin.org Ori Enhances Chemotherapeutic
Sensitivity Through the Activation of
Apoptosis Scale bars: 10 µM. (C,D) Cells were stained with LC3 and DAPI and images
were captured with confocal microscope. The LC3 fluorescence intensity was analyzed by Image J. Scale bars: 20 µM. (E–H) Western blots and statistical results
showed the protein levels of P62, LC3, Atg3, P-AMPK, AMPK, P-Akt, Akt, P-mTOR, and mTOR. The results showed the average of three independent experiments. *p < 0.05 and **p < 0.01 vs. the control group. #p < 0.05 and ##p < 0.01 vs. the Ori group. +p < 0.05 and ++p < 0.01 vs. the Cis group. FIGURE 4 | Ori enhances chemotherapeutic sensitivity through the modulation of autophagy and AMPK/Akt/mTOR pathways. A549 cells were exposed to Ori
(20 µM) in the absence or presence of cisplatin (20 µM) for 18 h. (A,B) Cells were stained with an autophagosome specific green reagent and examined by
fluorescence microscope. The green fluorescence intensity was analyzed by Image J. Scale bars: 10 µM. (C,D) Cells were stained with LC3 and DAPI and images
were captured with confocal microscope. The LC3 fluorescence intensity was analyzed by Image J. Scale bars: 20 µM. (E–H) Western blots and statistical results
showed the protein levels of P62, LC3, Atg3, P-AMPK, AMPK, P-Akt, Akt, P-mTOR, and mTOR. The results showed the average of three independent experiments. *p < 0.05 and **p < 0.01 vs. the control group. #p < 0.05 and ##p < 0.01 vs. the Ori group. +p < 0.05 and ++p < 0.01 vs. the Cis group. LC3II, Atg3, P62 and the accumulation of autophagosomes
under confocal and general fluorescence microscopy. The
increased levels of LC3 and P62 tend to block autophagosomal
maturation and degradation (suppression of autophagy at its
late stage), therefore we combined Ori and CQ to examine the
induced changes in autophagy flux. As expected, Ori induced
increased expression of LC3II and acidic autophagosomes in cells treated with CQ. Thus, these results suggested that Ori
inhibited autophagy flux and exerted an effect similar to that
of CQ to cause the activation of autophagosomes (Figure 1). Then, the anti-tumor potential and autophagy initiation of Ori
were weakened by the addition of 3-MA, so we considered
that Ori-induced autophagosome accumulation caused lung
cancer cell death (Figure 2). Based on the abovementioned August 2019 | Volume 9 | Article 769 Frontiers in Oncology | www.frontiersin.org 8 Oridonin Sensitizes Cisplatin in A549 Cells Yang et al. Ori Enhances Chemotherapeutic
Sensitivity Through the Activation of
Apoptosis Oridonin Sensitizes Cisplatin in A549 Cells
| Ori enhances chemotherapeutic sensitivity through the activation of apoptosis. (A) A549 cells were treated with different doses (10, 20, and 30 µM) of Ori
nce or presence of cisplatin (10, 20, and 30 µM) for 18 h. Cell viability was calculated as the percentage relative to untreated cells which were considered
B) Data was added into CalcuSyn and combination index (CI) was analyzed in this experiment. CI < 1 indicates synergism, while CI>1 indicates
. A549 cells were exposed to Ori (20 µM) in the absence or presence of cisplatin (20 µM) for 18 h. (C,D) The percentages of apoptotic and necrotic cells
mined by flow cytometry. (E,F) Western blots and statistical results showed the protein levels of caspase3, Bax, Bcl2, and PARP. The results showed the
three independent experiments. *p < 0.05 and **p < 0.01 vs. the control group. #p < 0.05 and ##p < 0.01 vs. the Ori group. +p < 0.05 and ++p <
e Cis group. FIGURE 5 | Ori enhances chemotherapeutic sensitivity through the activation of apoptosis. (A) A549 cells were treated with different doses (10, 20, and 30 µM) of Ori
in the absence or presence of cisplatin (10, 20, and 30 µM) for 18 h. Cell viability was calculated as the percentage relative to untreated cells which were considered
as 100%. (B) Data was added into CalcuSyn and combination index (CI) was analyzed in this experiment. CI < 1 indicates synergism, while CI>1 indicates
antagonism. A549 cells were exposed to Ori (20 µM) in the absence or presence of cisplatin (20 µM) for 18 h. (C,D) The percentages of apoptotic and necrotic cells
were determined by flow cytometry. (E,F) Western blots and statistical results showed the protein levels of caspase3, Bax, Bcl2, and PARP. The results showed the
average of three independent experiments. *p < 0.05 and **p < 0.01 vs. the control group. #p < 0.05 and ##p < 0.01 vs. the Ori group. +p < 0.05 and ++p <
0.01 vs. the Cis group. cisplatin
sensitivity
by
the
inhibition
of
autophagy
flux
(Figure 4). results, we investigated the role of Ori in cisplatin-treated
cells. Frontiers in Oncology | www.frontiersin.org Ori Enhances Chemotherapeutic
Sensitivity Through the Activation of
Apoptosis Additionally, it has been demonstrated that a
prominent hallmark of platinum resistance is the evasion of
apoptosis. Apoptotic signaling maintains a balance between cell
death and cell survival, however, the dysfunction of pro- and
anti-apoptotic proteins contributes to platinum resistance by
reducing apoptosis. More evidence shows that agents activating
apoptosis by targeting the Bcl-2 family, P53 and caspases can have a protective effect on sensitizing resistant cells (38–41). Therefore, we observed the effect of Ori on the above pathways
and found that Ori enhanced the levels of AMPK, Bax, caspase3
and PARP and suppressed Akt, mTOR and Bcl2 expression
in A549 cells (Figure 1). In cisplatin-treated cells, Ori had
more obvious regulatory effects on apoptosis and Akt/mTOR
(Figures 4, 5). Therefore, we considered that Ori increased
the sensitivity of cisplatin by regulating apoptosis, AMPK
and Akt/mTOR. AMPK is activated during situations in which the cellular level of
ATP is decreased and therefore will inhibit tumor cell growth and
enhance chemotherapy effect (17). For instance, the expression
of AMPK-α was decreased in human breast cancer tissues and
the AMPK activator metformin could have a positive impact on
the effect of chemotherapy (34, 35). In addition, the Akt/mTOR
pathway and apoptosis also participate in cisplatin resistance
in various cancers. Recent studies indicate that inhibition of
autophagy by andrographolide re-sensitizes cisplatin-resistant
non-small cell lung carcinoma cells via activation of the
Akt/mTOR pathways (36). While silencing long non-coding
RNA ROR improves sensitivity of non-small-cell lung cancer
to cisplatin resistance by inhibiting PI3K/Akt/mTOR signaling
pathway (37). Additionally, it has been demonstrated that a
prominent hallmark of platinum resistance is the evasion of
apoptosis. Apoptotic signaling maintains a balance between cell
death and cell survival, however, the dysfunction of pro- and
anti-apoptotic proteins contributes to platinum resistance by
reducing apoptosis. More evidence shows that agents activating
apoptosis by targeting the Bcl-2 family, P53 and caspases can Research has shown that AMPK and autophagy frequently
refer to the upstream regulators of apoptosis (17, 42). So
we investigated the correlation among these pathways using
compound c (AMPK inhibitor) and 3-MA (autophagy inhibitor). Results indicated that the overexpression of autophagy and
apoptosis related proteins by Ori were reversed by 3-MA
(Figure 3),
and
Ori-induced
Akt/mTOR,
autophagy
and
apoptosis-relevant changes were all impaired by compound
c (Figure 2). In keeping with these results, LC3 puncta
activated by Ori were reduced in the pretreatment of these
two inhibitors. Ori Enhances Chemotherapeutic
Sensitivity Through the Activation of
Apoptosis Ori showed increased expression of LC3II, Atg3 and
P62
and
decreased
cell
viability
after
cotreatment
with
cisplatin, and we reached the conclusion that Ori increased In the relevant pathways of cisplatin resistance, AMPK,
Akt/mTOR and apoptosis also play an important role (18, 19, 33). August 2019 | Volume 9 | Article 769 Frontiers in Oncology | www.frontiersin.org 9 Oridonin Sensitizes Cisplatin in A549 Cells Yang et al. FIGURE 6 | Ori alleviates cisplatin-induced nephrotoxicity in vivo. (A) Experimental design of Ori’s therapeutic effects on cisplatin-induced kidney injury. After fasting
for 12 h, intraperitoneal injection of cisplatin (20 mg/kg) was performed to establish an acute kidney injury model. Ori (20 mg/kg) was administered to the enterocoelia
for 3 consecutive days. (B,C) Body and kidney weights were calculated and the kidney index was shown as kidney weight divided by body weight. (D,E) Blood was
collected to detect the levels of BUN and CRE. The results showed the average of three independent experiments. **p < 0.01 vs. the control group. ##p < 0.01 vs. the Cis group. FIGURE 6 | Ori alleviates cisplatin-induced nephrotoxicity in vivo. (A) Experimental design of Ori’s therapeutic effects on cisplatin-induced kidney injury. After fasting
for 12 h, intraperitoneal injection of cisplatin (20 mg/kg) was performed to establish an acute kidney injury model. Ori (20 mg/kg) was administered to the enterocoelia
for 3 consecutive days. (B,C) Body and kidney weights were calculated and the kidney index was shown as kidney weight divided by body weight. (D,E) Blood was
collected to detect the levels of BUN and CRE. The results showed the average of three independent experiments. **p < 0.01 vs. the control group. ##p < 0.01 vs. the Cis group. AMPK is activated during situations in which the cellular level of
ATP is decreased and therefore will inhibit tumor cell growth and
enhance chemotherapy effect (17). For instance, the expression
of AMPK-α was decreased in human breast cancer tissues and
the AMPK activator metformin could have a positive impact on
the effect of chemotherapy (34, 35). In addition, the Akt/mTOR
pathway and apoptosis also participate in cisplatin resistance
in various cancers. Recent studies indicate that inhibition of
autophagy by andrographolide re-sensitizes cisplatin-resistant
non-small cell lung carcinoma cells via activation of the
Akt/mTOR pathways (36). While silencing long non-coding
RNA ROR improves sensitivity of non-small-cell lung cancer
to cisplatin resistance by inhibiting PI3K/Akt/mTOR signaling
pathway (37). Frontiers in Oncology | www.frontiersin.org DATA AVAILABILITY that Ori induced apoptosis by AMPK/Akt/mTOR-dependent
autophagosome accumulation and increased cisplatin resistance
in NSCLC cells. In addition, with the use of chemotherapy
drugs,
cisplatin-induced
nephrotoxicity
is
observed
(43). However, the protective effect of Ori as anti-tumor agent
should not be interfered by the side effect of cisplatin. So
we constructed a renal injury mouse model induced by
cisplatin and investigated whether Ori could have a therapeutic
effect. As
expected,
Ori
could
alleviate
cisplatin-induced
high levels of BUN and CRE, weight loss and kidney index
(Figure 6). The raw data supporting the conclusions of this manuscript will
be made available by the authors, without undue reservation, to
any qualified researcher. Ori Enhances Chemotherapeutic
Sensitivity Through the Activation of
Apoptosis In summary, we could made conclusions August 2019 | Volume 9 | Article 769 Frontiers in Oncology | www.frontiersin.org 10 Yang et al. Oridonin Sensitizes Cisplatin in A549 Cells FIGURE 7 | Schematic model of the increased cisplatin sensitivity by Ori in A549 cells. Ori induced apoptosis and enhanced the chemotherapeutic efficacy of cisplatin
via AMPK/Akt/mTOR-dependent impaired autophagosome accumulation (black lines). And the same binding targets between Ori and Cis were also exhibited
(green lines). FIGURE 7 | Schematic model of the increased cisplatin sensitivity by Ori in A549 cells. Ori induced apoptosis and enhanced the chemotherapeutic efficacy of cisplatin
via AMPK/Akt/mTOR-dependent impaired autophagosome accumulation (black lines). And the same binding targets between Ori and Cis were also exhibited
(green lines). ETHICS STATEMENT All animal studies were reviewed and approved by the Animal
Welfare and Research Ethics Committee of Jilin University. AUTHOR CONTRIBUTIONS In summary, Ori displayed anti-tumor bioactivity and
decreased cisplatin resistance by apoptotic signaling activation,
which
was
modulated
by
AMPK/Akt/mTOR-dependent
autophagy inhibition. Besides, Ori had a therapeutic effect
on cisplatin-induced nephrotoxicity (Figure 7). Our results
suggested
that
Ori,
as
a
specific
autophagy
modulator,
could be potentially developed as an adjuvant for further
cancer treatment and chemotherapy resistance. Additionally,
these
findings
highlighted
the
role
of
autophagosome
activation and the related AMPK/Akt/mTOR pathways and
apoptosis, thereby providing new ideas for the treatment of
lung cancer. HY, XC, and LP designed the research. HY, YG, XF, and XL
performed the research. YG, XF, and XL helped to analyze the
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conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Copyright © 2019 Yang, Gao, Fan, Liu, Peng and Ci. This is an open-access article
distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the
original author(s) and the copyright owner(s) are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these
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АГРОЭКОЛОГИЧЕСКОЕ ОБОСНОВАНИЕ ВНЕСЕНИЯ УДОБРЕНИЙ ПОД ОЗИМУЮ ПШЕНИЦУ СОРТА «МИРОНОВСКАЯ 61»
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Совместно с:
Институт морехозяйства и предпринимательства
При научной поддержке:
Экономическая академия им.Д.А.Ценова (Болгария)
Московский государственный университет путей сообщения (МИИТ)
Украи
инская госуд
дарств
вен
нна
ая ака
адем
мия
я железзнод
дорож
жного тра
ансп
портта
Нау
учно
о-иссле
едова
ате
ельский
й прое
екттно
о-констр
рукто
орски
ий инс
ститтут морс
скогго флотта
Луганский государственный медицинский университет
Харьковская медицинская академия последипломного образования
Бельцкий Го
осударс
стве
енн
ный Ун
ниве
ерси
итетт «Алеку Руссо»
Инс
ститу
ут во
одны
ых проб
блем и мелио
орац
ции На
ацион
нальной
й академии аггра
арны
ых наукк
Одесский нау
учно
о-исслед
дова
ател
льский
й ин
нсти
итутт связзи
Входит в международные наукометрические базы
РИНЦ
INDEXCOPERNICUS (ICI 88.47)
Н
Международное периодическое научное издание
Взгляд в
АУЧНЫЙ
Будущее
International periodic scientific journal
SCIENTIFIC LOOK INTO THE FUTURE
НАУКО
ОВИЙ ПОГЛ
ЛЯД У МА
АЙБУТНЄ
Є
Выпуск №13, апрель 2019
Issue №13, April 2019
Том 2
Part 2
Одессаа
Куприенко СВ
2019
УДК 08
ББК 94
Н 347
ISSN 2415-766Х (Print)
ISSN 2415-7538 (Online)
Главный редактор:
Шибаев Александр Григорьевич, доктор технических наук, профессор, Академик
Головний редактор:
Шибаєв Олександр Григорович, доктор технічних наук, професор, Академік
Chief Editor:
Shibaev Alexander Grigoryevich, Doctor of Technical Sciences, Professor, Academician
Заместитель Главного редактора:
Яценко Александр Владимирович, кандидат технических наук, профессор, Ректор ИМП
Заступник головного редактора:
Яценко Олександр Володимирович, кандидат технічних наук, професор, Ректор ІМП
Deputy Chief Editor:
Yatsenko Alexander Vladimirovich, Candidate of Technical Sciences, Professor, Rector of ISE
Редакционный Совет:
Более 150 докторов наук. Полный список представлен на страницах 3-5.
Редакційна Рада:
Понад 150 докторів наук. Повний список представлений на сторінках 3-5.
Editorial Board:
More than 150 doctors of sciences. The full list is available on pages 3-5.
Н 347 Научный взгляд в будущее. – Выпуск 13. Том 2. – Одесса:
КУПРИЕНКО СВ, 2019 – 134 с.
Журнал предназначается для научных работников, аспирантов, студентов старших курсов,
преподавателей, предпринимателей.
The journal is intended for researchers, graduate students, senior students, teachers and entrepreneurs.
Published quarterly.
УДК 08
ББК 94
DOI: 10.30888/2415-7538.2019-13-02
© Коллектив авторов, научные тексты 2019
© Куприенко С.В., оформление 2019
Выпуск 13
Том 2
Редакционный Совет
Попов Виктор Сергеевич, доктор технических наук, профессор, Россия
Рокочинский Анатолий Николаевич, доктор технических наук, профессор,
Украина
Ромащенко Михаил Иванович, доктор технических наук, профессор, Украина
Семенцов Георгий Никифорович, доктор технических наук, профессор,
Украина
Сухенко Юрий Григорьевич, доктор технических наук, профессор, Украина
Устенко Сергей Анатольевич, доктор технических наук, доцент, Украина
Хабибуллин Рифат Габдулхакович, доктор технических наук, профессор,
Россия
Червоный Иван Федорович, доктор технических наук, профессор, Украина
Шайко-Шайковский Александр Геннадьевич, доктор технических наук,
профессор, Украина
Щербань Игорь Васильевич, доктор технических наук, доцент, Россия
Бушуева Инна Владимировна, доктор фармацевтических наук, профессор,
Украина
Волох Дмитрий Степанович, доктор фармацевтических наук, профессор,
Украина
Георгиевский Геннадий Викторович, доктор фармацевтических наук,
старший научный сотрудник, Украина
Гудзенко Александр Павлович, доктор фармацевтических наук, профессор,
Украина
Тихонов Александр Иванович, доктор фармацевтических наук, профессор,
Украина
Шаповалов Валерий Владимирович, доктор фармацевтических наук,
профессор, Украина
Шаповалова Виктория Алексеевна, доктор фармацевтических наук,
профессор, Украина
Блатов Игорь Анатольевич, доктор физико-математических наук, профессор,
Россия
Кондратов Дмитрий Вячеславович, доктор физико-математических наук,
доцент, Россия
Лялькина Галина Борисовна, доктор физико-математических наук,
профессор, Россия
Малахов А В , доктор физико-математических наук, профессор, Украина
Ворожбитова Александра Анатольевна, доктор филологических наук,
профессор, Россия
Лыткина Лариса Владимировна, доктор филологических наук, доцент,
Россия
Попова Таисия Георгиевна, доктор филологических наук, профессор, Россия
Коваленко Елена Михайловна, доктор философских наук, профессор, Россия
Липич Тамара Ивановна, доктор философских наук, доцент, Россия
Майданюк Ирина Зиновиевна, доктор философских наук, доцент, Украина
Светлов Виктор Александрович, доктор философских наук, профессор,
Россия
Стовпец А В , доктор философских наук, доцент, Украина
Антрапцева Надежда Михайловна, доктор химических наук, профессор,
Украина
Бажева Рима Чамаловна, доктор химических наук, профессор, Россия
Гризодуб Александр Иванович, доктор химических наук, профессор,
Украина
Ермагамбет Болат Толеуханович, доктор химических наук, профессор,
Казахстан
Максин Виктор Иванович, доктор химических наук, профессор, Украина
Ангелова Поля Георгиева, доктор экономических наук, профессор, Болгария
Безденежных Татьяна Ивановна, доктор экономических наук, профессор,
Россия
Бурда Алексей Григорьевич, доктор экономических наук, профессор, Россия
Грановская Людмила Николаевна, доктор экономических наук, профессор,
Украина
Дорохина Елена Юрьевна, доктор экономических наук, доцент, Россия
Климова Наталья Владимировна, доктор экономических наук, профессор,
Россия
Кочинев Юрий Юрьевич, доктор экономических наук, доцент, Россия
Курмаев Петр Юрьевич, доктор экономических наук, профессор, Украина
Лапкина Инна Александровна, доктор экономических наук, профессор,
Украина
Мельник Алёна Алексеевна, доктор экономических наук, доцент, Украина
Миляева Лариса Григорьевна, доктор экономических наук, профессор,
Россия
Пахомова Елена Анатольевна, доктор экономических наук, доцент, Россия
Резников Андрей Валентинович, доктор экономических наук, доцент, Россия
Савельева Нелли Александровна, доктор экономических наук, профессор,
Россия
Соколова Надежда Геннадьевна, доктор экономических наук, доцент, Россия
Стрельцова Елена Дмитриевна, доктор экономических наук, доцент, Россия
Батыргареева Владислава Станиславовона, доктор юридических наук, ,
Украина
Гетьман Анатолий Павлович, доктор юридических наук, профессор, Украина
Кафарский Владимир Иванович, доктор юридических наук, профессор,
Украина
Кириченко Александр Анатольевич, доктор юридических наук, профессор,
Украина
Степенко Валерий Ефремович, доктор юридических наук, доцент, Россия
Тонков Евгений Евгеньевич, доктор юридических наук, профессор, Россия
Шепитько Валерий Юрьевич, доктор юридических наук, профессор, Украина
Шишка Роман Богданович, доктор юридических наук, профессор, Украина
Яровенко Василий Васильевич, доктор юридических наук, профессор, Россия
Кантарович Ю Л , кандидат искусствоведения, , Украина
Волгирева Галина Павловна, кандидат исторических наук, доцент, Россия
Токарева Наталья Геннадьевна, кандидат медицинских наук, доцент, Россия
Демидова В Г , кандидат педагогических наук, доцент, Украина
Могилевская И М , кандидат педагогических наук, профессор, Украина
Лебедева Лариса Александровна, кандидат психологических наук, доцент,
Россия
Шаповалов Валентин Валерьевич, кандидат фармацевтических наук, доцент,
Украина
Стовпец В Г , кандидат филологических наук, доцент, Украина
Зубков Руслан Сергеевич, доктор экономических наук, доцент, Украина
Толбатов Андрей Владимирович, кандидат технических наук, доцент,
Украина
Шарагов Василий Андреевич, доктор химических наук, доцент, Молдова
Бухарина Ирина Леонидовна, доктор биологических наук, профессор, Россия
Гребнева Надежда Николаевна, доктор биологических наук, профессор,
Россия
Гриценко Светлана Анатольевна, доктор биологических наук, доцент, Россия
Каленик Татьяна Кузьминична, доктор биологических наук, профессор,
Россия
Князева Ольга Александровна, доктор биологических наук, доцент, Россия
Кухар Елена Владимировна, доктор биологических наук, доцент, Казахстан
Моисейкина Людмила Гучаевна, доктор биологических наук, профессор,
Россия
Нефедьева Елена Эдуардовна, доктор биологических наук, доцент, Россия
Сентябрев Николай Николаевич, доктор биологических наук, профессор,
Россия
Стародубцев Владимир Михайлович, доктор биологических наук, профессор,
Украина
Тестов Борис Викторович, доктор биологических наук, профессор, Россия
Тунгушбаева Зина Байбагусовна, доктор биологических наук, , Казахстан
Фатеева Надежда Михайловна, доктор биологических наук, профессор,
Россия
Ахмадиев Габдулахат Маликович, доктор ветеринарных наук, профессор,
Россия
Шевченко Лариса Васильевна, доктор ветеринарных наук, профессор,
Украина
Анимица Евгений Георгиевич, доктор географических наук, профессор,
Россия
Сухова Мария Геннадьевна, доктор географических наук, доцент, Россия
Иржи Хлахула, доктор геолого-минералогических наук, профессор, Чехия
Федоришин Дмитро Дмитрович, доктор геолого-минералогических наук,
профессор, Украина
Кокебаева Гульжаухар Какеновна, доктор исторических наук, профессор,
Казахстан
Отепова Гульфира Елубаевна, доктор исторических наук, профессор,
Казахстан
Тригуб Петр Никитович, доктор исторических наук, профессор, Украина
Элезович М Далибор , доктор исторических наук, доцент, Сербия
Визир Вадим Анатольевич, доктор медицинских наук, профессор, Украина
Федянина Людмила Николаевна, доктор медицинских наук, профессор,
Россия
Орлов Николай Михайлович, доктор наук государственного управления,
доцент, Украина
Величко Степан Петрович, доктор педагогических наук, профессор, Украина
Гавриленко Наталия Николаевна, доктор педагогических наук, доцент,
Россия
Гилев Геннадий Андреевич, доктор педагогических наук, профессор, Россия
Дорофеев Андрей Викторович, доктор педагогических наук, доцент, Россия
Карпова Наталия Константиновна, доктор педагогических наук, профессор,
Россия
Мишенина Татьяна Михайловна, доктор педагогических наук, профессор,
Украина
Николаева Алла Дмитриевна, доктор педагогических наук, профессор,
Россия
Растрыгина Алла Николаевна, доктор педагогических наук, профессор,
Украина
Сидорович Марина Михайловна, доктор педагогических наук, профессор,
Украина
Смирнов Евгений Иванович, доктор педагогических наук, профессор, Россия
Фатыхова Алевтина Леонтьевна, доктор педагогических наук, доцент, Россия
Федотова Галина Александровна, доктор педагогических наук, профессор,
Россия
Ходакова Нина Павловна, доктор педагогических наук, доцент, Россия
Чигиринская Наталья Вячеславовна, доктор педагогических наук, профессор,
Россия
Чурекова Татьяна Михайловна, доктор педагогических наук, профессор,
Россия
Латыгина Наталья Анатольевна, доктор политологических наук, профессор,
Украина
Сирота Наум Михайлович, доктор политологических наук, профессор,
Россия
Хребина Светлана Владимировна, доктор психологических наук, профессор,
Россия
Вожегова Раиса Анатольевна, доктор сельскохозяйственных наук, профессор,
Украина
Денисов Сергей Александрович, доктор сельскохозяйственных наук,
профессор, Россия
Жовтоног Ольга Игоревна, доктор сельскохозяйственных наук, , Украина
Костенко Василий Иванович, доктор сельскохозяйственных наук, профессор,
Украина
Котляров Владимир Владиславович, доктор сельскохозяйственных наук,
профессор, Россия
Морозов Алексей Владимирович, доктор сельскохозяйственных наук,
профессор, Украина
Патыка Николай Владимирович, доктор сельскохозяйственных наук,
профессор, Украина
Ребезов Максим Борисович, доктор сельскохозяйственных наук, профессор,
Россия
Тарарико Юрий Александрович, доктор сельскохозяйственных наук,
профессор, Украина
Мальцева Анна Васильевна, доктор социологических наук, доцент, Россия
Стегний Василий Николаевич, доктор социологических наук, профессор,
Россия
Тарасенко Лариса Викторовна, доктор социологических наук, профессор,
Россия
Аверченков Владимир Иванович, доктор технических наук, профессор,
Россия
Антонов Валерий Николаевич, доктор технических наук, профессор, Украина
Быков Юрий Александрович, доктор технических наук, профессор, Россия
Гончарук Сергей Миронович, доктор технических наук, профессор, Россия
Захаров Олег Владимирович, доктор технических наук, профессор, Россия
Калайда Владимир Тимофеевич, доктор технических наук, профессор, Россия
Капитанов Василий Павлович, доктор технических наук, профессор, Украина
Кириллова Елена Викторовна, доктор технических наук, доцент, Украина
Коваленко Петр Иванович, доктор технических наук, профессор, Украина
Копей Богдан Владимирович, доктор технических наук, профессор, Украина
Косенко Надежда Федоровна, доктор технических наук, доцент, Россия
Круглов Валерий Михайлович, доктор технических наук, профессор, Россия
Кудерин Марат Крыкбаевич, доктор технических наук, профессор, Казахстан
Лебедев Анатолий Тимофеевич, доктор технических наук, профессор, Россия
Ломотько Денис Викторович, доктор технических наук, профессор, Украина
Макарова Ирина Викторовна, доктор технических наук, профессор, Россия
Морозова Татьяна Юрьевна, доктор технических наук, профессор, Россия
Павленко Анатолий Михайлович, доктор технических наук, профессор,
Украина
Парунакян Ваагн Эмильевич, доктор технических наук, профессор, Украина
Пачурин Герман Васильевич, доктор технических наук, профессор, Россия
Першин Владимир Федорович, доктор технических наук, профессор, Россия
Пиганов Михаил Николаевич, доктор технических наук, профессор, Россия
Поляков Андрей Павлович, доктор технических наук, профессор, Украина
Научный взгляд в будущее
3
ISSN 2415-766Х (P) / 2415-7538 (O)
Выпуск 13
Том 2
Редакційна Рада
Першин Володимир Федорович, доктор технічних наук, професор, Росія
Піганов Михайло Миколайович, доктор технічних наук, професор, Росія
Поляков Андрій Павлович, доктор технічних наук, професор, Україна
Попов Віктор Сергійович, доктор технічних наук, професор, Росія
Рокочінскій Анатолій Миколайович, доктор технічних наук, професор,
Україна
Ромащенко Михайло Іванович, доктор технічних наук, професор, Україна
Семенцов Георгій Никифорович, доктор технічних наук, професор, Україна
Сухенко Юрій Григорович, доктор технічних наук, професор, Україна
Устенко Сергій Анатолійович, доктор технічних наук, доцент, Україна
Хабібуллін Рифат Габдулхакович, доктор технічних наук, професор, Росія
Червоний Іван Федорович, доктор технічних наук, професор, Україна
Шайко-Шайковскій Олександр Геннадійович, доктор технічних наук,
професор, Україна
Щербань Ігор Васильович, доктор технічних наук, доцент, Росія
Бушуєва Інна Володимирівна, доктор фармацевтичних наук, професор,
Україна
Волох Дмитро Степанович, доктор фармацевтичних наук, професор, Україна
Георгієвський Геннадій Вікторович, доктор фармацевтичних наук, старший
науковий співробітник, Україна
Гудзенко Олександр Павлович, доктор фармацевтичних наук, професор,
Україна
Тихонов Олександр Іванович, доктор фармацевтичних наук, професор,
Україна
Шаповалов Валерій Володимирович, доктор фармацевтичних наук,
професор, Україна
Шаповалова Вікторія Олексіївна, доктор фармацевтичних наук, професор,
Україна
Блатов Ігор Анатолійович, доктор фізико-математичних наук, професор,
Росія
Кондратов Дмитро В'ячеславович, доктор фізико-математичних наук, доцент,
Росія
Лялькіна Галина Борисівна, доктор фізико-математичних наук, професор,
Росія
Малахов А В , доктор фізико-математичних наук, професор, Україна
Ворожбітова Олександра Анатоліївна, доктор філологічних наук, професор,
Росія
Литкіна Лариса Володимирівна, доктор філологічних наук, доцент, Росія
Попова Таїсія Георгіївна, доктор філологічних наук, професор, Росія
Коваленко Олена Михайлівна, доктор філософських наук, професор, Росія
Липич Тамара Іванівна, доктор філософських наук, доцент, Росія
Майданюк Ірина Зіновіївна, доктор філософських наук, доцент, Україна
Светлов Віктор Олександрович, доктор філософських наук, професор, Росія
Стовпец А В , доктор філософських наук, доцент, Україна
Антрапцева Надія Михайлівна, доктор хімічних наук, професор, Україна
Бажев Риму Чамаловна, доктор хімічних наук, професор, Росія
Гризодуб Олександр Іванович, доктор хімічних наук, професор, Україна
Ермагамбет Болат Толеухановіч, доктор хімічних наук, професор, Казахстан
Максін Віктор Іванович, доктор хімічних наук, професор, Україна
Ангелова Поля Георгієва, доктор економічних наук, професор, Болгарія
Безденежних Тетяна Іванівна, доктор економічних наук, професор, Росія
Бурда Олексій Григорович, доктор економічних наук, професор, Росія
Грановська Людмила Миколаївна, доктор економічних наук, професор,
Україна
Дорохіна Олена Юріївна, доктор економічних наук, доцент, Росія
Климова Наталія Володимирівна, доктор економічних наук, професор, Росія
Кочинев Юрій Юрійович, доктор економічних наук, доцент, Росія
Курман Петро Юрійович, доктор економічних наук, професор, Україна
Лапкіна Інна Олександрівна, доктор економічних наук, професор, Україна
Мельник Олена Олексіївна, доктор економічних наук, доцент, Україна
Міляєва Лариса Григорівна, доктор економічних наук, професор, Росія
Пахомова Олена Анатоліївна, доктор економічних наук, доцент, Росія
Резніков Андрій Валентинович, доктор економічних наук, доцент, Росія
Савельєва Неллі Олександрівна, доктор економічних наук, професор, Росія
Соколова Надія Геннадіївна, доктор економічних наук, доцент, Росія
Стрельцова Олена Дмитрівна, доктор економічних наук, доцент, Росія
Батиргареева Владислава Станіславовона, доктор юридичних наук, , Україна
Гетьман Анатолій Павлович, доктор юридичних наук, професор, Україна
Кафарський Володимир Іванович, доктор юридичних наук, професор,
Україна
Кириченко Олександр Анатолійович, доктор юридичних наук, професор,
Україна
Степенко Валерій Єфремович, доктор юридичних наук, доцент, Росія
Тонков Євген Євгенович, доктор юридичних наук, професор, Росія
Шепітько Валерій Юрійович, доктор юридичних наук, професор, Україна
Шишка Роман Богданович, доктор юридичних наук, професор, Україна
Яровенко Василь Васильович, доктор юридичних наук, професор, Росія
Кантаровіч Ю Л , кандидат мистецтвознавства, , Україна
Волгірева Галина Павлівна, кандидат історичних наук, доцент, Росія
Токарєва Наталія Геннадіївна, кандидат медичних наук, доцент, Росія
Демидова В Г , кандидат педагогічних наук, доцент, Україна
Могилевська І М , кандидат педагогічних наук, професор, Україна
Лебедєва Лариса Олександрівна, кандидат психологічних наук, доцент, Росія
Шаповалов Валентин Валерійович, кандидат фармацевтичних наук, доцент,
Україна
Стовпец В Г , кандидат філологічних наук, доцент, Україна
Зубков Руслан Сергійович, доктор економічних наук, доцент, Україна
Толбатов Андрій Володимирович, кандидат технічних наук, доцент, Україна
Шарагов Василь Андрійович, доктор хімічних наук, доцент, Молдова
Бухаріна Ірина Леонідівна, доктор біологічних наук, професор, Росія
Гребньова Надія Миколаївна, доктор біологічних наук, професор, Росія
Гриценко Світлана Анатоліївна, доктор біологічних наук, доцент, Росія
Каленик Тетяна Кузьмівна, доктор біологічних наук, професор, Росія
Князєва Ольга Олександрівна, доктор біологічних наук, доцент, Росія
Кухар Олена Володимирівна, доктор біологічних наук, доцент, Казахстан
Моісейкіна Людмила Гучаевна, доктор біологічних наук, професор, Росія
Нефедьева Олена Едуардівна, доктор біологічних наук, доцент, Росія
Сентябрьов Микола Миколайович, доктор біологічних наук, професор, Росія
Стародубцев Володимир Михайлович, доктор біологічних наук, професор,
Україна
Тестів Борис Вікторович, доктор біологічних наук, професор, Росія
Тунгушбаева Зіна Байбагусовна, доктор біологічних наук, , Казахстан
Фатєєва Надія Михайлівна, доктор біологічних наук, професор, Росія
Ахмадієв Габдулахат Маликович, доктор ветеринарних наук, професор, Росія
Шевченко Лариса Василівна, доктор ветеринарних наук, професор, Україна
Аніміца Євген Георгійович, доктор географічних наук, професор, Росія
Сухова Марія Геннадіївна, доктор географічних наук, доцент, Росія
Іржі Хлахула, доктор геолого-мінералогічних наук, професор, Чехія
Федоришин Дмитро Дмитрович, доктор геолого-мінералогічних наук,
професор, Україна
Кокебаева Гульжаухар Какеновна, доктор історичних наук, професор,
Казахстан
Отепова Гульфира Елубаевна, доктор історичних наук, професор, Казахстан
Тригуб Петро Микитович, доктор історичних наук, професор, Україна
Елезовіч М Далібор, доктор історичних наук, доцент, Сербія
Візир Вадим Анатолійович, доктор медичних наук, професор, Україна
Федяніна Людмила Миколаївна, доктор медичних наук, професор, Росія
Орлов Микола Михайлович, доктор наук з державного управління, доцент,
Україна
Величко Степан Петрович, доктор педагогічних наук, професор, Україна
Гавриленко Наталія Миколаївна, доктор педагогічних наук, доцент, Росія
Гилев Геннадій Андрійович, доктор педагогічних наук, професор, Росія
Дорофєєв Андрій Вікторович, доктор педагогічних наук, доцент, Росія
Карпова Наталія Костянтинівна, доктор педагогічних наук, професор, Росія
Мішеніна Тетяна Михайлівна, доктор педагогічних наук, професор, Україна
Миколаєва Алла Дмитрівна, доктор педагогічних наук, професор, Росія
Растригіна Алла Миколаївна, доктор педагогічних наук, професор, Україна
Сидорович Марина Михайлівна, доктор педагогічних наук, професор,
Україна
Смирнов Євген Іванович, доктор педагогічних наук, професор, Росія
Фатихова Алевтина Леонтіївна, доктор педагогічних наук, доцент, Росія
Федотова Галина Олександрівна, доктор педагогічних наук, професор, Росія
Ходакова Ніна Павлівна, доктор педагогічних наук, доцент, Росія
Чигиринська Наталія В'ячеславівна, доктор педагогічних наук, професор,
Росія
Чурекова Тетяна Михайлівна, доктор педагогічних наук, професор, Росія
Латигіна Наталія Анатоліївна, доктор політологічних наук, професор,
Україна
Сирота Наум Михайлович, доктор політологічних наук, професор, Росія
Хребіна Світлана Володимирівна, доктор психологічних наук, професор,
Росія
Вожегова Раїса Анатоліївна, доктор сільськогосподарських наук, професор,
Україна
Денисов Сергій Олександрович, доктор сільськогосподарських наук,
професор, Росія
Жовтоног Ольга Ігорівна, доктор сільськогосподарських наук, , Україна
Костенко Василь Іванович, доктор сільськогосподарських наук, професор,
Україна
Котляров Володимир Владиславович, доктор сільськогосподарських наук,
професор, Росія
Морозов Олексій Володимирович, доктор сільськогосподарських наук,
професор, Україна
Патика Микола Володимирович, доктор сільськогосподарських наук,
професор, Україна
Ребезов Максим Борисович, доктор сільськогосподарських наук, професор,
Росія
Тараріко Юрій Олександрович, доктор сільськогосподарських наук,
професор, Україна
Мальцева Анна Василівна, доктор соціологічних наук, доцент, Росія
Стегній Василь Миколайович, доктор соціологічних наук, професор, Росія
Тарасенко Лариса Вікторівна, доктор соціологічних наук, професор, Росія
Аверченко Володимир Іванович, доктор технічних наук, професор, Росія
Антонов Валерій Миколайович, доктор технічних наук, професор, Україна
Биков Юрій Олександрович, доктор технічних наук, професор, Росія
Гончарук Сергій Миронович, доктор технічних наук, професор, Росія
Захаров Олег Володимирович, доктор технічних наук, професор, Росія
Калайда Володимир Тимофійович, доктор технічних наук, професор, Росія
Капітанів Василь Павлович, доктор технічних наук, професор, Україна
Кирилова Олена Вікторівна, доктор технічних наук, доцент, Україна
Коваленко Петро Іванович, доктор технічних наук, професор, Україна
Копей Богдан Володимирович, доктор технічних наук, професор, Україна
Косенко Надія Федорівна, доктор технічних наук, доцент, Росія
Круглов Валерій Михайлович, доктор технічних наук, професор, Росія
Кудерін Марат Крикбаевіч, доктор технічних наук, професор, Казахстан
Лебедєв Анатолій Тимофійович, доктор технічних наук, професор, Росія
Ломотько Денис Вікторович, доктор технічних наук, професор, Україна
Макарова Ірина Вікторівна, доктор технічних наук, професор, Росія
Морозова Тетяна Юріївна, доктор технічних наук, професор, Росія
Павленко Анатолій Михайлович, доктор технічних наук, професор, Україна
Парунакян Ваагн Емільович, доктор технічних наук, професор, Україна
Пачурін Герман Васильович, доктор технічних наук, професор, Росія
Научный взгляд в будущее
4
ISSN 2415-766Х (P) / 2415-7538 (O)
Выпуск 13
Том 2
Editorial board
Ustenko Sergey Anatol'yevich, Doctor of Technical Sciences, assistant professor,
Ukraine
Khabibullin Rifat Gabdulkhakovich, Doctor of Technical Sciences, Professor,
Russia
Chervonyy Ivan Fedorovich, Doctor of Technical Sciences, Professor, Ukraine
Shayko-Shaykovskiy Aleksandr Gennad'yevich, Doctor of Technical Sciences,
Professor, Ukraine
Shibayev Aleksandr Grigor'yevich, Doctor of Technical Sciences, Professor,
Ukraine
Shcherban' Igor' Vasil'yevich, Doctor of Technical Sciences, assistant professor,
Russia
Bushuyeva Inna Vladimirovna, Doctor of Pharmaceutical Sciences, Professor,
Ukraine
Volokh Dmitriy Stepanovich, Doctor of Pharmaceutical Sciences, Professor,
Ukraine
Georgiyevskiy Gennadiy Viktorovich, Doctor of Pharmaceutical Sciences, Senior
Researcher, Ukraine
Gudzenko Aleksandr Pavlovich, Doctor of Pharmaceutical Sciences, Professor,
Ukraine
Tikhonov Aleksandr Ivanovich, Doctor of Pharmaceutical Sciences, Professor,
Ukraine
Shapovalov Valeriy Vladimirovich, Doctor of Pharmaceutical Sciences, Professor,
Ukraine
Shapovalova Viktoriya Alekseyevna, Doctor of Pharmaceutical Sciences,
Professor, Ukraine
Blatov Igor' Anatol'yevich, Doctor of Physical and Mathematical Sciences,
Professor, Russia
Kondratov Dmitriy Vyacheslavovich, Doctor of Physical and Mathematical
Sciences, assistant professor, Russia
Lyal'kina Galina Borisovna, Doctor of Physical and Mathematical Sciences,
Professor, Russia
Malakhov A V , Doctor of Physical and Mathematical Sciences, Professor,
Ukraine
Vorozhbitova Aleksandra Anatol'yevna, doctor of philology, Professor, Russia
Lytkina Larisa Vladimirovna, doctor of philology, assistant professor, Russia
Popova Taisiya Georgiyevna, doctor of philology, Professor, Russia
Kovalenko Yelena Mikhaylovna, doctor of philosophical science, Professor,
Russia
Lipich Tamara Ivanovna, doctor of philosophical science, assistant professor,
Russia
Maydanyuk Irina Zinoviyevna, doctor of philosophical science, assistant professor,
Ukraine
Svetlov Viktor Aleksandrovich, doctor of philosophical science, Professor, Russia
Stovpets A V , doctor of philosophical science, assistant professor, Ukraine
Antraptseva Nadezhda Mikhaylovna, Doctor of Chemical Sciences, Professor,
Ukraine
Bazheva Rima Chamalovna, Doctor of Chemical Sciences, Professor, Russia
Grizodub Aleksandr Ivanovich, Doctor of Chemical Sciences, Professor, Ukraine
Yermagambet Bolat Toleukhanovich, Doctor of Chemical Sciences, Professor,
Kazakhstan
Maksin Viktor Ivanovich, Doctor of Chemical Sciences, Professor, Ukraine
Angelova Polya Georgiyeva, Doctor of Economic Sciences, Professor, Bulgaria
Bezdenezhnykh Tat'yana Ivanovna, Doctor of Economic Sciences, Professor,
Russia
Burda Aleksey Grigor'yevich, Doctor of Economic Sciences, Professor, Russia
Granovskaya Lyudmila Nikolayevna, Doctor of Economic Sciences, Professor,
Ukraine
Dorokhina Yelena Yur'yevna, Doctor of Economic Sciences, assistant professor,
Russia
Klimova Natal'ya Vladimirovna, Doctor of Economic Sciences, Professor, Russia
Kochinev Yuriy Yur'yevich, Doctor of Economic Sciences, assistant professor,
Russia
Kurmayev Petr Yur'yevich, Doctor of Economic Sciences, Professor, Ukraine
Lapkina Inna Aleksandrovna, Doctor of Economic Sciences, Professor, Ukraine
Mel'nik Alona Alekseyevna, Doctor of Economic Sciences, assistant professor,
Ukraine
Milyayeva Larisa Grigor'yevna, Doctor of Economic Sciences, Professor, Russia
Pakhomova Yelena Anatol'yevna, Doctor of Economic Sciences, assistant
professor, Russia
Reznikov Andrey Valentinovich, Doctor of Economic Sciences, assistant
professor, Russia
Savel'yeva Nelli Aleksandrovna, Doctor of Economic Sciences, Professor, Russia
Sokolova Nadezhda Gennad'yevna, Doctor of Economic Sciences, assistant
professor, Russia
Strel'tsova Yelena Dmitriyevna, Doctor of Economic Sciences, assistant professor,
Russia
Batyrgareyeva Vladislava Stanislavovona, doctor of law, , Ukraine
Get'man Anatoliy Pavlovich, doctor of law, Professor, Ukraine
Kafarskiy Vladimir Ivanovich, doctor of law, Professor, Ukraine
Kirichenko Aleksandr Anatol'yevich, doctor of law, Professor, Ukraine
Stepenko Valeriy Yefremovich, doctor of law, assistant professor, Russia
Tonkov Yevgeniy Yevgen'yevich, doctor of law, Professor, Russia
Shepit'ko Valeriy Yur'yevich, doctor of law, Professor, Ukraine
Shishka Roman Bogdanovich, doctor of law, Professor, Ukraine
Yarovenko Vasiliy Vasil'yevich, doctor of law, Professor, Russia
Kantarovich YU L , Ph D in History of Arts, , Ukraine
Volgireva Galina Pavlovna, Candidate of Historical Sciences, assistant professor,
Russia
Tokareva Natal'ya Gennad'yevna, Candidate of Medical Sciences, assistant
professor, Russia
Demidova V G , Candidate of Pedagogical Sciences, assistant professor, Ukraine
Mogilevskaya I M , Candidate of Pedagogical Sciences, Professor, Ukraine
Lebedeva Larisa Aleksandrovna, Candidate of Psychological Sciences, assistant
professor, Russia
Yatsenko Oleksandr Volodymyrovych, Candidate of Technical Sciences,
Professor, Ukraine
Shapovalov Valentin Valer'yevich, Candidate of Pharmaceutical Sciences,
assistant professor, Ukraine
Stovpets V G , Candidate of Philology, assistant professor, Ukraine
Ruslan Zubkov, Doctor of Economics, Associate Professor, Ukraine
Tolbatov Andrey Vladimirovich, candidate of technical sciences, associate
professor, Ukraine
Sharagov Vasily Andreevich, Doctor of Chemistry, Associate Professor, Moldova
Bukharina Irina Leonidovna, Doctor of Biological Sciences, Professor, Russia
Grebneva Nadezhda Nikolayevna, Doctor of Biological Sciences, Professor,
Russia
Gritsenko Svetlana Anatol'yevna, Doctor of Biological Sciences, assistant
professor, Russia
Kalenik Tat'yana Kuz'minichna, Doctor of Biological Sciences, Professor, Russia
Knyazeva Ol'ga Aleksandrovna, Doctor of Biological Sciences, assistant professor,
Russia
Kukhar Yelena Vladimirovna, Doctor of Biological Sciences, assistant professor,
Kazakhstan
Moiseykina Lyudmila Guchayevna, Doctor of Biological Sciences, Professor,
Russia
Nefed'yeva Yelena Eduardovna, Doctor of Biological Sciences, assistant professor,
Russia
Sentyabrev Nikolay Nikolayevich, Doctor of Biological Sciences, Professor,
Russia
Starodubtsev Vladimir Mikhaylovich, Doctor of Biological Sciences, Professor,
Ukraine
Testov Boris Viktorovich, Doctor of Biological Sciences, Professor, Russia
Tungushbayeva Zina Baybagusovna, Doctor of Biological Sciences, , Kazakhstan
Fateyeva Nadezhda Mikhaylovna, Doctor of Biological Sciences, Professor, Russia
Akhmadiyev Gabdulakhat Malikovich, Doctor of Veterinary Science, Professor,
Russia
Shevchenko Larisa Vasil'yevna, Doctor of Veterinary Science, Professor, Ukraine
Animitsa Yevgeniy Georgiyevich, Doctor of Geographical Sciences, Professor,
Russia
Sukhova Mariya Gennad'yevna, Doctor of Geographical Sciences, assistant
professor, Russia
Irzhi Khlakhula, Doctor of Geological and Mineralogical Sciences, Professor,
Czech Republic
Fedorishin Dmitro Dmitrovich, Doctor of Geological and Mineralogical Sciences,
Professor, Ukraine
Kokebayeva Gul'zhaukhar Kakenovna, Doctor of Historical Sciences, Professor,
Kazakhstan
Otepova Gul'fira Yelubayevna, Doctor of Historical Sciences, Professor,
Kazakhstan
Trigub Petr Nikitovich, Doctor of Historical Sciences, Professor, Ukraine
Elezovich M Dalibor, Doctor of Historical Sciences, assistant professor, Serbia
Vizir Vadim Anatol'yevich, Doctor of Medical Sciences, Professor, Ukraine
Fedyanina Lyudmila Nikolayevna, Doctor of Medical Sciences, Professor, Russia
Orlov Nikolay Mikhaylovich, Doctor of Science in Public Administration, assistant
professor, Ukraine
Velichko Stepan Petrovich, doctor of pedagogical sciences, Professor, Ukraine
Gavrilenko Nataliya Nikolayevna, doctor of pedagogical sciences, assistant
professor, Russia
Gilev Gennadiy Andreyevich, doctor of pedagogical sciences, Professor, Russia
Dorofeyev Andrey Viktorovich, doctor of pedagogical sciences, assistant
professor, Russia
Karpova Nataliya Konstantinovna, doctor of pedagogical sciences, Professor,
Russia
Mishenina Tat'yana Mikhaylovna, doctor of pedagogical sciences, Professor,
Ukraine
Nikolayeva Alla Dmitriyevna, doctor of pedagogical sciences, Professor, Russia
Rastrygina Alla Nikolayevna, doctor of pedagogical sciences, Professor, Ukraine
Sidorovich Marina Mikhaylovna, doctor of pedagogical sciences, Professor,
Ukraine
Smirnov Yevgeniy Ivanovich, doctor of pedagogical sciences, Professor, Russia
Fatykhova Alevtina Leont'yevna, doctor of pedagogical sciences, assistant
professor, Russia
Fedotova Galina Aleksandrovna, doctor of pedagogical sciences, Professor, Russia
Khodakova Nina Pavlovna, doctor of pedagogical sciences, assistant professor,
Russia
Chigirinskaya Natal'ya Vyacheslavovna, doctor of pedagogical sciences, Professor,
Russia
Churekova Tat'yana Mikhaylovna, doctor of pedagogical sciences, Professor,
Russia
Latygina Natal'ya Anatol'yevna, Doctor of Political Sciences, Professor, Ukraine
Sirota Naum Mikhaylovich, Doctor of Political Sciences, Professor, Russia
Khrebina Svetlana Vladimirovna, Doctor of Psychology, Professor, Russia
Vozhegova Raisa Anatol'yevna, doctor of agricultural sciences, Professor, Ukraine
Denisov Sergey Aleksandrovich, doctor of agricultural sciences, Professor, Russia
Zhovtonog Ol'ga Igorevna, doctor of agricultural sciences, , Ukraine
Kostenko Vasiliy Ivanovich, doctor of agricultural sciences, Professor, Ukraine
Kotlyarov Vladimir Vladislavovich, doctor of agricultural sciences, Professor,
Russia
Morozov Aleksey Vladimirovich, doctor of agricultural sciences, Professor,
Ukraine
Patyka Nikolay Vladimirovich, doctor of agricultural sciences, Professor, Ukraine
Rebezov Maksim Borisovich, doctor of agricultural sciences, Professor, Russia
Tarariko Yuriy Aleksandrovich, doctor of agricultural sciences, Professor, Ukraine
Mal'tseva Anna Vasil'yevna, Doctor of Sociology, assistant professor, Russia
Stegniy Vasiliy Nikolayevich, Doctor of Sociology, Professor, Russia
Tarasenko Larisa Viktorovna, Doctor of Sociology, Professor, Russia
Averchenkov Vladimir Ivanovich, Doctor of Technical Sciences, Professor, Russia
Antonov Valeriy Nikolayevich, Doctor of Technical Sciences, Professor, Ukraine
Bykov Yuriy Aleksandrovich, Doctor of Technical Sciences, Professor, Russia
Goncharuk Sergey Mironovich, Doctor of Technical Sciences, Professor, Russia
Zakharov Oleg Vladimirovich, Doctor of Technical Sciences, Professor, Russia
Kalayda Vladimir Timofeyevich, Doctor of Technical Sciences, Professor, Russia
Kapitanov Vasiliy Pavlovich, Doctor of Technical Sciences, Professor, Ukraine
Kirillova Yelena Viktorovna, Doctor of Technical Sciences, assistant professor,
Ukraine
Kovalenko Petr Ivanovich, Doctor of Technical Sciences, Professor, Ukraine
Kopey Bogdan Vladimirovich, Doctor of Technical Sciences, Professor, Ukraine
Kosenko Nadezhda Fedorovna, Doctor of Technical Sciences, assistant professor,
Russia
Kruglov Valeriy Mikhaylovich, Doctor of Technical Sciences, Professor, Russia
Kuderin Marat Krykbayevich, Doctor of Technical Sciences, Professor,
Kazakhstan
Lebedev Anatoliy Timofeyevich, Doctor of Technical Sciences, Professor, Russia
Lomot'ko Denis Viktorovich, Doctor of Technical Sciences, Professor, Ukraine
Makarova Irina Viktorovna, Doctor of Technical Sciences, Professor, Russia
Morozova Tat'yana Yur'yevna, Doctor of Technical Sciences, Professor, Russia
Pavlenko Anatoliy Mikhaylovich, Doctor of Technical Sciences, Professor,
Ukraine
Parunakyan Vaagn Emil'yevich, Doctor of Technical Sciences, Professor, Ukraine
Pachurin German Vasil'yevich, Doctor of Technical Sciences, Professor, Russia
Pershin Vladimir Fedorovich, Doctor of Technical Sciences, Professor, Russia
Piganov Mikhail Nikolayevich, Doctor of Technical Sciences, Professor, Russia
Polyakov Andrey Pavlovich, Doctor of Technical Sciences, Professor, Ukraine
Popov Viktor Sergeyevich, Doctor of Technical Sciences, Professor, Russia
Rokochinskiy Anatoliy Nikolayevich, Doctor of Technical Sciences, Professor,
Ukraine
Romashchenko Mikhail Ivanovich, Doctor of Technical Sciences, Professor,
Ukraine
Sementsov Georgiy Nikiforovich, Doctor of Technical Sciences, Professor,
Ukraine
Sukhenko Yuriy Grigor'yevich, Doctor of Technical Sciences, Professor, Ukraine
Научный взгляд в будущее
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ISSN 2415-766Х (P) / 2415-7538 (O)
Выпуск 13
Том 2
О журнале
Международный научный периодический журнал "Научный взгляд в будущее" получил большое признание
среди отечественных и зарубежных интеллектуалов. Сегодня в журнале публикуются авторы из России,
Украины, Молдовы, Казахстана, Беларуси, Чехии, Болгарии, Литвы Польши и других государств.
Учрежден в 2015 году. Периодичность выхода: ежеквартально.
Основными целями журнала "Научный взгляд в будущее" являются:
• cодействие обмену знаниями в научном сообществе;
• помощь молодым ученым в информировании научной общественности об их научных достижениях;
• cоздание основы для инноваций и новых научных подходов, а также открытий в неизвестных областях;
• содействие объединению профессиональных научных сил и формирование нового поколения ученыхспециалистов в разных сферах.
Журнал целенаправленно знакомит читателя с оригинальными исследованиями авторов в различных областях
науки, лучшими образцами научной публицистики.
Публикации журнала "Научный взгляд в будущее" предназначены для широкой читательской аудитории – всех
тех, кто любит науку. Материалы, публикуемые в журнале, отражают актуальные проблемы и затрагивают
интересы всей общественности.
Каждая статья журнала включает обобщающую информацию на английском языке.
Журнал зарегистрирован в РИНЦ SCIENCE INDEX и INDEXCOPERNICUS.
Про журнал
Міжнародний науковий періодичний журнал "Науковий погляд у майбутнє" отримав велике визнання серед
вітчизняних і зарубіжних інтелектуалів. Сьогодні в журналі публікуються автори з Росії, України, Молдови,
Казахстану, Білорусі, Чехії, Болгарії, Литви, Польщі та інших держав.
Дата заснування в 2015 році. Періодичність виходу: щоквартально
Основними цілями журналу є:
• cприяння обміну знаннями в науковому співтоваристві;
• допомога молодим вченим в інформуванні наукової громадськості про їх наукові досягнення;
• створення основи для інновацій і нових наукових підходів, а також відкриттів в невідомих областях;
• сприяння об'єднанню фахових наукових сил і формування нового покоління вчених-фахівців в різних
сферах.
Журнал цілеспрямовано знайомить читача з оригінальними дослідженнями авторів в різних областях науки,
кращими зразками наукової публіцистики.
Публікації журналу призначені для широкої читацької аудиторії - усіх тих, хто любить науку. Матеріали, що
публікуються в журналі, відображають актуальні проблеми і зачіпають інтереси всієї громадськості.
Кожна стаття журналу включає узагальнюючу інформацію англійською мовою.
Журнал зареєстрований в РИНЦ SCIENCE INDEX і INDEXCOPERNICUS.
About the journal
The International Scientific Periodical Journal "Scientific look into the future" has gained considerable recognition
among domestic and foreign researchers and scholars. Today, the journal publishes authors from Russia, Ukraine,
Moldova, Kazakhstan, Belarus, Czech Republic, Bulgaria, Lithuania, Poland and other countries.
Journal Established in 2015. Рeriodicity of publication: Quarterly
The journal activity is driven by the following objectives:
• Broadcasting young researchers and scholars outcomes to wide scientific audience
• Fostering knowledge exchange in scientific community
• Promotion of the unification in scientific approach
• Creation of basis for innovation and new scientific approaches as well as discoveries in unknown domains
The journal purposefully acquaints the reader with the original research of authors in various fields of science, the best
examples of scientific journalism.
Publications of the journal are intended for a wide readership - all those who love science. The materials published in
the journal reflect current problems and affect the interests of the entire public.
Each article in the journal includes general information in English.
The journal is registered in the RISC SCIENCE INDEX and INDEXCOPERNICUS.
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Том 2
Требования к статьям
Статьи должны соответствовать тематическому профилю журнала, отвечать международным стандартам научных
публикаций и быть оформленными в соответствии с установленными правилами. Они также должны представлять собой
изложение результатов оригинального авторского научного исследования, быть вписанными в контекст отечественных и
зарубежных исследований по этой тематике, отражать умение автора свободно ориентироваться в существующем
библиографическом контексте по затрагиваемым проблемам и адекватно применять общепринятую методологию
постановки и решения научных задач.
Все тексты должны быть написаны литературным языком, отредактированы и соответствовать научному стилю речи.
Некорректность подбора и недостоверность приводимых авторами фактов, цитат, статистических и социологических
данных, имен собственных, географических названий и прочих сведений может стать причиной отклонения присланного
материала (в том числе – на этапе регистрации).
Все таблицы и рисунки в статье должны быть пронумерованы, иметь заголовки и ссылки в тексте. Если данные
заимствованы из другого источника, на него должна быть дана библиографическая ссылка в виде примечания.
Название статьи, ФИО авторов, учебные заведения (кроме основного языка текста) должны быть представлены и на
английском языке.
Статьи должны сопровождаться аннотацией и ключевыми словами на языке основного текста и обязательно на английском
языке. Аннотация должна быть выполнена в форме краткого текста, который раскрывает цель и задачи работы, ее структуру
и основные полученные выводы. Аннотация представляет собой самостоятельный аналитический текст и должна давать
адекватное представление о проведенном исследовании без необходимости обращения к статье. Аннотация на английском
(Abstract) должна быть написана грамотным академическим языком.
Приветствуется наличие УДК, ББК, а также (для статей по Экономике) код JEL (https://www.aeaweb.org/jel/guide/jel.php)
Принятие материала к рассмотрению не является гарантией его публикации. Зарегистрированные статьи рассматриваются
редакцией и при формальном и содержательном соответствии требованиям журнала направляются на экспертное
рецензирование, в том числе через открытое обсуждение с помощью веб-ресурса www.sworld.education.
В журнале могут быть размещены только ранее неопубликованные материалы.
Вимоги до статей
Статті повинні відповідати тематичному профілю журналу, відповідати міжнародним стандартам наукових публікацій і
бути оформленими відповідно до встановлених правил. Вони також повинні представляти собою виклад результатів
оригінального авторського наукового дослідження, бути вписаними в контекст вітчизняних і зарубіжних досліджень з цієї
тематики, відображати вміння автора вільно орієнтуватися в існуючому бібліографічному контексті по піднятим проблемам
і адекватно застосовувати загальноприйняту методологію постановки і вирішення наукових завдань.
Всі тексти повинні бути написані літературною мовою, відредаговані і відповідати науковому стилю мовлення.
Некоректність підбору і недостовірність наведених авторами фактів, цитат, статистичних та соціологічних даних, власних
імен, географічних назв та інших відомостей може стати причиною відхилення надісланого матеріалу (в тому числі - на
етапі реєстрації).
Всі таблиці і рисунки в статті повинні бути пронумеровані, мати заголовки і посилання в тексті. Якщо дані запозичені з
іншого джерела, на нього повинні бути бібліографічні посилання у вигляді примітки.
Назва статті, ПІБ авторів, навчальні заклади (крім основної мови тексту) повинні бути представлені і на англійській мові.
Статті повинні супроводжуватися анотацією та ключовими словами на мові основного тексту і обов'язково англійською
мовою. Анотація повинна бути виконана у формі короткого тексту, який розкриває мету і завдання роботи, її структуру та
основні отримані висновки. Анотація представляє собою самостійний аналітичний текст і повинна давати адекватне
уявлення про проведене дослідження без необхідності звернення до статті. Анотація англійською (Abstract) повинна бути
написана грамотною академічною мовою.
Заохочується наявність УДК, ББК, а також (для статей по Економіці) код JEL (https://www.aeaweb.org/jel/guide/jel.php)
Ухвалення матеріалу до розгляду не є гарантією його публікації. Зареєстровані статті розглядаються редакцією і при
формальному і змістовному відповідно до вимог журналу направляються на експертне рецензування, в тому числі через
відкрите обговорення за допомогою веб-ресурсу www.sworld.education.
У журналі можуть бути розміщені тільки раніше неопубліковані матеріали.
Requirements for articles
Articles should correspond to the thematic profile of the journal, meet international standards of scientific publications and be
formalized in accordance with established rules. They should also be a presentation of the results of the original author's scientific
research, be inscribed in the context of domestic and foreign research on this topic, reflect the author's ability to freely navigate in the
existing bibliographic context on the problems involved and adequately apply the generally accepted methodology of setting and
solving scientific problems.
All texts should be written in literary language, edited and conform to the scientific style of speech. Incorrect selection and
unreliability of the facts, quotations, statistical and sociological data, names of own, geographical names and other information cited
by the authors can cause the rejection of the submitted material (including at the registration stage).
All tables and figures in the article should be numbered, have headings and links in the text. If the data is borrowed from another
source, a bibliographic reference should be given to it in the form of a note.
The title of the article, the full names of authors, educational institutions (except the main text language) should be presented in
English.
Articles should be accompanied by an annotation and key words in the language of the main text and must be in English. The abstract
should be made in the form of a short text that reveals the purpose and objectives of the work, its structure and main findings. The
abstract is an independent analytical text and should give an adequate idea of the research conducted without the need to refer to the
article. Abstract in English (Abstract) should be written in a competent academic language.
The presence of UDC, BBK
Acceptance of the material for consideration is not a guarantee of its publication. Registered articles are reviewed by the editorial
staff and, when formally and in substance, the requirements of the journal are sent to peer review, including through an open
discussion using the web resource www.sworld.education
Only previously unpublished materials can be posted in the journal.
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Том 2
Положение об этике публикации научных данных и ее нарушениях
Редакция журнала осознает тот факт, что в академическом сообществе достаточно широко распространены
случаи нарушения этики публикации научных исследований. В качестве наиболее заметных и вопиющих
можно выделить плагиат, направление в журнал ранее опубликованных материалов, незаконное присвоение
результатов чужих научных исследований, а также фальсификацию данных. Мы выступаем против подобных
практик.
Редакция убеждена в том, что нарушения авторских прав и моральных норм не только неприемлемы с
этической точки зрения, но и служат преградой на пути развития научного знания. Потому мы полагаем, что
борьба с этими явлениями должна стать целью и результатом совместных усилий наших авторов, редакторов,
рецензентов, читателей и всего академического сообщества. Мы призываем всех заинтересованных лиц
сотрудничать и участвовать в обмене информацией в целях борьбы с нарушением этики публикации научных
исследований.
Со своей стороны редакция готова приложить все усилия к выявлению и пресечению подобных неприемлемых
практик. Мы обещаем принимать соответствующие меры, а также обращать пристальное внимание на любую
предоставленную нам информацию, которая будет свидетельствовать о неэтичном поведении того или иного
автора.
Обнаружение нарушений этики влечет за собой отказ в публикации. Если будет выявлено, что статья содержит
откровенную клевету, нарушает законодательство или нормы авторского права, то редакция считает себя
обязанной удалить ее с веб-ресурса и из баз цитирования. Подобные крайние меры могут быть применены
исключительно при соблюдении максимальной открытости и публичности.
Положення про етику публікації наукових даних і її порушеннях
Редакція журналу усвідомлює той факт, що в академічній спільноті досить широко поширені випадки
порушення етики публікації наукових досліджень. В якості найбільш помітних можна виділити плагіат,
відправлення в журнал раніше опублікованих матеріалів, незаконне привласнення результатів чужих наукових
досліджень, а також фальсифікацію даних. Ми виступаємо проти подібних практик.
Редакція переконана в тому, що порушення авторських прав і моральних норм не тільки неприйнятні з етичної
точки зору, але і служать перешкодою на шляху розвитку наукового знання. Тому ми вважаємо, що боротьба з
цими явищами повинна стати метою і результатом спільних зусиль наших авторів, редакторів, рецензентів,
читачів і усієї академічної спільноти. Ми закликаємо всіх зацікавлених осіб співпрацювати і брати участь в
обміні інформацією з метою боротьби з порушенням етики публікації наукових досліджень.
Зі свого боку редакція готова докласти всіх зусиль до виявлення та припинення подібних неприйнятних
практик. Ми обіцяємо вживати відповідних заходів, а також звертати пильну увагу на будь-яку надану нам
інформацію, яка буде свідчити про неетичну поведінку того чи іншого автора.
Виявлення порушень етики тягне за собою відмову в публікації. Якщо буде виявлено, що стаття містить
відвертий наклеп, порушує законодавство або норми авторського права, то редакція вважає себе зобов'язаною
видалити її з веб-ресурсу і з баз цитування. Подібні крайні заходи можуть бути застосовані виключно при
дотриманні максимальної відкритості і публічності.
Regulations on the ethics of publication of scientific data and its violations
The editors of the journal are aware of the fact that in the academic community there are quite widespread cases of
violation of the ethics of the publication of scientific research. As the most notable and egregious, one can single out
plagiarism, the posting of previously published materials, the misappropriation of the results of foreign scientific
research, and falsification of data. We oppose such practices.
The editors are convinced that violations of copyrights and moral norms are not only ethically unacceptable, but also
serve as a barrier to the development of scientific knowledge. Therefore, we believe that the fight against these
phenomena should become the goal and the result of joint efforts of our authors, editors, reviewers, readers and the
entire academic community. We encourage all stakeholders to cooperate and participate in the exchange of information
in order to combat the violation of the ethics of publication of scientific research.
For its part, the editors are ready to make every effort to identify and suppress such unacceptable practices. We promise
to take appropriate measures, as well as pay close attention to any information provided to us, which will indicate
unethical behavior of one or another author.
Detection of ethical violations entails refusal to publish. If it is revealed that the article contains outright slander,
violates the law or copyright rules, the editorial board considers itself obliged to remove it from the web resource and
from the citation bases. Such extreme measures can be applied only with maximum openness and publicity.
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Том 2
https://www.scilook.eu/index.php/slif/article/view/slif13-008
DOI: 10.30888/2415-7538.2019-13-02-008
УДК 631.81
AGROECOLOGICAL GROUND of TOP-DRESSING UNDER WINTER
WHEAT Of SORT «MURONIVSKA 61»
АГРОЕКОЛОГІЧНЕ ОБГРУНТУВАННЯ ВНЕСЕННЯ ДОБРИВ ПІД ОЗИМУ
ПШЕНИЦЮ СОРТУ « МИРОНІВСЬКА 61»
Kudriawytzka A.N./Кудрявицька А.М.
c.a.s. ., as.prof./ к.с.-г.н., доц.
SPIN: 7001-1956
Yanik К. O./Яник К.О.
National University of Life and Environmental Sciences of Ukraine
Kyiv,street of Heroes of defensive,17,03041
Національний університет біоресурсів і природокористування України.
м. Київ, вул. Героїв оборони, 17, 03041
Анотація. Дослідженнями на лучно-чорноземному карбонатному ґрунті встановлено,
що тривале застосування мінеральних добрив на фоні післядії органічних, забезпечує
приріст урожаю зерна районованого сорту ярої пшениці Миронівська яра на 1,73 т/га.
Урожайність і якість зерна ярої пшениці підвищуються при внесенні полуторної норми
мінеральних добрив на фоні післядії органічних, з відповідно високими показниками якості :
збору білку- 0,64 т/га та збору «сирої» клейковини-1,36 т/га.
Ключові слова: пшениця, урожайність, добрива, доза, білок, «сира» клейковина, сорт,
ґрунт, сівозміна.
Питання про вплив умов вирощування, біологічних особливостей сортів,
впливу різних доз мінеральних та органічних добрив, на врожай зерна озимої
пшениці має теоретичне та практичне значення [1,2].
Питання про агроекологічне обґрунтування внесення добрив, застосування
різних доз мінеральних та органічних добрив на врожай зерна озимої пшениці
має теоретичне та практичне значення [2,3].
Об’єктом дослідження є характер зміни вмісту елементів живлення в
рослинах озимої пшениці сорту Миронівська 61 та ярої пшениці сорту
Миронівська яра, врожайність нових сортів озимої та ярої пшениці за
викорисання традиційних видів добрив.
Предметом дослідження є вміст макроелементів в рослинах пшениці,
врожай та якість зерна районованого сорту пшениці озимої Миронівська 61 та
ярої Миронівська яра.
Об’єктом дослідження була озима пшениця сорту Миронівська 61,
попередником якої є конюшина. Дослід має трьохкратну повторність. Площа
облікової ділянки 100 м2, посівної 175 м2.
Стаціонарний дослід має 12 варіантів. Робочими варіантами в в зернобуряковій сівозміні були наступні:
Контроль
Післядія гною - фон
Фон + P80
Фон + P80K80
Фон + N60P80K80
Фон + N75P120K120
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Выпуск 13
Том 2
N60P80K80
В результаті досліджень на лучно-чорноземному грубопилувато легкосуглинковому ґрунті встановлено, що тривале застосування мінеральних
добрив на фоні післядії гною, сприяє підвищенню врожаю зерна озимої
пшениці сорту Миронівська 61 на 11,4-26,3 ц/га (табл.1), при урожаї на
контролі відповідно 32,7 ц/га.
Озима пшениця має чітко виражені закономірності в нагромадженні
вмісту білку і клейковини (табл.2).
Таблиця 1
Вплив добрив на врожайність зерна озимої пшениці, ц/га
Приріст до контролю
Варіант досліду
Врожайність, ц/га
ц/га
%
Без добрив
32,7
100
(контроль)
Післядія
30 т/га
4,1
11,4
34,8
гною (фон)
Фон+Р80
45,5
12,8
39,1
Фон+Р80К80
46,2
13,5
41,3
Фон+N80Р80К80
54,9
22,2
67,8
Фон+N110Р120К120
59,0
26,3
80,4
N80Р80К80
46,1
13,4
40,9
Таблиця 2
Вплив тривалого застосування добрив на якість зерна озимої пшениці
Збір «сирої»
Збір білка
клейковини
«Сира»
Білок,
приріст
приріст
клейкови
Варіант досліду
%
до
до
на, %
ц/га
ц/га
контролю,
контролю,
ц/га
ц/га
Без
добрив
9,2
3,0
20,1
6,6
(контроль)
Післядія 30 т/га
11,6
5,1
2,1
21,2
9,3
2,7
гною (фон)
Фон+Р80
11,9
5,4
2,4
21,5
9,8
3,2
Фон+Р80К80
12,1
5,6
2,6
21,1
9,7
3,1
Фон+N80Р80К80
12,4
6,8
3,8
25,6
14,0
7,4
Фон+N110Р120К120
12,7
7,5
4,5
27,2
16,0
9,4
N80Р80К80
12,1
5,6
2,6
24,1
11,1
4,5
НІР05,%
0,64
1,25
Висновки
1. Внесення мінеральних добрив на фоні післядії органічних підвищувало
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Том 2
урожай зерна озимої пшениці до 26,3 ц\га, при урожаї на контролі 2,7ц/га.
Найвищі врожаї зерна озимої і пшениці отримали при внесенні полуторної
норми мінеральних добрив на фоні післядії органічних, відповідно 59,0 ц/га.
2. Озима пшениця має чітко виражені закономірності в нагромадженні
вмісту білку і клейковини. Мінеральні добрива внесені на фоні післядії
органічних підвищували вміст білку в озимій пшениці на 2,4-3,5% і клейковини
на 1,1-7,1% при вмісті їх на контролі 9,2% і 20,1%.
Література:
1. Агрохімічний аналіз: підр. для студ вищих навч. закл. / М.М. Городній,
А.П. Лісовал, А.В. Бикін та ін.; – К.: Арістей. 2005. – 468с.
2. Антонова А.А. Відтворення родючості чорноземів. / Антонова А.А.,
Головінов А.А. // Агрохімічний вісник. - 2001. - №4. - С. 40–52.
3. Габібов М.А. Післядія мінеральних добрив при вирощуванні озимої
пшениці / Габібов М.А. // Зернові культури. - 2001. - №1. - С. 11–19.
References:
1. Agrohіmіchnij analіz: pіdr. dlya stud vishchih navch. zakl. / M.M. Gorodnіj, A.P. Lіsoval,
A.V. Bikіn ta іn.; – K.: Arіstej. 2005. – 468s.
2. Antonova A.A. Vіdtvorennya rodyuchostі chornozemіv. / Antonova A.A., Golovіnov A.A.
// Agrohіmіchnij vіsnik. - 2001. - №4. - S. 40–52.
3. Gabіbov M.A. Pіslyadіya mіneral'nih dobriv pri viroshchuvannі ozimoї pshenicі / Gabіbov
M.A. // Zernovі kul'turi. - 2001. - №1. - S. 11–19.
Abstract. On black carbonate soil it is set researches, that the protracted application of
mineral fertilizers is on a background the afteraction of organic, the Миронівська furious provides
the increase of harvest of grain of the districted sort of furious wheat on 1,73 t/he. The productivity
and quality of grain of furious wheat rise at bringing of one-and-a-half norm of mineral fertilizers
on a background the afteraction of organic, from accordingly by the high indexes of quality : of
collection of albumen of 0,64 т/and and to collection of "raw" gluten of 1,36 t/he.
Key words: wheat, productivity, fertilizers, dose, albumen, "raw" gluten, sort, soil, crop
rotation.
Стаття відправлена 02.04.2019 г.
© Кудрявицька А.М.
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Абдулвелеева Рауза Рашитовна, кандидат педагогических наук, доцент, Оренбургский государственный университет, Россия
Артюхина Марина Владимировна, кандидат экономических наук, доцент, Славянский государственный педагогический
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Афинская Зоя Николаевна, кандидат филологических наук, доцент, Московский государственный университет имени М.В.
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Башлай Сергей Викторович, кандидат экономических наук, доцент, Украинская академия банковского дела, Украина
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СОДЕРЖАНИЕ / CONTENTS
Экономика и торговля
Economy and trade
Економіка і торгівля
https://www.scilook.eu/index.php/slif/article/view/slif13-020
9
ESSENCE, FUNCTIONS AND MAIN TASKS OF RETAIL TRADE
ENTERPRISES
СУТНІСТЬ, ФУНКЦІЇ ТА ОСНОВНІ ЗАВДАННЯ ДІЯЛЬНОСТІ ПІДПРИЄМСТВ
РОЗДРІБНОЇ ТОРГІВЛІ
Chechui A.V./ Чечуй А. В.
https://www.scilook.eu/index.php/slif/article/view/slif13-023
16
POTENTIAL OF THE TRADE ENTERPRISE: GENERAL APPROACHES TO
FORMATION AND REALIZATION
ПОТЕНЦІАЛ ТОРГОВЕЛЬНОГО ПІДПРИЄМСТВА: ЗАГАЛЬНОНАУКОВІ ПІДХОДИ ДО
ФОРМУВАННЯ ТА РЕАЛІЗАЦІЇ
Zhuvahina I.O. / Жувагіна І.О., Zamaraikina T.S. / Замарайкіна Т.С.
https://www.scilook.eu/index.php/slif/article/view/slif13-033
22
FRAGMENTATION IN MANUFACTURE OF AUTOMOBILE COMPONENTS
IN UKRAINE: EXPIRIENCE FOR BELARUS
ФРАГМЕНТАЦІЯ У ВИРОБНИЦТВІ АВТОКОМПОНЕНТІВ В УКРАЇНІ: ДОСВІД ДЛЯ
БІЛОРУСІ
Kostenko N.V. / Костенко Н.В.
https://www.scilook.eu/index.php/slif/article/view/slif13-043
27
FUTURE AND PROSPECTS OF GLOBALIZATION AND ENVIRONMENTAL
INTEGRATION PROCESS
СУПЕРЕЧНОСТІ ТА ПЕРСПЕКТИВИ ПРОЦЕСУ ГЛОБАЛІЗАЦІЇ ТА
ПРИРОДООХОРОННОЇ ІНТЕГРАЦІЇ
Rogach S.M. / Рогач С.М.
Менеджмент и маркетинг
Management and marketing
Менеджмент і маркетинг
https://www.scilook.eu/index.php/slif/article/view/slif13-031
33
MANAGEMENT COMMUNICATION IN SCIENCE AND THE INFORMATION
ENVIRONMENT
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Makovetska I. M. / Маковецька І.М.
https://www.scilook.eu/index.php/slif/article/view/slif13-038
38
POLITICAL AND ADMINISTRATIVE DECHOTOMY - MYTH OR REALITY
ПОЛИТИКО-АДМИНИСТРАТИВНАЯ ДИХОТОМИЯ – МИФ ИЛИ РЕАЛЬНОСТЬ
Fetescu C. P. / Фетеску Ч. П.
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FEATURES OF DEVELOPMENT OF THE RURAL SETTLEMENT OF
MORDOVO-ISHUTKINA OF M. R. ISAKLINSKIY SAMARA REGION
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СОСТАВЕ М.Р. ИСАКЛИНСКИЙ САМАРСКОЙ ОБЛАСТИ
Kuznetsov M.V. / Кузнецов М.В., Bulankina E.V. / Буланкина Е. В.
Образование и педагогика
Education and pedagogy
Освіта і педагогіка
https://www.scilook.eu/index.php/slif/article/view/slif13-005
50
TOLERANCE AS THE BASE PRINCIPLE OF THE ORGANIZATION OF
ACTIVITY OF INTERNATIONAL STUDENT'S ASSOCIATIONS
ТОЛЕРАНТНОСТЬ КАК БАЗОВЫЙ ПРИНЦИП ОРГАНИЗАЦИИ ДЕЯТЕЛЬНОСТИ
ИНТЕРНАЦИОНАЛЬНЫХ СТУДЕНЧЕСКИХ ОБЪЕДИНЕНИЙ
Vorobjeva G.V./ Воробьёва Г.В., Baturina L.A. / Батурина Л.А., Lepikhov N.V./ Лепихов Н.В.
https://www.scilook.eu/index.php/slif/article/view/slif13-006
54
SOME PROBLEMS OF MATHEMATICAL TRAINING OF ECONOMISTS
НЕКОТОРЫЕ ВОПРОСЫ МАТЕМАТИЧЕСКОЙ ПОДГОТОВКИ ЭКОНОМИСТОВ
Belova T.B. / Белова Т.Б., Mikhin M.N. / Михин М.Н.
https://www.scilook.eu/index.php/slif/article/view/slif13-017
58
THE ROLE OF STUDENTS INDEPENDENT WORK
IN STUDYING INFORMATICS AT UNIVERSITY
РОЛЬ САМОСТОЯТЕЛЬНОЙ РАБОТЫ СТУДЕНТОВ ПРИ ИЗУЧЕНИИ
ИНФОРМАТИКИ В ВУЗЕ
Eroshenko A.V. / Ерошенко А.В.
https://www.scilook.eu/index.php/slif/article/view/slif13-024
ТЕХНОЛОГІЯ ФОРМУВАННЯ НАВИЧОК ГРАМОТИ У ДІТЕЙ
62
З РОЗЛАДАМИ АУТИСТИЧНОГО СПЕКТРА
Dmitriyeva I.V. / Дмитрієва І.В., Burlachenko A.G. / Бурлаченко А.Г.
https://www.scilook.eu/index.php/slif/article/view/slif13-032
66
THE EXPEDIENCY OF USING AUTHENTIC TEXTS IN TEACHING FOREIGN
LANGUAGE
ЦЕЛЕСООБРАЗНОСТЬ ИСПОЛЬЗОВАНИЯ АУТЕНТИЧНЫХ ТЕКСТОВ ПРИ
ОБУЧЕНИИ ИНОСТРАННОМУ ЯЗЫКУ
Zaitseva S.E. / Зайцева С.Е.
https://www.scilook.eu/index.php/slif/article/view/slif13-035
72
THE QUESTION OF EFFICIENCY OF TEACHING LECTURE
К ВОПРОСУ ЭФФЕКТИВНОСТИ ЛЕКЦИОННОГО ОБУЧЕНИЯ
Emchenko E.A. / Емченко Е.А.
Научный взгляд в будущее
131
ISSN 2415-766Х (P) / 2415-7538 (O)
https://www.scilook.eu/index.php/slif/article/view/slif13-037
78
PROFESSIONALLY ORIENTED TRAINING IN THE FRAMEWORK OF THE
COMPETENCE APPROACH
ПРОФЕССИОНАЛЬНО ОРИЕНТИРОВАННОЕ ОБУЧЕНИЕ В РАМКАХ
КОМПЕТЕНТНОСТНОГО ПОДХОДА
Belova N.M. / Белова Н.М., Mikhin M.N. / Михин М.Н.
Физическое воспитание и спорт
Physical education and sport
Фізичне виховання і спорт
https://www.scilook.eu/index.php/slif/article/view/slif13-007
83
FORMATION OF A HEALTHY LIFESTYLE OF YOUNGER STUDENTS IN THE
CONDITIONS OF EXTRACURRICULAR ACTIVITIES
ФОРМИРОВАНИЕ ЗДОРОВОГО ОБРАЗА ЖИЗНИ МЛАДШИХ ШКОЛЬНИКОВ В
УСЛОВИЯХ ВНЕКЛАССНОЙ РАБОТЫ
Vilgelm K.I. / Вильгельм К.И., Sevenuk S.A. / Севенюк С.А.
https://www.scilook.eu/index.php/slif/article/view/slif13-039
87
PHYSICAL EDUCATION OF SCHOOLCHILDREN IN RURAL KAZAKH
SCHOOL
ФИЗИЧЕСКОЕ ВОСПИТАНИЕ ШКОЛЬНИКОВ В КАЗАХСКОЙ СЕЛЬСКОЙ ШКОЛЕ
Dzhusupov U.S./ Джусупов У.С., Issayeva A.U. / Исаева А.У.
Психология и социология
Psychology and sociology
Психологія і соціологія
https://www.scilook.eu/index.php/slif/article/view/slif13-044
93
YOGOTSENTRISM YOUTH SCHOOL LEVEL IN THE TEXT OF THE
DECLARATION
УРОВЕНЬ ЭГОЦЕНТРИЗМА МЛАДШЕГО ШКОЛЬНИКА В ПРОЦЕССЕ
ПОНИМАНИЯ ТЕКСТА
PerepeliykT.D./ Перепелюк Т.Д.
Философия
Philosophy
Філософія
https://www.scilook.eu/index.php/slif/article/view/slif13-046
98
SPECIFICATION OF «FROZEN» CONFLICTS: SOCIAL AND PHILOSOPHICAL
ASPECTS
СПЕЦИФІКА «ЗАМОРОЖЕНИХ» КОНФЛІКТІВ: СОЦІАЛЬНО-ФІЛОСОФСЬКІ
АСПЕКТИ
Авер’янова Н.М. / Averianova N.M., Воропаєва Т.С. / Voropayeva T.S.
Научный взгляд в будущее
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Юридические и политические науки
Legal and political sciences
Юридичні і політичні науки
https://www.scilook.eu/index.php/slif/article/view/slif13-013
105
INTERNATIONAL COMMUNICATIONS OF UKRAINE WITH THE REPUBLIC
OF POLAND: HISTORICAL ASPECTS AND DEVELOPMENT PERSPECTIVES
МІЖНАРОДНІ ЗВ’ЯЗКИ УКРАЇНИ З РЕСПУБЛІКОЮ ПОЛЬЩА: ІСТОРИЧНИЙ
АСПЕКТ ТА ПЕРСПЕКТИВИ РОЗВИТКУ
Vanina O.Y. / Ваніна О.Ю.
https://www.scilook.eu/index.php/slif/article/view/slif13-019
110
INTERNATIONAL RELATIONS OF UKRAINE WITH GREECE
МІЖНАРОДНІ ВІДНОСИНИ УКРАЇНИ З ГРЕЦІЄЮ
Sekunova Y.V. / Сєкунова Ю.В., Shevchuck I.Y./ Шевчук І.Ю.
https://www.scilook.eu/index.php/slif/article/view/slif13-045
114
THE CRISIS OF INTERNATIONAL RELATIONS OF UKRAINE AND POLAND
МІЖНАРОДНІ ВІДНОСИНИ УКРАЇНИ ТА РЕСПУБЛІКИ ПОЛЬЩА: ПРОБЛЕМНІ
СТОРІНКИ ІСТОРІЇ ТА ПЕРСПЕКТИВИ РОЗВИТКУ
Дзуг М.С./ Dzuh M. S., Сєкунова Ю.В./ Sekunova Y.V.
Сельское, лесное, рыбное и водное хозяйство
Agriculture, forestry, fishery and water management
Сільське, лісове, рибне та водне господарство
https://www.scilook.eu/index.php/slif/article/view/slif13-008
119
AGROECOLOGICAL GROUND of TOP-DRESSING UNDER WINTER WHEAT
Of SORT «MURONIVSKA 61»
АГРОЕКОЛОГІЧНЕ ОБГРУНТУВАННЯ ВНЕСЕННЯ ДОБРИВ ПІД ОЗИМУ ПШЕНИЦЮ
СОРТУ « МИРОНІВСЬКА 61»
Kudriawytzka A.N./Кудрявицька А.М., Yanik К. O./Яник К.О.
https://www.scilook.eu/index.php/slif/article/view/slif13-028
122
USE OF GROWTH RATE INDICATORS FOR PUREBRED
LARGE WHITE BREEDS AND THEIR HYBRIDS WITH LANDRACE, DUROC
AND PIETRAIN BREEDS FOR COMPARATIVE ANALYSIS OF THE GROWTH
OF ANIMALS
ВИКОРИСТАННЯ ПОКАЗНИКІВ ІНТЕНСИВНОСТІ РОСТУ ЧИСТОПОРОДНИХ
СВИНЕЙ ВЕЛИКОЇ БІЛОЇ ПОРОДИ ТА ЇХ ПОМІСЕЙ ІЗ ПОРОДАМИ ЛАНДРАС,
ДЮРОК ТА П’ЄТРЕН ДЛЯ ПОРІВНЯЛЬНОГО АНАЛІЗУ РОСТУ ТВАРИН
Barkar Ye.V. / Баркарь Є.В., Dekhtiar Yu.F. / Дехтяр Ю.Ф., Kot S.P. / Кот С.П.
Научный взгляд в будущее
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ISSN 2415-766Х (P) / 2415-7538 (O)
Научное издание
Международный периодический рецензируемый научный журнал
НАУЧНЫЙ ВЗГЛЯД В БУДУЩЕЕ
Выпуск 13
Том 2
На украинском, русском и английском языках
Входит в международные наукометрические базы (высокий импактфактор):
РИНЦ, INDEXCOPERNICUS (ICI 88.47)
Научные достижения Авторов были также представлены для открытого
обсуждения на международной научной конференции «Практическое значение
современных научных исследований ‘2019» (с 8 по 9 апреля 2019 г.)
Решением международной научной конференции работы, которые получили
положительную оценку, были рекомендованы к изданию в журнале.
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The Trouble with “Female Sexuality”
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The Trouble with “Female Sexuality” Sarah Salih • King’s College London Recommended citation: Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different
Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. Using medieval western art to speak of female sexuality is difficult. Karma Lochrie
argues that medieval women’s sexuality was organised in ways so alien to current
categories that it requires careful excavation: Medieval hybrids that are incomprehensible today, such as ‘chaste marriage’
or even a kind of ‘willful virginity,’ were not only practised during the Middle
Ages, but they suggest a much more diffused and complex interaction of
categories than we are used to. Armed only with the heterosexual/homosexual
divide and a presumption of heteronormativity, we cannot even begin to sort
out such categories as Amazons, female masculinity, or even virginity.[1] Lochrie’s book, like most studies of medieval sexuality, is primarily concerned with
textual sources. Can the visual arts contribute to this work of categorisation? This
brief overview will suggest that such a focus tends if anything to find more
uncertainties of various kinds; to indicate that “Female sexuality [in the visual arts] …
wasn’t.” The encounters of women, the visual arts and eros, that is, are so
heterogenous and their boundaries so unclear as to make the category elusive. Of
course women in the Middle Ages had sexual experiences, desires, fantasies,
pleasures and pains; and of course we cannot have direct access to the experiences
of the long dead, though we can converse about them. But the very nature of artistic
representation, whether visual or textual, means that such desires and pleasures
become shared property, which cannot be said to belong to women more than to men – or indeed to the medieval rather than to the modern. Hans Belting argues
that “The human being is the natural locus of images, a living organ for images”;
thus a contemporary viewer, assessing the sexual content or impact of a medieval
image, must put their own bodies and sensibilities forward as substitutes for those of
medieval viewers.[2] men – or indeed to the medieval rather than to the modern. h, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016).
org/10 61302/IRRS2726 Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016).
https://doi.org/10.61302/IRRS2726. Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016).
https://doi.org/10.61302/IRRS2726. h, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016).
/10 61302/IRRS2726 The Trouble with “Female Sexuality” Hans Belting argues
that “The human being is the natural locus of images, a living organ for images”;
thus a contemporary viewer, assessing the sexual content or impact of a medieval
image, must put their own bodies and sensibilities forward as substitutes for those of
medieval viewers.[2] “Sexuality” itself, of course, is a post-medieval term, which is nevertheless, with
appropriate caveats, regularly used. I do not propose not using it, but would note to
begin with that it seems unlikely that its range quite matches any medieval domain
of knowledge. Medieval textual sources tell us a number of quite different things
about female sexuality: that a woman’s desire may be directed to men, women,
herself, or lifeless things; that women have an insatiable desire to be penetrated; that
they are naturally inclined towards chastity; that their reluctance can be overcome by
violence or seduction.[3] Visual sources are no more consistent. I might identify both
a painting of St. Catherine of Siena’s stigmatisation by a crucifix (fig. 1) and a female
exhibitionist figure (fig. 2) as illustrating aspects of female sexuality, but there is no
evidence that these images spoke to one another in any known medieval context:
their conjunction is a product of my framing category. The split between sacred and
profane desires is so pronounced that the category does not cohere. 2 2 Giovanni di Paolo, Stigmatization of St Catherine of Siena, tempera and gold on wood, 1460–1465. New York, Metropolitan Museum of Art, inv. no. 1997.117.3. (Met open use policy via ArtStor) Fig. 1. Giovanni di Paolo, Stigmatization of St Catherine of Siena, tempera and gold on wood, 1460–1465. New York, Metropolitan Museum of Art, inv. no. 1997.117.3. (Met open use policy via ArtStor) 3 Fig. 2. Female exhibitionist figure, stone, date unknown, Old Parish Church, Llandrindod Wells. Llandrindod Wells, Radnorshire Museum (Photo: John Harding) Fig. 2. Female exhibitionist figure, stone, date unknown, Old Parish Church, Llandrindod Wells. Llandrindod Wells, Radnorshire Museum (Photo: John Harding) The problem of sexual identities is not quite, as it is sometimes put, that the Middle
Ages preceded them. Lochrie argues persuasively that the absence of a homosexual
identity means that the presumption of a heteronormative framework must be
questioned.[4] But the contemporary division of homo and heterosexualities is not
the only way to map desires. Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016).
https://doi.org/10.61302/IRRS2726. The Trouble with “Female Sexuality” Medieval women did not lack sexual identities
(although men’s are harder to pin down): as maidens, wives, professed virgins and
whores their presumed sexual activity was the foundational category that Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. 4 determined their dress, demeanour, social position and material location. Thanks to
the work of social historians on women’s lifecycles, we know quite a lot about these
identities, their local variations and their slippages. These sexual identity categories,
however, do not necessarily tell us about women’s sexual pleasures, pains and
desires, or the extent to which they felt at one with or alienated from such identity
categories. The Book of Margery Kempe shows its protagonist at various periods of
her life as a sexually satisfied young wife, who had “ful many delectabyl thowtys,
fleschly lustys, and inordinate lovys” of her husband’s body, as a wife later repulsed
from marital sex as from “the mukke in the chanel,” and as the Bride of a Christ who
claims the right of a husband to “be homly wyth the and lyn in thi bed wyth the.”[5]
But Kempe’s book, despite its seeming frankness, is not a transparent witness but a
crafted tale of a penitent sinner; meanwhile other desires, such as female
homoeroticism, hardly break discursive cover at all.[6] The topic of “female sexuality in medieval art” includes two overlapping fields of
activity: women as sexualised addressees and consumers of art, and the sexualised
representation of women in art. Both raise so many problems of identification and
interpretation that further examination shows them to be almost nothing but
problems. It might seem more promising to begin with female sexual subjects; that is, female
spectators of sexualised art, since, as Sherry Lindquist shows in this volume, it is
well-known that devout women had intense affective responses to artefacts such as
crucifixes. The phenomenon of ‘sex without sex’ between chaste people might be
mediated through visual art.[7] The image of St. Catherine mentioned above depicts
one of the most famous of such events, showing the saint’s ardor as she opens her
arms to receive the stigmata from the crucifix. Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016).
https://doi.org/10.61302/IRRS2726. The Trouble with “Female Sexuality” However, I would caution that art
cannot be isolated from other triggers, that sexuality in such responses cannot be
isolated from other drives and affects, and that women’s responses cannot be
isolated from men’s; indeed, that the shared ground of devotional eros tends to
break down gender difference. For the anchoritic reader of “The Wooing of Our Lord,”
the crucifix is the site of union. The text directs her to speak to Christ in these words: My body hangs with your body nailed to the cross, enclosed on all sides within
four walls, and I will hang with you and never again come off my cross until I
die. For then I shall leap from the cross to rest, from sorrow to joy and to
eternal bliss. Oh Jesus, it is so sweet to hang with you, for when I see you
hanging beside me, your great sweetness totally frees me from pain.[8] Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. 5 While she spoke, she would surely fix her eyes on the crucifix in her cell and
incorporate that into this fulfilment of her union with Christ: the artefact would be
inextricably linked with the height of her pleasure. It is not, however, the sole cause
of it: this is a multi-media experience, combining words, the artefact, visionary
capacity, and probably movement, all of which are necessary constituents of the
pleasure. If it is complexly visual, it is also complexly erotic. To describe such practices
either as channelling sexual response into devotional purposes, or as smuggling
sexuality under cover of devotion, is to overclarify. The practice is designed to
produce sexual and devotional emotions together, and which was experienced as
the primary response would have varied from viewer to viewer, from moment to
moment. Gender, however, was not a significant factor in this variability; all the available
evidence suggests that women’s response to devotional art was not markedly
different from men’s. Men were if anything more often interpellated as passionate
spectators of crucifixes; Rupert of Deutz’s often-cited account of the passionate kiss
of a crucifix figure is one of many such encounters.[9] The illustrations of the
Rothschild Canticles are some of the most joyous images of mystical eros, as the
figure of the Bride cavorts with a unicorn (fol. 51r; fig. 3), or reaches up to her Heavenly
Bridegroom (fol. h, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016).
org/10 61302/IRRS2726 Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016).
https://doi.org/10.61302/IRRS2726. Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016).
https://doi.org/10.61302/IRRS2726. h, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016).
/10 61302/IRRS2726 Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016).
https://doi.org/10.61302/IRRS2726. The Trouble with “Female Sexuality” 66r; fig. 4). Jeffrey Hamburger’s extensive study of the manuscript
sites it in the context of feminine spirituality, arguing “[t]hat the Rothschild Canticles
was intended for a woman is virtually certain.”[10] Nevertheless, as Sarah Bromberg
points out, the manuscript also includes images of men as lovers of Christ, and male
readers would not have been excluded from identification with the Bride: “[a] male
viewer of the Rothschild Canticles might be . . . accustomed to using the visually
feminized symbol of the Sponsa in order to act out his own supplication in seeking
divine union.”[11] If the position of the Bride is that of female sexuality, then it is not
restricted to women. 6 6 Fig. 3. Bride and unicorn, Rothschild Canticles, Rhineland, c. 1300. New Haven, Yale Beinecke Rare Book
and Manuscript Library, MS 404, fol. 51r (Beinecke Rare Book & Manuscript Library: fair use policy) Fig. 3. Bride and unicorn, Rothschild Canticles, Rhineland, c. 1300. New Haven, Yale Beinecke Rare Book
and Manuscript Library, MS 404, fol. 51r (Beinecke Rare Book & Manuscript Library: fair use policy) 7 7 Fig. 4. Spiritual marriage, Rothschild Canticles, Rhineland, c. 1300. New Haven, Yale Beinecke Rare Book
and Manuscript Library, MS 404, fol. 66r (Beinecke Rare Book & Manuscript Library: fair use policy) Fig. 4. Spiritual marriage, Rothschild Canticles, Rhineland, c. 1300. New Haven, Yale Beinecke Rare Book
and Manuscript Library, MS 404, fol. 66r (Beinecke Rare Book & Manuscript Library: fair use policy) 8 8 Such positions are formed in networks of persons, places and material objects.[12]
The anchoress contemplating the crucifix in “The Wooing of our Lord” speaks a script
written for her by her spiritual director, gazes at a crucifix crafted by an artisan to a
model lent in a workshop, perhaps donated by a patron or relative.[13] Her cell, the
site of what Michelle Sauer calls “erotic enclosure,” was a social and material space
marked out by lay and clerical practices.[14] The anchoress may be the point at which
art meets eros, but this meeting is enabled by the network that provides and vivifies
the crucifix figure and gives the anchoress the space to contemplate it. Women’s art
was never exclusively so. Books made for specific elite patrons would also have been
accessible to their families and household intimates. Imagery in nunnery churches
would frequently have been visible to priests and visitors. Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016).
https://doi.org/10.61302/IRRS2726. The Trouble with “Female Sexuality” And some images made for
women, as Madeline Caviness famously argued, might be better regarded as having
been made against women: she read the sexual aspects of the illustrative
programme of the Hours of Jeanne d’Evreux as a coercive interpellation of the young
queen into wifely subordination, arguing that “the aggressive phallic forms … may
well have frightened the adolescent Jeanne.”[15] The book produces its reader as
sexual subject, but offers her no pleasures. As Caviness goes on to say, however, the intent does not fix the meaning, and we
must surmise a variety of responses: “I do not propose to substitute a monolithic
personal reading but to allow the work a multiplicity of affective possibilities that
might be accounted for by differences of mood, life stage, and gender in the readers
of this particular book; it not only may have looked different to Jeanne in her celibate
widowhood, but certainly must have done so to Charles V when he inherited it.”[16]
Paula Gerson confirms that women might take an active interest in sexual imagery,
arguing that a woman “not unlike Chaucer’s Wife of Bath” most likely commissioned
similar bawdy imagery in her Book of Hours because she liked it.[17] Gerson’s
identification of female agency in this manuscript is plausible, but it is harder to be
sure of the importance of specifically sexual response; the emphasis on “visual
attacks on male genitalia and buttocks” suggests that aggressive impulses were
mixed in with sexual ones.[18] All we can say is that sexualised imagery was
addressed to women, but how it affected their fears, pleasures and desires would
depend on a multitude of factors. As these examples show, images that have sexual content cannot necessarily be
assumed to have stimulated sexual responses. Indeed, this seems more typically to
be a negative relation. As Martha Easton has argued, “the most sexually explicit
images are found in religious spaces like churches, cathedrals, and devotional
manuscripts, and depending on the context, can be read as censorious rather than
celebratory of eroticism and love”; and my own previous study found that “[s]exual Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. 9 form, sexual content and sexual affect so frequently diverge in medieval art that they
must be assessed separately.”[19] Visual images of sexuality are often more
connotative than explicit. The Trouble with “Female Sexuality” The presence of a rabbit may instruct the viewer to read
erotic import into an otherwise innocuous conversation scene, because the animal’s
French name, “con,” puns on the word for female genitals.[20] Any sexual pleasure
from contemplating the organ at this double remove would be inextricably bound
up with the pleasure of successful decoding of the signifying chain.[21] Meanwhile,
apparently sexual images might mean something completely different; a late
fifteenth-century drawing of a couple embracing (fig. 5) is to be understood as
encoding an alchemical process. Sexually explicit images such as the exhibitionist
figures on churches are usually more grotesque than alluring. Many were probably
intended to be anti-erotic, a warning against sexual sin; others have been argued to
be fertility images or apotropaic charms.[22] form, sexual content and sexual affect so frequently diverge in medieval art that they
must be assessed separately.”[19] Visual images of sexuality are often more
connotative than explicit. The presence of a rabbit may instruct the viewer to read
erotic import into an otherwise innocuous conversation scene, because the animal’s
French name, “con,” puns on the word for female genitals.[20] Any sexual pleasure
from contemplating the organ at this double remove would be inextricably bound
up with the pleasure of successful decoding of the signifying chain.[21] Meanwhile,
apparently sexual images might mean something completely different; a late
fifteenth-century drawing of a couple embracing (fig. 5) is to be understood as
encoding an alchemical process. Sexually explicit images such as the exhibitionist
figures on churches are usually more grotesque than alluring. Many were probably
intended to be anti-erotic, a warning against sexual sin; others have been argued to
be fertility images or apotropaic charms.[22] Fig. 5. Alchemical process, Miscellaneous Treatises on Alchemy, England, 2nd half of the 15th century. London, British Library, MS Harley 2407, fol. 57v, detail (© British Library Board) Fig. 5. Alchemical process, Miscellaneous Treatises on Alchemy, England, 2nd half of the 15th century. London, British Library, MS Harley 2407, fol. 57v, detail (© British Library Board) Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. 10 Towards the end of the period it becomes easier to identify images that better fit
modern expectations of erotic art. h, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016).
org/10 61302/IRRS2726 The Trouble with “Female Sexuality” Paula Nuttall establishes that non-narrative
images of nude women, for the viewing pleasure either of men or mixed audiences,
were produced in northern art of the late fifteenth century. The major themes seem
to have been voyeuristic gazing on women as they bathe, and male enthrallment to
female sexual allure; they may be combined, as in the picture known as “The Love
Charm” (fig. 6).[23] But this of course says nothing very directly about female
sexuality itself, though it does show what kinds of representations of women’s bodies
were desirable to some viewers. Viewing pleasure, however, is rarely pure. Van Eyck’s
lost picture of bathing women, for example, is known only from this description of its
content as: women of uncommon beauty emerging from the bath, the more intimate
parts of the body being with excellent modesty veiled in fine linen, and of one
of them he has shown only the face and breast but has then represented the
hind parts of her body in a mirror painted on the wall opposite, so you may see
her back as well as her breast. In the same picture there is a lantern in the
bath chamber, just like one lit, and an old woman seemingly sweating, a
puppy lapping up water, and also horses, minute figures of men, mountains,
groves, hamlets and castles, carried out with such skill you would believe one
was fifty miles distant from another.[24] women of uncommon beauty emerging from the bath, the more intimate
parts of the body being with excellent modesty veiled in fine linen, and of one
of them he has shown only the face and breast but has then represented the
hind parts of her body in a mirror painted on the wall opposite, so you may see
her back as well as her breast. In the same picture there is a lantern in the
bath chamber, just like one lit, and an old woman seemingly sweating, a
puppy lapping up water, and also horses, minute figures of men, mountains,
groves, hamlets and castles, carried out with such skill you would believe one
was fifty miles distant from another.[24] 11 11 Fig. 6. The Love Charm, oil on panel, Germany or Lower Rhine, 1470–1480. Leipzig, Museum der
Bildenden Künste (Public domain image) Fig. 6. The Love Charm, oil on panel, Germany or Lower Rhine, 1470–1480. h, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016).
org/10 61302/IRRS2726 Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016).
https://doi.org/10.61302/IRRS2726. The Trouble with “Female Sexuality” Leipzig, Museum der
Bildenden Künste (Public domain image) 12 Fig. 7. Vulva figure depicted as a pilgrim with pilgrim’s staff and rosary, lead tin, Arnemuiden, the
Netherlands, 1425–1475. Family Van Beuningen, Langbroek, Inv.nr. 4462 [cat. HP3, afb. 3026] (photo:
Medieval Badges Foundation) Fig. 7. Vulva figure depicted as a pilgrim with pilgrim’s staff and rosary, lead tin, Arnemuiden, the
Netherlands, 1425–1475. Family Van Beuningen, Langbroek, Inv.nr. 4462 [cat. HP3, afb. 3026] (photo:
Medieval Badges Foundation) This account suggests that enjoyment of this picture’s painterly playfulness and
verisimilitude was inseparable from enjoyment of its images of female flesh. It
rehearses a process of viewing that registers bodies, textures, surfaces, light and
shade as problems of representation to which an elegant solution has been found. A
patron’s pleasure in such art would surely also include an element of pride in the
ownership of such a prestigious and innovative piece. Sexuality is inextricably bound
up with other pleasures. I would argue that sexuality is not absent from medieval art, but that there are
reasons why it is hard to find. In a variety of genres, media and motifs, sexuality is
shown to be impersonal: disembodied, dispersed, alienated and alienating. It is not
securely attached to gendered bodies or gendered subjectivities; it continually
exceeds, attacks or distorts the entity that experiences it. One example of such a Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. 13 representation is the corpus of sexual badges, featuring male and/or female sexual
organs, usually in motion. The meanings of the badges are obscure, though they are
plainly genital in form, and most critics agree that they are not erotically enticing. One of the best-known, the pilgrim-vulva (fig. 7), has been interpreted as a
misogynist satire on women pilgrims.[25] Drawing on the deep-rooted division
between the good woman in her household and the bad woman wandering the
world, these badges say that a female pilgrim, identifiable here by her staff, hat and
rosary, is nothing but a vulva on legs. To depict a person in this reductive fashion, as
only a gaping organ, is to register desire as a force that unbalances the self, makes
one nothing but sexuality. The same goes for the exhibitionist images on churches,
also depicting both male and female organs, which share some forms with the
badges. The Trouble with “Female Sexuality” Such an image, then, might convey the subjective experience of living in a
sexed body; it is an image of the perceptual distortions of desire, working on similar
principles to the modern image of the sensory homunculus (fig. 8), which maps the
body according to the sensitivity of each part, producing a representation more
truthful to the subjective experience of being in the body than to its outward
appearance. To recognise oneself depicted in such an image, repellent and reductive
as it is, would be a profoundly alienating experience in which identification would
alternate with disavowal. representation is the corpus of sexual badges, featuring male and/or female sexual
organs, usually in motion. The meanings of the badges are obscure, though they are
plainly genital in form, and most critics agree that they are not erotically enticing. One of the best-known, the pilgrim-vulva (fig. 7), has been interpreted as a
misogynist satire on women pilgrims.[25] Drawing on the deep-rooted division
between the good woman in her household and the bad woman wandering the
world, these badges say that a female pilgrim, identifiable here by her staff, hat and
rosary, is nothing but a vulva on legs. To depict a person in this reductive fashion, as
only a gaping organ, is to register desire as a force that unbalances the self, makes
one nothing but sexuality. The same goes for the exhibitionist images on churches,
also depicting both male and female organs, which share some forms with the
badges. Such an image, then, might convey the subjective experience of living in a
sexed body; it is an image of the perceptual distortions of desire, working on similar
principles to the modern image of the sensory homunculus (fig. 8), which maps the
body according to the sensitivity of each part, producing a representation more
truthful to the subjective experience of being in the body than to its outward
appearance. To recognise oneself depicted in such an image, repellent and reductive
as it is, would be a profoundly alienating experience in which identification would
alternate with disavowal. Fig. 8. Sensory Homunculus. This is all over the web with no obvious ownership. Fig. 8. Sensory Homunculus. This is all over the web with no obvious ownership. Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016).
https://doi.org/10.61302/IRRS2726. The Trouble with “Female Sexuality” Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. 14 Fig. 9. Adam and Eve, Cædmon Manuscript, England, c. 1000. Bodleian Library, Oxford, MS Junius 11, p. 11
(© Oxford, Bodleian Library) Fig. 9. Adam and Eve, Cædmon Manuscript, England, c. 1000. Bodleian Library, Oxford, MS Junius 11, p. 11
(© Oxford, Bodleian Library) Anne Marie Rasmussen points out how often the genital badges are images of
mobility: they walk, they ride, they fly, and some even crew ships. She connects them
with comic stories about genitalia which leave their owners to wander the world: a
penis attempts to hide in a nunnery, or a stray vagina is mistaken for a toad.[26] The Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. 15 sexual organ, that is, is detachable from the person and from gender: it is imagined
as a force which wanders the world under its own steam. And the badges
themselves, of course, are mobile, in that, pinned to people’s clothing, they moved
around the world, their meanings no doubt shifting as they appeared in different
material and social spaces. This means that they may well not be firmly gendered: we
have no information at all about who wore them and when, but there is no reason to
suppose that they had to be gender-appropriate to their wearers. If “female sexuality”
is located in a badge of a vagina-pilgrim, then female sexuality is a quality which is
mobile, detachable, not owned by female persons. A motif such as the phallus tree,
shared by badges and other media, has a similar concept of sexuality as something
which is out there in the world, only temporarily appropriated by individuals,
something which one might pick off a tree. Such images show coherent personhood and sexuality to be mutually exclusive, and
desire as a disruptive force, a perception that is quite consistent with the Christian
theorisation of the condition of post-lapsarian humanity. As St Augustine explained
the effects of the Fall: “They experienced an unprecedented movement of their own
disobedient flesh as punishment in kind, as it were, for their own disobedience. Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016).
https://doi.org/10.61302/IRRS2726. The Trouble with “Female Sexuality” The
soul, in fact, delighting now in its own freedom to do wickedness and scorning to
serve God, was stripped of the former subjection of the body, and because it had
wilfully deserted its own higher master, no longer kept its lower servant subject to its
will.”[27] The experience of desire is an experience of splitting: body and soul, God
and humanity, are recognised as distinct through the operation of desire. A human
subject experiencing desire is registering its fallenness, its distance from its
prelapsarian perfection, its non-self-identity. One does not have a sexuality because
one is not one in these conditions, but a disordered collection of jostling entities. The
prelapsarian Adam and Eve in the Anglo-Saxon Junius MS (fig. 9) anticipate this
moment at which sexual awakening is registered at the cost of personal
disintegration. They stand fully naked and barely distinguishable, with smooth
groins, but their sexual parts are externalised to the genital-like forms of the plants
that each grasps firmly. In the visual arts, this idea is not, however, limited to the
theological or moral register, but is expressed also in popular and comic art and
performance. Images of sexuality informed by such perspectives are likely to be
uncomfortably placed in the category of “female sexuality,” both because this
Augustinian model imagines female desire to be exactly analogous to male desire,
and because sexuality is so disruptive of the subject that no-one can lay claim to one
of their own. In such images and texts anything we might want to call “female
sexuality” is already fraught, complicated and disrupted; there is no coherent
package of desires, behaviours and relations that belongs to female subjects. 16 References References
1
Karma Lochrie, Heterosyncracies: Female Sexuality when Normal Wasn’t
(Minneapolis: University of Minnesota Press, 2005), xv. 2
Hans Belting, An Anthropology of Images: Picture, Medium, Body, trans. Thomas
Dunlap (Princeton: Princeton University Press, 2011), 37. https://doi.org/10.1515/9781400839780
3
Ancrene Wisse: A Corrected Edition of the Text in Cambridge, Corpus Christi
College, MS 402, ed. Bella Millett, Early English Text Society, Original Series 325
(Oxford: Oxford University Press, 2005), 121; Ruth Mazo Karras, Sexuality in
Medieval Europe: Doing unto Others (New York: Routledge, 2005), 79; Christine
de Pizan, The Book of the City of Ladies, trans. Rosalind Brown-Grant
(Harmondsworth: Penguin, 1999), 144; Kathryn Gravdal, Ravishing Maidens:
Writing Rape in Old French Literature and Law (Philadelphia: University of
Pennsylvania Press, 1991), 111. Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016).
https://doi.org/10.61302/IRRS2726. The Trouble with “Female Sexuality” https://doi.org/10.9783/9780812200331
4
Lochrie, Heterosyncracies, xiv-xv. 5
The Book of Margery Kempe, ed. Barry Windeatt (Cambridge: D.S. Brewer, 2000),
332, 62, 196. 6
Jacqueline Murray, “Twice Marginal and Twice Invisible: Lesbians in the Middle
Ages,” in Handbook of Medieval Sexuality, ed. Vern L. Bullough and James A. Brundage (New York: Garland, 2000), 191-222. 7
“Sex without sex” is the theme of a special issue of the Journal of the History of
Sexuality 19, no. 1 (2010), ed. Sally N. Vaughn and Christina Christoforatou. 8
Þe Wohunge of ure Lauerd, ed. W. Meredith Thompson, EETS Original Series 241
(London: Oxford University Press, 1958), ll. 590-602; my translation. 9
Sara Lipton, “‘The Sweet Lean of his Head’: Writing about Looking at the Crucifix
in the High Middle Ages,” Speculum 80, no. 4 (2005): 1172-1208. https://doi.org/10.1017/S003871340000138X
10
Jeffrey F. Hamburger, The Rothschild Canticles: Art and Mysticism in Flanders
and the Rhineland circa 1300 (New Haven: Yale University Press, 1990), 3. 11
Sarah Bromberg, “Gendered and Ungendered Readings of the Rothschild
Canticles,” Different Visions: A Journal of New Perspectives on Medieval Art 1
(2008), https://doi.org/10.61302/PMSR4045 Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016). https://doi.org/10.61302/IRRS2726. 17 12
The concept of network here is indebted to Bruno Latour, Reassembling the
Social: An Introduction to Actor-Network Theory (Oxford: Oxford University Press,
2007). 13
I have previously discussed the scripting of the meditation in Sarah Salih,
“Transvestism in the Anchorhold,” in The Milieu and Context of the Wohunge
Group, ed. Susannah M. Chewning (Cardiff: University of Wales Press, 2009),
148-64. 14
Michelle Sauer, “Uncovering Difference: Encoded Homoerotic Anxiety within the
Christian Eremetic Tradition in Medieval England,” Journal of the History of
Sexuality 19, no. 1 (2010), 133-52, at 143. https://doi.org/10.1353/sex.0.0075
15
Madeline H. Caviness, “Patron or Matron? A Capetian Bride and a Vade Mecum
for Her Marriage Bed,”Speculum 68, no. 2 (1993), 333-62, at 355. https://doi.org/10.2307/2864556
16
Caviness, “Patron or Matron,” 356. 17
Paula Gerson, “Margins for Eros,” Romance Languages Annual 5 (1993): 47-53, at
50. 18
Gerson, “Margins for Eros,” 52. 19
Martha Easton, “‘Was it Good for You Too?’ Medieval Erotic Art and it Audiences,”
Different Visions: A Journal of New Perspectives on Medieval Art 1 (2008),
https://doi.org/10.61302/BUIO3522, 14; Sarah Salih, “Erotica,” in A Cultural History
of Sexuality in the Middle Ages,ed. Ruth Evans(Oxford: Berg, 2010), 181-212, 186. Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016).
https://doi.org/10.61302/IRRS2726. h, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016).
org/10 61302/IRRS2726 Sarah Salih, “The Trouble with ‘Female Sexuality,’” Different Visions: New Perspectives on Medieval Art 5 (2016).
https://doi.org/10.61302/IRRS2726. The Trouble with “Female Sexuality” https://doi.org/10.5040/9781350049673-ch-009
20
Michael Camille, The Medieval Art of Love: Objects and Subjects of Desire (New
York: Harry N. Abrams, 1998), 96. 21
A point previously made in Salih, “Erotica,” 189. 22
Anthony Weir and James Jerman, Images of Lust: Sexual Carvings on Medieval
Churches (London: Routledge, 1999); Marian Bleeke, “Sheelas, Sex, and
Significance in Romanesque Sculpture: The Kilpeck Corbel Series,” Studies in
Iconography 26 (2005): 1-26; Eamonn Kelly, “Irish Sheela-na-gigs and Related
Figures with Reference to the Collections of the National Museums of Ireland,” in
Medieval Obscenities, ed. Nicola McDonald (Woodbridge: York Medieval Press,
2006), 124-37. 18 23
Paula Nuttall, “Reconsidering the Nude: Northern Tradition and Venetian
Invention,” The Meanings of Nudity in Medieval Art, ed. Sherry C.M. Lindquist
(Farnham, UK; Burlington, VT: Ashgate, 2012), 299-318. https://doi.org/10.4324/9781315085975-12
24
Quoted in Keith Christiansen, “The View from Italy,” in From Van Eyck to Bruegel:
Early Netherlandish Painting in the Metropolitan Museum of Art, ed. Maryan
Wynn Ainsworth and Keith Christiansen (New York: Metropolitan Museum of Art,
1998), 39-62, 39. 25
Malcolm Jones, The Secret Middle Ages: Discovering the Real Medieval World
(Stroud: Sutton, 2002), 256. 26
Anne Marie Rasmussen, Wandering Genitalia: Sexuality and the Body in
German Culture between the Late Middle Ages and Early Modernity (London:
King’s College London Medieval Studies, 2009), 8-9. 27
Augustine of Hippo, The City of God Against the Pagans, 7 vols, trans. George E. McCracken, Philip Levine, William M. Green, Loeb Classical Library (London:
Heinemann; Cambridge Massachusetts: Harvard University Press, 1966), Book 13,
ch. 13. 23 19
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Toxicological Study of<i>Ocimum sanctum</i>Linn Leaves: Hematological, Biochemical, and Histopathological Studies
|
Journal of toxicology
| 2,014
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cc-by
| 5,807
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Hindawi Publishing Corporation
Journal of Toxicology
Volume 2014, Article ID 135654, 9 pages
http://dx.doi.org/10.1155/2014/135654 Hindawi Publishing Corporation
Journal of Toxicology
Volume 2014, Article ID 135654, 9 pages
http://dx.doi.org/10.1155/2014/135654 Hindawi Publishing Corporation
Journal of Toxicology
Volume 2014, Article ID 135654, 9 pages
http://dx.doi.org/10.1155/2014/135654 Hindawi Publishing Corporation
Journal of Toxicology
Volume 2014, Article ID 135654, 9 pages
http://dx.doi.org/10.1155/2014/135654 M. K. Gautam and R. K. Goel Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India Correspondence should be addressed to R. K. Goel; rkgoelbhu50@gmail.com Received 31 July 2013; Revised 28 November 2013; Accepted 28 November 2013; Published 29 January 2014 Academic Editor: Lucio Guido Costa Copyright © 2014 M. K. Gautam and R. K. Goel. This is an open access article distributed under the Creative Commons Attr
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properl Copyright © 2014 M. K. Gautam and R. K. Goel. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The present study was aimed to study the acute and subacute toxicity studies with orally administered 50% ethanolic leaves extract
of Ocimum sanctum Linn (OSE). In acute toxicity tests, four groups of mice (𝑛= 6/group/sex) were orally treated with doses of
200, 600, and 2000 mg/kg, and general behavior, adverse effects, and mortality were recorded for up to 14 days. In subacute toxicity
study, rats received OSE by gavage at the doses of 200, 400, and 800 mg/kg/day (𝑛= 6/group/sex) for 28 days, and biochemical,
hematological, and histopathological changes in tissues (liver, kidney, spleen, heart, and testis/ovary) were determined. OSE did
not produce any hazardous symptoms or death and CNS and ANS toxicities in the acute toxicity test. Subacute treatment with OSE
did not show any change in body weight, food and water consumption, and hematological and biochemical profiles. In addition, no
change was observed both in macroscopic and microscopic aspects of vital organs in rats. Our result showed that Ocimum sanctum
extract could be safe for human use. 1. Introduction Ocimum sanctum Linn (Labiatae), known as holy basil,
is a commonly used home remedy and has been advocated
for various ailments like cold, fever, dysentery, hemorrhage
and dyspepsia, glucoma, cataract, chronic conjunctivitis,
and other painful eye diseases, as well as gastric and
hepatic disorders in indigenous system of medicine [5]. The plant is endowed with a variety of pharmacological
properties including antistress, antifertility, immunoregu-
latory, hypoglycemic, antibacterial, antifungal, antiinflam-
matory, anti-carcinogenic, antioxidant, and cyclooxygenase
inhibitory [6]. OS has been reported to show gastroduo-
denal ulcer protective, antisecretory, and gastric mucosal
defense enhancing activities [7]. The leaves of O. sanctum
(OS) contain a volatile oil composed of limonene, bor-
neol, copaene, caryophyllene, and elemol, phenolic com-
pounds (rosmarinic acid, apigenin, cirsimaritin, and isothy-
musin), flavonoids (orientin and vicenin), and aromatic
compounds (methyl chavicol and methyl eugenol) [6]. Eugenol forms the major active constituent of OS, even
though other minor constituents like fixed oils and flavones
have also been reported to have pharmacological activities
[5]. Plants have always been an important source of drugs. A large
number of the world’s populations, especially in developing
countries, depend upon medicinal plants as an alternative
and complimentary drugs therapy for various ailments. Some
of the most common practices involve the use of crude plant
extracts, which may contain a broad diversity of molecules
with often unknown biological effects [1]. Since the medicinal
plants are being used indiscriminately without notifying to
their possible unhealthy or toxic effects, the World Health
Organization has recommended that traditional plants used
for the treatment of diseases need further scientific investiga-
tion on their toxic side effects [2]. Plants produce bioactive
compounds which act as defense mechanisms against any
disease, and at the same time, may be toxic in nature [3]. However, the general acceptability of herbal medicines has
been limited by a lack of defined chemical characterization,
dose regimen, and adequate toxicity data to evaluate their
safety [4]. Therefore, it has become essential to assess the
safety of plants used for medicinal purposes for possible
toxicity. 2 Journal of Toxicology 2
Journal of Toxicology
(a)
(b)
(c)
(d)
Figure 1: Histology of liver (H&E, 100x) of control and OSE-treated animals. (a) Section of liver from control animals revealed normal
architecture and hepatic cells with granulated cytoplasm; ((b), (c), and (d)) liver from OSE (200, 400, and 800 mg/kg)-treated animals
exhibited normal architecture of hepatocytes and hepatic cells with granulated cytoplasm. 1. Introduction (a) (b) (b) (a) (d) (c) (d) (c) Figure 1: Histology of liver (H&E, 100x) of control and OSE-treated animals. (a) Section of liver from control animals revealed normal
architecture and hepatic cells with granulated cytoplasm; ((b), (c), and (d)) liver from OSE (200, 400, and 800 mg/kg)-treated animals
exhibited normal architecture of hepatocytes and hepatic cells with granulated cytoplasm. As to the best of our knowledge, there is no reference
about the safe dosage of Ocimum sanctum Linn in traditional
medicine so it was thought worthwhile to do the acute toxicity
(mortality and CNS/ANS toxicities) in mice and subacute
toxicity (biochemical, hematological, and histopathological)
in rats. Hindu University) were collected during October-December
and identified with the standard sample preserved in the
Department of Dravyaguna, Institute of Medical Sciences,
Banaras Hindu University, Varanasi. 50% ethanolic extract of
OS (OSE) was prepared by adding 500 g of dried, crushed,
and powdered leaves of OS in 1000 mL of 50% ethanol in
a round bottom flask and was kept at room temperature
for 3 days in shade. The extract was filtered and the above
procedure was repeated twice. The extract filtrate so obtained
was pooled and evaporated on water bath till it dried. The
yield of OSE was about 5.00% (w/w). 2.2. Plant Material and Preparation of Extract. The leaves
of Ocimum sanctum (OS) (Ayurvedic Gardens, Banaras 2. Materials and Methods 2.1. Experimental Animals. Inbred Charles-Foster albino rats
(160–180 g) and Swiss albino mice (20–25 g) of either sex
were obtained from the central animal house of the Institute
of Medical Sciences, Banaras Hindu University, Varanasi. They were kept in the departmental animal house at 26
± 20∘C and relative humidity of 44–56%, with light and
dark cycles of 10 and 14 h, respectively, for one week before
and during the experiments. Animals were provided with
standard rodent pellet diet (Pashu Aahar Vihar, Ramnagar,
Varanasi) and water ad libitum. “Principles of laboratory
animal care” (NIH publication number 82-23, revised 1985)
guidelines were followed. Approval from the Central Animal
Ethical Committee of the University was taken prior to the
experimental work (Notification number-Dean/2010-11/173
dated 23.07.2010). 2.3. Acute Toxicity Study. Adult Swiss albino mice of either
sex, weighing between 20 and 25 g, fasted overnight and
were used for acute toxicity study, as per the Organization
for Economic Co-Operation and Development (OECD 423)
guideline [8]. Four groups of mice (𝑛= 6) of both sexes
was fasted overnight. The first control group mice received
0.5% carboxymethyl cellulose (CMC) suspension in distilled
water while the other three groups received OSE suspended
in 0.5% CMC at doses of 200, 600, and 2000 mg/kg. The above
doses were selected on the basis of our previous reported
work on OSE where 400 mg/kg was found to have good
ulcer protective effects [7]. Animals were observed closely
for first 4 hours, for any toxicity manifestation, like increased
motor activity, salivation, convulsion, coma, and death. Sub-
sequently observations were made at regular intervals for 2.3. Acute Toxicity Study. Adult Swiss albino mice of either
sex, weighing between 20 and 25 g, fasted overnight and
were used for acute toxicity study, as per the Organization
for Economic Co-Operation and Development (OECD 423)
guideline [8]. Four groups of mice (𝑛= 6) of both sexes
was fasted overnight. The first control group mice received
0.5% carboxymethyl cellulose (CMC) suspension in distilled
water while the other three groups received OSE suspended
in 0.5% CMC at doses of 200, 600, and 2000 mg/kg. The above
doses were selected on the basis of our previous reported
work on OSE where 400 mg/kg was found to have good
ulcer protective effects [7]. Animals were observed closely
for first 4 hours, for any toxicity manifestation, like increased
motor activity, salivation, convulsion, coma, and death. 2. Materials and Methods Sub-
sequently observations were made at regular intervals for 2.2. Plant Material and Preparation of Extract. The leaves
of Ocimum sanctum (OS) (Ayurvedic Gardens, Banaras 3 3 Journal of Toxicology (a)
(b)
(c)
(d)
Figure 2: Histology of kidney (H&E, 100x) of control and OSE-treated animals. (a) Section of kidney from control animal showed normal
size of glomeruli with normal tubules; ((b), (c), and (d)) kidney from OSE (200, 400, and 800 mg/kg)-treated animals exhibit normal size of
glomeruli with normal tubules. (b) (a) (a) (b) (d) (c) (d) (c) Figure 2: Histology of kidney (H&E, 100x) of control and OSE-treated animals. (a) Section of kidney from control animal showed normal
size of glomeruli with normal tubules; ((b), (c), and (d)) kidney from OSE (200, 400, and 800 mg/kg)-treated animals exhibit normal size of
glomeruli with normal tubules. 24 h. The animals were under further investigation up to a
period of 14 days and the number of mice that died within
the study period was noted [9]. 2.4.1. Haematological Parameters. Haemoglobin, total leuko-
cyte count, and differential leukocyte count (polymorph,
lymphocyte, and eosinophil) [11] were determined in control
and OSE-treated groups. 2.4. Subacute Toxicity Study. The repeated doses (28 days) for
oral toxicity studies were carried out in rats according to the
OECD test guideline 407 [10]. Rats were divided randomly
into 4 groups of 6 animals each (3 males and 3 females). After
an overnight fast, animals in group I received 0.5% CMC
suspension (control group), whereas rats in groups II, III, and
IV (test groups) received OSE suspended in 0.5% CMC at the
doses of 200, 400, and 800 mg/kg body weight, respectively. Doses of OSE and CMC were administered daily by oral
gavage in the volume of 10 mL/kg body weight, once daily for
28 consecutive days. The animals were observed daily for any
abnormal clinical signs and death during the study period. Body weights were measured and recorded at the beginning
and then after every week of the experiment. At the end
of the study, all animals fasted overnight (water ad libitum)
and, on 29th day, the animals were weighed. Blood was
collected from retroorbital technique with or without EDTA
for haematological and biochemical analysis, respectively. The animals were sacrificed with an overdose of ether and
other body organs were taken out for detailed weight and
histopathological changes. 2.4.2. Biochemical Estimations. 2. Materials and Methods The serum was carefully aspi-
rated with a Pasteur pipette into sample bottles for the various
biochemical assays. Assay kits (Span diagnostic reagent kit
and Precichem diagnostic kit) were employed for aspartate
transaminase (AST), alanine transaminase (ALT), alkaline
phosphate (ALP), creatinine, blood glucose, total protein, and
total cholesterol and bilirubin analysis was determined in the
serum following the procedure described in the kits. 2.4.2. Biochemical Estimations. The serum was carefully aspi-
rated with a Pasteur pipette into sample bottles for the various
biochemical assays. Assay kits (Span diagnostic reagent kit
and Precichem diagnostic kit) were employed for aspartate
transaminase (AST), alanine transaminase (ALT), alkaline
phosphate (ALP), creatinine, blood glucose, total protein, and
total cholesterol and bilirubin analysis was determined in the
serum following the procedure described in the kits. 2.4.3. Organs Weight and Histology. The rats were quickly dis-
sected and the liver, kidneys, stomach, spleen, lung, heart, and
testis or ovary were excised and weighed. The specimens for
histopathology were fixed in 10% neutral, buffered formalin
for 18 h at 4∘C. 3-4 𝜇m in thickness of each specimen of liver,
kidney, heart, spleen, and testis/ovary was cut and stained
with hematoxylin and eosin stain following the standard
laboratory procedures. The stained sections were examined
under microscope for any cellular damage or change in
morphology of that particular tissue. Journal of Toxicology (a)
(b)
(c)
(d)
Figure 3: Histology of heart (H&E, 100x) of control and OSE-treated animals. (a) Section of heart from control animal showed normal muscle
fibers with acidophilic cytoplasm and centrally located nuclei; ((b), (c), and (d)) heart from OSE (200, 400, and 800 mg/kg)-treated animals
exhibit normal muscle fibers with acidophilic cytoplasm and centrally located nuclei. (b) (a) (a) (b) (d) (c) (d) (c) Figure 3: Histology of heart (H&E, 100x) of control and OSE-treated animals. (a) Section of heart from control animal showed normal muscle
fibers with acidophilic cytoplasm and centrally located nuclei; ((b), (c), and (d)) heart from OSE (200, 400, and 800 mg/kg)-treated animals
exhibit normal muscle fibers with acidophilic cytoplasm and centrally located nuclei. control group after 28 days of study period in rats (Tables 1
and 2). 2.5. Statistical Analysis. Statistical comparison was per-
formed using one way analysis of variance (ANOVA) fol-
lowed by Dunnett’s test for multiple comparisons. All statisti-
cal analysis was performed using SPSS statistical version 16.0
software package (SPSS Inc., USA). 3.2.3. Hematological Studies. 2. Materials and Methods Hematological parameters like
mean haemoglobin content, WBC, RBC, and differential cell
counts were not significantly different with OSE-treated rats
from control group (Table 3). 3. Result 3.1. Acute Toxicity Study. The limit dose of 2000 mg/kg did
not cause death or any toxic signs in treated male and female
mice. All six mice were normal throughout the study and
survived until the end of the 14-day experiment period. 3.2.4. Biochemical Analysis. Biochemical parameters for liver
and kidney function test like aspartate transaminase (AST),
alanine transaminase (ALT), alkaline phosphatase (ALP),
creatinine, albumin, blood glucose, total protein, total choles-
terol, and bilirubin did not show any difference with the above
doses of OSE compared to control group (Table 3). 3.2. Subacute Toxicity Study OSE 400
160.6 ± 5.38
162.5 ± 4.22
171.2 ± 6.39
188.8 ± 3.75
206.3 ± 5.95
222.6 ± 5.21
OSE 800
163.1 ± 5.58
166.3 ± 4.97
173.8 ± 3.75
190.3 ± 3.71
204.4 ± 3.46
223.8 ± 2.45
Values are expressed as mean ± SEM of 6 rats in each group. (a)
(b)
(c)
(d)
Figure 4: Histology of spleen (H&E, 100x) of control and OSE-treated animals. (a) Section of spleen from control animal showed normal
granular hemosiderin pigment predominantly within macrophages in the red pulp; ((b), (c), and (d)) spleen from OSE (200, 400, and
800 mg/kg)-treated animals exhibit normal hemosiderin pigment predominantly within macrophages in the red pulp with normal structure. (b) (a) (b) (a) (d) (c) (d) (c) Figure 4: Histology of spleen (H&E, 100x) of control and OSE-treated animals. (a) Section of spleen from control animal showed normal
granular hemosiderin pigment predominantly within macrophages in the red pulp; ((b), (c), and (d)) spleen from OSE (200, 400, and
800 mg/kg)-treated animals exhibit normal hemosiderin pigment predominantly within macrophages in the red pulp with normal structure. 3.2. Subacute Toxicity Study 3.2.1. General Observations. Oral administration of OSE at
doses of 200, 400, or 800 mg/kg body weight daily for 28 day,
did not produce any mortality. All the treated and control
mice were normal throughout the study. The animals did not
show any changes in general behavior or other physiological
activities. 3.2.5. Organs Weight and Histology. The organs like liver,
kidney, heart, spleen, and testis or ovary isolated in various
groups did not reveal any abnormalities in their gross exam-
inations and difference in their mean weights both in treated
and control groups (Table 4). The histological studies with
liver, spleen, kidney, heart, and testis/ovary did not reveal any
pathological changes after treatment even with higher dose of
800 mg dose of OSE when administered for 28 days (Figures
1, 2, 3, 4, 5, and 6). 3.2.5. Organs Weight and Histology. The organs like liver,
kidney, heart, spleen, and testis or ovary isolated in various
groups did not reveal any abnormalities in their gross exam-
inations and difference in their mean weights both in treated
and control groups (Table 4). The histological studies with
liver, spleen, kidney, heart, and testis/ovary did not reveal any
pathological changes after treatment even with higher dose of
800 mg dose of OSE when administered for 28 days (Figures
1, 2, 3, 4, 5, and 6). 3.2.2. Physical Parameters. Little or no change was observed
in body weight, food consumption, and water intake in OSE
(200, 400, and 800 mg/kg)-treated groups compared with 5 Journal of Toxicology Table 1: Effect on body weight after 28 days oral administration of OSE. Table 1: Effect on body weight after 28 days oral administration of OSE. ft
Oral treatment (mg/kg, od)
Body weight (g)
0 day
3 days
7 days
14 days
21 days
28 days
Control 0.5% CMC
162.5 ± 6.34
166.3 ± 5.32
173.4 ± 5.46
185.0 ± 5.09
199.4 ± 5.78
215.0 ± 5.00
OSE 200
164.4 ± 6.01
165.0 ± 6.12
171.3 ± 4.20
186.9 ± 3.53
203.1 ± 7.49
220.0 ± 4.72
OSE 400
160.6 ± 5.38
162.5 ± 4.22
171.2 ± 6.39
188.8 ± 3.75
206.3 ± 5.95
222.6 ± 5.21
OSE 800
163.1 ± 5.58
166.3 ± 4.97
173.8 ± 3.75
190.3 ± 3.71
204.4 ± 3.46
223.8 ± 2.45
Values are expressed as mean ± SEM of 6 rats in each group. 4. Discussion and safe in mice. Subacute toxicity study in rats with 200, 400,
and 800 mg/kg of OSE when administered for 28 days did
not produce any mortality, change in food and water intake,
body and organ weights, or histopathological changes in the
organs like liver, kidney, spleen, heart, testis or ovary further
strengthen the safety profile of OSE. The administration of herbal preparations without any stan-
dard dosage, coupled with a scarcity of adequate scientific
studies on their safety, has raised concerns regarding their
toxicity [12]. Recently we reported teratological effects of
Asparagus racemosus in rats which have been advocated
in indigenous system of medicine during pregnancy and
lactation [13], which thus indicated that herbal drugs are not
safe as thought otherwise. To the best of our knowledge, there
are no published studies on Ocimum sanctum toxicological
profile following subacute exposure. In oral acute toxicity
study, as high dose of OSE at 2000 mg/kg did not show any
observable toxic effects in the mice in terms of any deaths
or abnormal symptoms which points to its being nontoxic i
Subacute studies in rats did not show any change in
hematological, liver functions and spleen with 800 mg/kg
OSE when administered for 28 days. The hematopoietic
system/bone marrow is one of the most sensitive targets for
toxic compounds and an important index of physiological
and pathological status in man and animal [14]. Analysis of
blood parameters is relevant to risk evaluation as the changes
in the hematological system have a higher predictive value
for human toxicity, when the data are translated from animal 6 Journal of Toxicology 6 Table 2: Effect on food intake and water intake after 28 days of the oral administration of OSE. 4. Discussion Oral treatment (mg/kg, od)
Food intake (g/day)
Water intake (mL/day)
0 day
3 days
7 days
14 days
21 days
28 days
0 day
3 days
7 days
14 days
21 days
28 days
Control 0.5% CMC
11.4 ± 0.70
11.63 ± 0.55
12.2 ± 0.61
12.9 ± 0.66
13.9 ± 0.79
15.2 ± 0.90
12.3 ± 0.46
12.5 ± 0.62
13.1 ± 0.74
14.2 ± 0.63
14.6 ± 0.76
15.5 ± 0.99
OSE 200
11.5 ± 0.77
12.2 ± 0.69
12.7 ± 0.59
13.8 ± 0.76
14.5 ± 0.67
15.6 ± 0.82
13.1 ± 0.5
13.7 ± 0.58
14.1 ± 0.76
14.9 ± 0.74
16.0 ± 0.85
16.4 ± 0.91
OSE 400
11.6 ± 0.63
12.1 ± 0.49
13.0 ± 0.57
13.7 ± 0.65
14.5 ± 0.78
15.9 ± 0.86
12.9 ± 0.55
13.7 ± 0.65
14.1 ± 0.67
15.2 ± 0.77
16.1 ± 0.91
17.2 ± 0.98
OSE 800
10.9 ± 0.27
11.4 ± 0.44
13.1 ± 0.77
13.3 ± 0.99
14.5 ± 0.94
15.2 ± 1.10
13.3 ± 0.53
13.6 ± 0.60
14.3 ± 0.72
15.2 ± 0.76
16.1 ± 0.83
16.1 ± 0.95
Values are expressed as mean ± SEM of 6 rats in each group. Table 2: Effect on food intake and water intake after 28 days of the oral administration of OSE. 4. Discussion Oral treatment (mg/kg, od)
Food intake (g/day)
Water intake (mL/day)
0 day
3 days
7 days
14 days
21 days
28 days
0 day
3 days
7 days
14 days
21 days
28 days
Control 0.5% CMC
11.4 ± 0.70
11.63 ± 0.55
12.2 ± 0.61
12.9 ± 0.66
13.9 ± 0.79
15.2 ± 0.90
12.3 ± 0.46
12.5 ± 0.62
13.1 ± 0.74
14.2 ± 0.63
14.6 ± 0.76
15.5 ± 0.99
OSE 200
11.5 ± 0.77
12.2 ± 0.69
12.7 ± 0.59
13.8 ± 0.76
14.5 ± 0.67
15.6 ± 0.82
13.1 ± 0.5
13.7 ± 0.58
14.1 ± 0.76
14.9 ± 0.74
16.0 ± 0.85
16.4 ± 0.91
OSE 400
11.6 ± 0.63
12.1 ± 0.49
13.0 ± 0.57
13.7 ± 0.65
14.5 ± 0.78
15.9 ± 0.86
12.9 ± 0.55
13.7 ± 0.65
14.1 ± 0.67
15.2 ± 0.77
16.1 ± 0.91
17.2 ± 0.98
OSE 800
10.9 ± 0.27
11.4 ± 0.44
13.1 ± 0.77
13.3 ± 0.99
14.5 ± 0.94
15.2 ± 1.10
13.3 ± 0.53
13.6 ± 0.60
14.3 ± 0.72
15.2 ± 0.76
16.1 ± 0.83
16.1 ± 0.95
Values are expressed as mean ± SEM of 6 rats in each group. Table 2: Effect on food intake and water intake after 28 days of the oral administration of OSE. 4. Discussion Oral treatment (mg/kg, od)
Food intake (g/day)
Water intake (mL/day)
0 day
3 days
7 days
14 days
21 days
28 days
0 day
3 days
7 days
14 days
21 days
28 days
Control 0.5% CMC
11.4 ± 0.70
11.63 ± 0.55
12.2 ± 0.61
12.9 ± 0.66
13.9 ± 0.79
15.2 ± 0.90
12.3 ± 0.46
12.5 ± 0.62
13.1 ± 0.74
14.2 ± 0.63
14.6 ± 0.76
15.5 ± 0.99
OSE 200
11.5 ± 0.77
12.2 ± 0.69
12.7 ± 0.59
13.8 ± 0.76
14.5 ± 0.67
15.6 ± 0.82
13.1 ± 0.5
13.7 ± 0.58
14.1 ± 0.76
14.9 ± 0.74
16.0 ± 0.85
16.4 ± 0.91
OSE 400
11.6 ± 0.63
12.1 ± 0.49
13.0 ± 0.57
13.7 ± 0.65
14.5 ± 0.78
15.9 ± 0.86
12.9 ± 0.55
13.7 ± 0.65
14.1 ± 0.67
15.2 ± 0.77
16.1 ± 0.91
17.2 ± 0.98
OSE 800
10.9 ± 0.27
11.4 ± 0.44
13.1 ± 0.77
13.3 ± 0.99
14.5 ± 0.94
15.2 ± 1.10
13.3 ± 0.53
13.6 ± 0.60
14.3 ± 0.72
15.2 ± 0.76
16.1 ± 0.83
16.1 ± 0.95
Values are expressed as mean ± SEM of 6 rats in each group 7 Journal of Toxicology 7 Table 3: Effect on hematological and biochemical parameters after 28 days of the oral administration of OSE. 4. Discussion Parameters
Control group
OSE 200 mg/kg
OSE 400 mg/kg
OSE 800 mg/kg
RBC (million/mm3)
9.2 ± 1.1
9.1 ± 1.1
8.9 ± 0.8
9.1 ± 1.0
Hb (g/dL)
12.2 ± 1.2
12.2 ± 1.1
12.3 ± 0.7
12.1 ± 0.9
WBC (million/mm3)
8.8 ± 0.9
8.7 ± 1.2
8.9 ± 1.4
9.0 ± 1.1
Neutrophils %
22.35 ± 6.3
23.38 ± 9.3
22.58 ± 5.4
23.51 ± 8.7
Eosinophils %
2.37 ± 1.0
2.34 ± 1.3
2.82 ± 1.1
2.47 ± 1.4
Basophils %
0.18 ± 0.06
0.18 ± 0.08
0.15 ± 0.09
0.19 ± 0.06
Lymphocytes %
76.33 ± 2.7
74.46 ± 3.4
75.56 ± 2.5
73.35 ± 3.1
Monocytes %
3.38 ± 0.61
3.87 ± 0.72
4.04 ± 0.65
3.26 ± 0.73
AST (U/L)
195.90 ± 1.30
194.98 ± 1.90
196.85 ± 1.87
197.87 ± 1.08
ALT (U/L)
81.46 ± 1.23
80.72 ± 1.03
81.17 ± 2.07
83.5 ± 2.23
ALP (U/L)
232.28 ± 1.28
231.14 ± 1.60
232.26 ± 1.52
231.08 ± 1.07
Creatinine (mg/dL)
0.91 ± 0.07
0.90 ± 0.04
0.92 ± 0.02
0.92 ± 0.05
Albumin (g/dL)
2.84 ± 0.05
2.74 ± 0.07
2.83 ± 0.09
2.84 ± 0.08
Total protein (g/dL)
7.3 ± 1.3
7.2 ± 1.4
6.9 ± 1.5
7.1 ± 1.5
Glucose (mg/dL)
94.86 ± 4.63
89.53 ± 5.34
90.68 ± 6.39
91.53 ± 4.87
Total cholesterol (mg/dL)
122.8 ± 3.6
121.2 ± 4.4
118.9 ± 2.8
121.1 ± 3.9
Bilirubin total (mg/dL)
1.26 ± 0.21
1.23 ± 0.22
1.22 ± 0.31
1.21 ± 0.41
Bilirubin direct (mg/dL)
0.71 ± 0.02
0.68 ± 0.01
0.72 ± 0.01
0.70 ± 0.01
Values are expressed as mean ± SEM of 6 rats in each group. Table 3: Effect on hematological and biochemical parameters after 28 days of the oral administration of OSE. Values are expressed as mean ± SEM of 6 rats in each group. (a)
(b)
(c)
(d)
Figure 5: Histology of testis (H&E, 100x) of control and OSE-treated animals. (a) Section of testis from control animal showed well-layered
seminiferous tubules with germ cell; ((b), (c), and (d)) testis from OSE (200, 400, and 800 mg/kg)-treated animals exhibit normal seminiferous
tubules with germ cell. (b) (a) (a) (a) (b) (d) (c) (d) (c) Figure 5: Histology of testis (H&E, 100x) of control and OSE-treated animals. 4. Discussion Oral treatment (mg/kg, od)
Isolated organs weight/100 g body weight rat
Liver
Right kidney
Heart
Spleen
Testis
Ovary
Control 0.5% CMC
2.7 ± 0.09
0.338 ± 0.01
0.360 ± 0.03
0.327 ± 0.01
0.649 ± 0.26
0.265 ± 0.17
OSE 200
2.6 ± 0.07
0.345 ± 0.02
0.354 ± 0.02
0.319 ± 0.01
0.668 ± 0.27
0.257 ± 0.20
OSE 400
2.7 ± 0.12
0.343 ± 0.01
0.352 ± 0.00
0.326 ± 0.02
0.656 ± 0.28
0.261 ± 0.16
OSE 800
2.7 ± 0.16
0.319 ± 0.01
0.364 ± 0.02
0.320 ± 0.01
0.661 ± 0.28
0.263 ± 0.18
Values are expressed as mean ± SEM of 6 rats in each group. (b) (a) (b) (a) (c) (d) (d) (c) Figure 6: Histological of ovary (H&E, 100x) of control and experimental group of animals. (a) Section of ovary from control animal showed
normal small follicles and large follicles; ((b), (c) and (d)) ovary from OSE 200, 400, and 800 mg/kg treated exhibit normal small follicles and
large follicles in the histology. studies [15]. Subacute exposure of rat to the lower doses
of the OSE produced small and transient changes in some
biochemical and hematological parameters. of both transaminases in the blood [16]. In our study, both
AST and ALT did not show any treatment related increase
even at the 800 mg/kg dose compared to the control group. In addition, AST found in the serum is of both mitochondrial
and cytoplasmic origin and any rise can be taken as a first
sign of cell damage that leads to the outflow of the enzymes
into the serum [19]. Thus, no significant increases observed
in ALT and AST activities strongly suggest that the subacute
administration of OSE did not alter the hepatocytes and
consequently the metabolism in the rats. Further, OSE did not
show any histological changes in spleen and liver indicating
no effect on reticuloendothelial system.if Liver is the major site for metabolism including drugs. Liver is a site of cholesterol disposal or degradation and
its major site of synthesis. In the same perspective, it
controls glucose synthesis and generates free glucose from
hepatic glycogen stores [16]. Since no significant changes
were observed in glucose and cholesterol levels this study, it
suggests that OSE had no effect on the lipid and carbohydrate
metabolism in rats. 4. Discussion (a) Section of testis from control animal showed well-layered
seminiferous tubules with germ cell; ((b), (c), and (d)) testis from OSE (200, 400, and 800 mg/kg)-treated animals exhibit normal seminiferous
tubules with germ cell. Figure 5: Histology of testis (H&E, 100x) of control and OSE-treated animals. (a) Section of testis from control animal showed well-layered
seminiferous tubules with germ cell; ((b), (c), and (d)) testis from OSE (200, 400, and 800 mg/kg)-treated animals exhibit normal seminiferous
tubules with germ cell. 8 Journal of Toxicology 8 Table 4: Effect on isolated organs weight after 28 days of the oral administration of OSE. Oral treatment (mg/kg, od)
Isolated organs weight/100 g body weight rat
Liver
Right kidney
Heart
Spleen
Testis
Ovary
Control 0.5% CMC
2.7 ± 0.09
0.338 ± 0.01
0.360 ± 0.03
0.327 ± 0.01
0.649 ± 0.26
0.265 ± 0.17
OSE 200
2.6 ± 0.07
0.345 ± 0.02
0.354 ± 0.02
0.319 ± 0.01
0.668 ± 0.27
0.257 ± 0.20
OSE 400
2.7 ± 0.12
0.343 ± 0.01
0.352 ± 0.00
0.326 ± 0.02
0.656 ± 0.28
0.261 ± 0.16
OSE 800
2.7 ± 0.16
0.319 ± 0.01
0.364 ± 0.02
0.320 ± 0.01
0.661 ± 0.28
0.263 ± 0.18
Values are expressed as mean ± SEM of 6 rats in each group. (a)
(b)
(c)
(d)
Figure 6: Histological of ovary (H&E, 100x) of control and experimental group of animals. (a) Section of ovary from control animal showed
normal small follicles and large follicles; ((b), (c) and (d)) ovary from OSE 200, 400, and 800 mg/kg treated exhibit normal small follicles and
large follicles in the histology. Table 4: Effect on isolated organs weight after 28 days of the oral administration of OSE. Table 4: Effect on isolated organs weight after 28 days of the oral administration of OSE. 4. Discussion Further, drugs showing any toxicity in
liver affect the transaminases, (aspartate aminotransferase)
AST, and (alanine amino transferase) ALT which are well-
known enzymes used as good indicators of liver function [17]
and biomarkers predicting possible toxicity [18]. Generally,
any damage to the parenchymal liver cells results in elevations f
y
OSE neither showed any significant difference in the
weight of the organs or color of organs nor affected
the histopathological changes in organs like heart and
testis/ovary indicating least cumulative toxic effects on 9 Journal of Toxicology [8] OECD, “Acute Oral Toxicity: Acute Toxic Class Method,” Test
Guideline 423, 2001. reproductive and cardiac tissues. Further, no change was
also observed in kidney function as evidenced by little or
no change in serum creatinine level as well as histological
changes in treated kidneys. [9] M. K. Gautam, A. Singh, C. V. Rao, and R. K. Goel, “Toxico-
logical evaluation of Murraya paniculata (L.) leaves extract on
rodents,” The American Journal of Pharmacology and Toxicology,
vol. 7, no. 2, pp. 62–67, 2012. Conflict of Interests [17] J. El Hilaly, Z. H. Israili, and B. Lyoussi, “Acute and chronic
toxicological studies of Ajuga iva in experimental animals,”
Journal of Ethnopharmacology, vol. 91, no. 1, pp. 43–50, 2004.f The authors declare that they have no conflict of interests. [18] M. F. Rahman, “Effects of Vepacide (Azadirachta indica) on
aspartate and alanine aminotransferase profiles in a subchronic
study with rats,” Human and Experimental Toxicology, vol. 20,
no. 5, pp. 243–249, 2001. Abbreviations CNS: Central nervous system
ANS: Autonomous nervous system
RBC: Red blood cell
Hb:
Hemoglobin
WBC: White blood cell
AST:
Aspartate transaminase
ALT:
Alanine transaminase
ALP:
Alkaline phosphate. CNS: Central nervous system
ANS: Autonomous nervous system
RBC: Red blood cell
Hb:
Hemoglobin
WBC: White blood cell
AST:
Aspartate transaminase
ALT:
Alanine transaminase
ALP:
Alkaline phosphate. CNS: Central nervous system
ANS: Autonomous nervous system
RBC: Red blood cell
Hb:
Hemoglobin
WBC: White blood cell
AST:
Aspartate transaminase
ALT:
Alanine transaminase
ALP:
Alkaline phosphate. CNS: Central nervous system
ANS: Autonomous nervous system
RBC: Red blood cell
Hb:
Hemoglobin
WBC: White blood cell
AST:
Aspartate transaminase
ALT:
Alanine transaminase
ALP:
Alkaline phosphate. [14] J. T. Mukinda and J. A. Syce, “Acute and chronic toxicity of
the aqueous extract of Artemisia afra in rodents,” Journal of
Ethnopharmacology, vol. 112, no. 1, pp. 138–144, 2007. [15] H. Olson, G. Betton, D. Robinson et al., “Concordance of
the toxicity of pharmaceuticals in humans and in animals,”
Regulatory Toxicology and Pharmacology, vol. 32, no. 1, pp. 56–
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State of the Art: trxG Factor Regulation of Post-embryonic Plant Development
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Frontiers in plant science
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Keywords: trxG, PcG, development, chromatin, histone methylation, transcription, epigenetics, Arabidopsis Keywords: trxG, PcG, development, chromatin, histone methylation, transcription, epigenetics, Arabidopsis Reviewed by:
Isabel Bäurle,
University of Potsdam, Germany
Keqiang Wu,
National Taiwan University, Taiwan Reviewed by:
Isabel Bäurle,
University of Potsdam, Germany
Keqiang Wu,
National Taiwan University, Taiwan
*Correspondence:
Jennifer C. Fletcher
jfletcher@berkeley.edu State of the Art: trxG Factor
Regulation of Post-embryonic
Plant Development Jennifer C. Fletcher1,2* 1 Plant Gene Expression Center, United States Department of Agriculture – Agricultural Research Service, Albany, CA,
United States, 2 Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA, United States Multicellular organisms rely on the precise and consistent regulation of gene expression
to direct their development in tissue- and cell-type specific patterns. This regulatory
activity involves arrays of DNA-binding transcription factors and epigenetic factors
that modify chromatin structure. Among the chromatin modifiers, trithorax (trxG)
and Polycomb (PcG) group proteins play important roles in orchestrating the stable
activation and repression of gene expression, respectively. These proteins have generally
antagonistic functions in maintaining cell and tissue homeostasis as well as in mediating
widespread transcriptional reprogramming during developmental transitions. Plants
utilize multiple trxG factors to regulate gene transcription as they modulate their
development in response to both endogenous and environmental cues. Here, I will
discuss the roles of trxG factors and their associated proteins in post-embryonic plant
development. INTRODUCTION The development of multicellular organisms is driven by precise patterns of gene transcription
that are tightly regulated in a spatial and temporal manner. Establishing and sustaining specific
transcription states at gene loci are complex, multi-step processes. They require repertoires
of sequence-specific DNA-binding transcription factors as well as epigenetic factors that alter
chromatin structure and thereby affect accessibility by the transcriptional machinery. Epigenetic
regulators classified as trithorax group (trxG) and Polycomb group (PcG) factors are critical
for maintaining the stable transcription patterns at developmental regulatory loci by organizing
chromatin in an active or inactive state, respectively (Schwartz and Pirrota, 2007). trxG and
PcG factors generally act in large, multi-component complexes that function antagonistically to
generate and maintain a balanced state of gene expression (Piunti and Shilatifard, 2016). *Correspondence:
Jennifer C. Fletcher
jfletcher@berkeley.edu Specialty section:
This article was submitted to
Plant Evolution and Development,
a section of the journal
Frontiers in Plant Science
Received: 25 September 2017
Accepted: 24 October 2017
Published: 14 November 2017
Citation:
Fletcher JC (2017) State of the Art:
trxG Factor Regulation
of Post-embryonic Plant
Development. Front. Plant Sci. 8:1925. doi: 10.3389/fpls.2017.01925 Specialty section:
This article was submitted to
Plant Evolution and Development,
a section of the journal
Frontiers in Plant Science
Received: 25 September 2017
Accepted: 24 October 2017
Published: 14 November 2017 TrxG genes were first identified in Drosophila as positive regulators of PcG developmental
target genes (Ingham, 1988; Kennison, 1995), and their protein products operate in multiple
complexes that affect gene expression on a global scale (Piunti and Shilatifard, 2016). Because transcription activation involves numerous steps, trxG factors are heterogeneous
and fall into several functional categories: chromatin remodeling proteins, histone modifying
methyltransferase and demethylase proteins, and DNA-binding and accessory proteins
(Xiao et al., 2016) (Table 1). The chromatin remodeling proteins include members of
the SWI/SNF, ISWI, and CHD families that utilize ATP to alter nucleosome assembly
and distribution (Gentry and Hennig, 2014). The histone modifying enzymes deposit Edited by: Edited by:
Stefan A. Rensing,
Philipps University of Marburg,
Germany MINI REVIEW MINI REVIEW
published: 14 November 2017
doi: 10.3389/fpls.2017.01925 published: 14 November 2017
doi: 10.3389/fpls.2017.01925 Citation: November 2017 | Volume 8 | Article 1925 1 Frontiers in Plant Science | www.frontiersin.org trxG Factors in Plant Development Fletcher ROLE OF trxG FACTORS IN ROOT AND
SHOOT APICAL MERISTEM
MAINTENANCE H3K4me2/3
and/or
H3K36me2/3
marks
associated
with
transcription activation to counteract the activity of PcG
complexes such as POLYCOMB REPRESSIVE COMPLEX 2
(PRC2) that deposit H3K27me3 as the major repressive mark for
transcription (Piunti and Shilatifard, 2016). H3K4me2/3
and/or
H3K36me2/3
marks
associated
with
transcription activation to counteract the activity of PcG
complexes such as POLYCOMB REPRESSIVE COMPLEX 2
(PRC2) that deposit H3K27me3 as the major repressive mark for
transcription (Piunti and Shilatifard, 2016). Plants are sessile organisms that grow continuously and alter
their development in response to changes in their environment. Organogenesis occurs throughout the life cycle from specialized
structures at the growing shoot and root tips, called apical
meristems (Steeves and Sussex, 1989). Both the root and shoot
apical meristems (SAMs) contain small reservoirs of stem cells
that constantly replenish themselves as well as provide progeny
cells for continuous organ formation. The flexible regulation
of gene expression via chromatin remodeling is essential for
maintaining these pluripotent stem cell populations whose
progeny can assume different fates. Animal stem cells possess
special chromatin signatures (Bernstein et al., 2006) that permit
plasticity in stem cell dynamics, and much is known about
the epigenetic factors and mechanisms involved (Spivakov and
Fisher, 2007). In contrast, the roles of epigenetic factors in p
Plant trxG factors have been identified either by homology
to known trxG factors in animals or by genetic characterization
based on their ability to suppress PcG mutant phenotypes. Given their fundamental roles in the epigenetic regulation
of gene expression states, mutations in plant trxG genes
often cause pleiotrophic developmental phenotypes, including
defects in seedling growth, anther and ovule formation,
and
gametophyte
development
(Grini
et
al.,
2009;
Guo
et al., 2010; Carter et al., 2016; Chen et al., 2017). They
also
play
key
roles
during
developmental
transitions
when widespread gene reprogramming occurs. Here I will
summarize
our
current
understanding
of
trxG
protein
function in plant meristems, which drive post-embryonic
development. TABLE 1 | Biological functions of the trxG factors and their accessory proteins in post-embryonic development. ROLE OF trxG FACTORS IN ROOT AND
SHOOT APICAL MERISTEM
MAINTENANCE trxG factor
Biological function(s)
Reference
Chromatin remodelers
BRM
Maintains RAM and SAM activity;
Farrona et al., 2004; Yang et al., 2015
Represses floral transition;
Farrona et al., 2011; Li et al., 2015
Specifies floral organ identity
Farrona et al., 2004; Wu et al., 2012
CHR11
Promotes floral morphogenesis
Smaczniak et al., 2012
CHR17
Promotes floral morphogenesis
Smaczniak et al., 2012
PKL
Maintains RAM activity
Aichinger et al., 2011
SYD
Maintains SAM activity;
Kwon et al., 2005
Specifies floral organ identity
Wagner and Meyerowitz, 2002; Wu et al., 2012
Histone methyltransferases
ATX1/SDG27
Maintains RAM activity;
Napsucialy-Mendivil et al., 2014
Represses floral transition;
Pien et al., 2008
Specifies floral organ identity
Alvarez-Venegas et al., 2003
ATXR3/SDG2
Maintains RAM activity
Yao et al., 2013
ATXR7/SDG25
Represses floral transition
Berr et al., 2009; Tamada et al., 2009
SDG8
Represses floral transition;
Shafiq et al., 2014; Yang et al., 2014
Specifies floral organ identity
Grini et al., 2009
SDG26
Promotes floral transition
Berr et al., 2015
SDG701
Promotes floral transition in rice
Liu et al., 2017
SDG708
Promotes floral transition in rice
Liu et al., 2016
AtCOMPASS core components
ASH2R
Represses floral transition
Jiang et al., 2011
RBL
Represses floral transition
Jiang et al., 2011
WDR5
Represses floral transition
Jiang et al., 2009
Histone demethylases
ELF6
Represses floral transition
Yang et al., 2016
REF6/JMJ12
Promotes floral transition
Noh et al., 2004; Yang et al., 2016
DNA-binding and accessory proteins
ALP1
Restricts FM activity
Liang et al., 2015
NF-Y
Promotes floral transition
Hou et al., 2014
SEP3
Specifies floral organ identity
Pelaz et al., 2000
ULT1/2
Restricts SAM activity;
Carles et al., 2004, 2005
Restricts FM activity
Carles and Fletcher, 2009 Biological function(s) Biological function(s) November 2017 | Volume 8 | Article 1925 Frontiers in Plant Science | www.frontiersin.org Reference November 2017 | Volume 8 | Article 1925 Frontiers in Plant Science | www.frontiersin.org 2 trxG Factors in Plant Development Fletcher regulating plant stem cell activity are only beginning to be
revealed. Maintenance of stem cell reservoirs in Arabidopsis shoot
and floral meristems occurs via a spatial negative feedback
loop mediated by the WUSCHEL (WUS)-CLAVATA (CLV)
signal transduction pathway. The homeobox TF gene WUS is
expressed in the core of the meristem and confers stem cell
identity on the overlying cells (Laux et al., 1996). The stem
cell-inducing activity of WUS is antagonized by the secreted
polypeptide CLV3 (Fletcher et al., 1999; Rojo et al., 2002), which
is produced by the stem cells and activates a signal transduction
pathway in the meristem interior that limits the accumulation
of WUS-expressing cells (Brand et al., 2000). This feedback loop
mediates stem cell homeostasis, balancing the loss of stem cells to
organ formation with their replenishment via cell division. Recent genetic studies have uncovered roles for multiple
trxG factors in root apical meristem (RAM) maintenance. The
RAM generates the entire underground root system and has a
stereotypical organization. Four rarely dividing cells known as
the quiescent center (QC) act as a niche (van den Berg et al.,
1997) that maintains the surrounding cells as stem cells, aka
initial cells, which undergo asymmetric cell divisions to generate
the distinct root cell lineages. A gradient of the hormone auxin
across the root tip exists due to the activity of members of the
PIN-FORMED (PIN) family of auxin transport proteins (Blilou
et al., 2005; Grieneisen et al., 2007). The auxin concentration
maximum coincides with the QC and promotes the expression
of the PLETHORA (PLT) AP2 domain transcription factor (TF)
genes, which are essential for root stem cell niche maintenance
(Aida et al., 2004). Although little is known about trxG activity in the SAM,
the ratio of H3K4me3 active to H3K27me3 repressive marks
is known to be important for reproductive SAM development
in rice (Liu et al., 2015). The SWI2/SNF2 trxG factors BRM
and SYD both act to sustain SAM activity in Arabidopsis
(Farrona et al., 2004; Kwon et al., 2005), with SYD shown to
bind to the WUS promoter and elevate its transcription in
the SAM (Kwon et al., 2005) (Figure 1B). Reference In contrast, the
SAND domain-containing proteins ULTRAPETALA1 (ULT1)
and ULT2 restrict shoot and floral stem cell activity by limiting
the size of the WUS expression domain (Carles et al., 2004,
2005). The SAND domain occurs in a small number of eukaryotic
proteins including the human AIRE transcriptional regulator
that is implicated in autoimmune diseases (Abramson and
Goldfarb, 2016). ULT1 antagonizes the repressive activity of
PRC1 and PRC2 PcG complex components (Carles and Fletcher,
2009; Pu et al., 2013), and physically associates with the H3K4
methyltransferase ATX1 (Carles and Fletcher, 2009) as well
as the transcription factors KANADI1 (KAN1), KAN2 and
ULTRAPETALA1 INTERACTING FACTOR (UIF1) (Pires et al.,
2014; Moreau et al., 2016). Because both ATX1 and the AIRE
protein form complexes with RNA Pol II and RNA-processing
components (Abramson et al., 2010; Ding et al., 2012), the
ULT proteins may coordinate chromatin mark modification at
transcriptionally priming loci with basal transcription machinery
recruitment by stage- and tissue-specific transcription factors
(Carles and Fletcher, 2010; Engelhorn et al., 2014a). Two H3K4 histone methyltransferase trxG factors have been
implicated in Arabidopsis RAM maintenance (Figure 1A). The
SET domain protein SET DOMAIN GROUP 2 (SDG2) is the
major H3K4 trimethyltransferase in Arabidopsis and is necessary
for genome-wide H3K4me3 deposition (Guo et al., 2010). In
the RAM SDG2 is required to maintain the auxin gradient
and QC maximum, and to sustain cell identity and stem cell
activity in the QC and surrounding initial cells (Yao et al.,
2013). These functions correlate with a requirement for SDG2
to promote PLT1 expression and global H3K4me3 deposition in
root cells (Yao et al., 2013). The ARABIDOPSIS HOMOLOG
OF TRITHORAX1 (ATX1/SDG27) protein contributes ∼15%
of genome-wide H3K4 trimethylation (Alvarez-Venegas and
Avramova, 2005). ATX1 is needed for TATA binding protein
(TBP) and RNA Polymerase II recruitment to its target promoters
(Ding et al., 2011) and is also critical for H3K4me3 deposition
associated with transcription elongation (Ding et al., 2012). Like
SDG2, ATX1 is necessary for normal RAM organization, but also
restricts the expression of QC markers such as WOX5 to the
stem cell niche in an auxin-independent fashion (Napsucialy-
Mendivil et al., 2014), indicating that the two H3K4 histone
methyltransferases have distinct as well as shared roles in RAM
maintenance. The SWI2/SNF2 chromatin remodeling complex ATPase
genes PICKLE (PKL) and BRAHMA (BRM) also regulate
RAM activity in Arabidopsis. Reference PKL acts antagonistically to the
PRC2 PcG factor CURLY LEAF (CLF) to maintain RAM
stem cell activity (Aichinger et al., 2011). PKL does not
induce the activity of the root stem cell niche by affecting
auxin accumulation. Instead, PKL elevates the expression
levels of and limits CLF-mediated H3K27me3 deposition
at PLT1, PLT2, and WOX5 (Aichinger et al., 2011). BRM
likewise maintains the RAM stem cell niche by promoting
expression of PLT1 and PLT2 (Yang et al., 2015). However,
BRM unlike PKL affects auxin accumulation in the root tip
by directly binding to and up-regulating the transcription
of five PIN loci (Yang et al., 2015). Thus the evidence to
date suggests that the auxin-dependent and auxin-independent
regulatory pathways utilize distinct trxG factors to sustain RAM
activity. ROLES OF trxG FACTORS IN FLORAL
INDUCTION AT THE SHOOT APICAL
MERISTEM During vegetative development
FLC directly represses the transcription of the flowering time
integrators FLOWERING LOCUS T (FT), FLOWERING LOCUS
D (FD) and SOC1, which promote the transition to flowering
(Michaels and Amasino, 2001). FT protein is produced in the
rosette leaves and moves to the SAM, where it interacts with the
FD protein. The FT-FD complex then induces the floral transition
by activating the expression of TF genes such as SOC1, LEAFY
(LFY) and APETALA1 (AP1), which confer floral meristem (FM)
identity on the primordia that form on the flanks of the primary
SAM (Irish, 2010). In Arabidopsis, chromatin modifications at key regulatory loci
such as FLC, SOC1 and FT are crucial to the timing of the
floral transition, and a number of trxG factors are involved in
these processes (Figure 1C). Because the role of epigenetic factors
in FLC regulation during vernalization has been extensively
reviewed (Jarillo and Pineiro, 2011; Andres and Coupland, 2012;
He, 2012; Hepworth and Dean, 2015), I will focus here on
the control of flowering through other pathways. To prevent
premature flowering during vegetative growth, the FLC locus is
maintained in a transcriptionally active state marked by H3K36
tri-methylation (Zhao et al., 2005; Yang et al., 2014), which
inhibits accumulation of H3K27me3 repressive marks (Shafiq
et al., 2014). SDG8 is the major H3K36me3 methyltransferase in
Arabidopsis (Yang et al., 2014) and is required for H3K36me3
deposition at the FLC locus (Shafiq et al., 2014; Yang et al.,
2014). SDG8 associates with components of the transcription
machinery, including RNA Pol II and PAF1, as well as with the
H3K27 demethylase EARLY FLOWERING 6 (ELF6) (Yang et al.,
2016). These physical associations (Figure 2) couple removal of
repressive histone marks with deposition of active marks and
transcription initiation/elongation to sustain high levels of FLC
expression. ATXR7/SDG25 also represses the floral transition by
binding to the FLC promoter and augmenting both H3K36me3
and H3K4me3 accumulation (Berr et al., 2009; Tamada et al.,
2009), but whether SDG8 and ATXR7 function in concert to
induce FLC transcription is unknown. The chromatin remodeling ATPase BRM prevents premature
flowering by regulating FLC and FLC-related gene expression
(Farrona et al., 2011; Li et al., 2015). Loss of function
brm seedlings exhibit elevated H3K4me3 levels and reduced
H3K27me3 levels in the FLC promoter, indicating that BRM
imposes a repressive chromatin configuration at the FLC
locus (Farrona et al., 2011). ROLES OF trxG FACTORS IN FLORAL
INDUCTION AT THE SHOOT APICAL
MERISTEM The floral induction process directs the SAM to transition
from generating vegetative organs (leaves) to reproductive
organs (flowers). The timing of this dynamic meristem cell fate
switch is critical for plant reproductive success and occurs in
response to endogenous pathways such as the age, GA and
autonomous pathways (APs) as well as environmental cues
including photoperiod, vernalization and temperature (Amasino,
2010; Srikanth and Schmid, 2011; Andres and Coupland, 2012). The MADS domain TF FLOWERING LOCUS C (FLC) is
the main floral repressor in Arabidopsis seedlings (Michaels
and Amasino, 1999) and is a key target of both endogenous November 2017 | Volume 8 | Article 1925 Frontiers in Plant Science | www.frontiersin.org 3 trxG Factors in Plant Development Fletcher Fletcher FIGURE 1 | Regulatory targets of trxG factors in post-embryonic development. (A) Root apical meristem maintenance. (B) Shoot apical meristem maintenance. (C) Vegetative to reproductive meristem transition. (D) Floral meristem patterning. Gene targets shown in green type. Arrows indicate positive and bars indicate
negative regulatory interactions. The SAM image in (B) is reprinted from Fiume et al. (2010). No permission is required for its reproduction. FIGURE 1 | Regulatory targets of trxG factors in post-embryonic development. (A) Root apical meristem maintenance. (B) Shoot apical meristem maintenance. (C) Vegetative to reproductive meristem transition. (D) Floral meristem patterning. Gene targets shown in green type. Arrows indicate positive and bars indicate
negative regulatory interactions. The SAM image in (B) is reprinted from Fiume et al. (2010). No permission is required for its reproduction. ATXR7/SDG25 does act independently of the H3K4me3
methyltransferase ATX1 in repressing the flowering transition
(Tamada et al., 2009), although ATX1 also directly binds to
the FLC locus to deposit H3K4me3 and elevate expression of
the floral repressor gene (Pien et al., 2008). ATX1 physically
associates with WDR5a (Figure 2), a core component of
the Arabidopsis COMPASS-like complex (AtCOMPASS) that
also binds the FLC locus and promotes its expression by
elevating H3K4me3 levels (Jiang et al., 2009). Two other core
AtCOMPASS components, ASH2R and RbBP5-LIKE (RBL),
also repress the floral transition by enhancing FLC expression,
with ASH2R availability apparently being the rate-limiting
factor in H3K4 tri-methylation at FLC and other target loci
(Jiang et al., 2011). Therefore both H3K36me3 and H3K4me3
contribute to the maintenance of active FLC transcription in
Arabidopsis. and environmental signaling pathways (Sheldon et al., 2000;
Michaels and Amasino, 2001). ROLES OF trxG FACTORS IN FLORAL
INDUCTION AT THE SHOOT APICAL
MERISTEM REF6
also induces transcription of the floral activator genes SOC1
and FT in an FLC-independent fashion (Noh et al., 2004; Lu
et al., 2011). It is recruited by the nuclear factor Y (NF-Y)
transcription factor complex to demethylate the SOC1 locus in
response to the photoperiod and GA pathways (Hou et al., 2014),
indicating that REF6 is a component of both endogenous and
environmental signaling modules. Like REF6, the SDG26 histone
methyltransferase also binds to and induces SOC1 transcription,
augmenting the deposition of both H3K4me3 and H3K36me3 at
the locus to promote the floral transition (Berr et al., 2015). the identity of each floral organ – sepals, petals, stamens, and
carpels – from the outside to the inside of the flower (Coen
and Meyerowitz, 1991). The activation of the floral homeotic
genes at the onset of flower patterning requires counteracting
the PcG-mediated repressive state that has persisted throughout
vegetative development (Pu and Sung, 2015), an activity that is
associated with increases in H3K4me3 levels at PcG target genes
(Engelhorn et al., 2017) and involves multiple trxG factors. g
p
The plant specific TF LFY and the MADS domain TF
SEPALLATA3 (SEP3) play crucial roles in activating the
expression of MADS box-containing floral homeotic genes such
as APETALA3 (AP3) and AGAMOUS (AG) that specify petal,
stamen, and carpel identity in the developing flower (Weigel and
Meyerowitz, 1993; Pelaz et al., 2000). SYD and BRM physically
associate with the LFY and SEP3 proteins (Figure 2), which
recruit SYD to the AP3 and AG loci (Wu et al., 2012). At the onset
of flower patterning, SYD and BRM redundantly regulate floral
organ identity specification (Wagner and Meyerowitz, 2002;
Farrona et al., 2004) (Figure 1D) by activating AP3 and AG
expression, antagonizing CLF activity at the two loci by reducing
H3K27me3 deposition and enhancing H3K4me3 deposition (Wu
et al., 2012). ATX1 and SDG8 also specify floral organ identity
by maintaining floral homeotic gene expression levels (Alvarez-
Venegas et al., 2003; Grini et al., 2009), although the mechanistic
details are as yet unknown. Finally, REF6 and PKL as well
as two ISWI-type chromatin remodelers, CHR11 and CHR17,
occur in floral MADS domain protein complexes and affect
floral organ morphogenesis (Smaczniak et al., 2012). ROLES OF trxG FACTORS IN FLORAL
INDUCTION AT THE SHOOT APICAL
MERISTEM In addition, BRM directly
activates the expression of the FLC-related MADS box TF
gene SHORT VEGETATIVE PHASE (SVP) (Li et al., 2015). SVP forms a complex with FLC to repress flowering under
non-inductive conditions (Fujiwara et al., 2008; Li et al.,
2008). SVP expression is regulated by the AP, GA and
temperature pathways, and directly represses SOC1 and FT
transcription (Li et al., 2008). BRM represses flowering largely
by inducing SVP transcription in vegetative tissues, binding
to the SVP locus where it limits H3K27me3 accumulation
by restricting CLF occupancy and activity (Li et al., 2015). Thus the early flowering phenotype of brm mutants can be
accounted for by a reduction in SVP mRNA levels leading
to lower abundance of the FLC-SVP repressor complex,
resulting in elevated FT transcript levels that induce precocious
flowering. November 2017 | Volume 8 | Article 1925 Frontiers in Plant Science | www.frontiersin.org Frontiers in Plant Science | www.frontiersin.org 4 trxG Factors in Plant Development Fletcher FIGURE 2 | Association networks of trxG factors and interacting proteins. Solid bars designate physical associations between trxG factors (blue), transcription
factors (green), AtCOMPASS components (yellow), and transcription machinery components and accessory proteins (purple). FIGURE 2 | Association networks of trxG factors and interacting proteins. Solid bars designate physical associations between tr
factors (green), AtCOMPASS components (yellow), and transcription machinery components and accessory proteins (purple). works of trxG factors and interacting proteins. Solid bars designate physical associations between trxG factors (blue), transcription
S components (yellow), and transcription machinery components and accessory proteins (purple). FIGURE 2 | Association networks of trxG factors and interacting proteins. Solid bars designate physical associations between trxG factors (blue), transcription
factors (green), AtCOMPASS components (yellow), and transcription machinery components and accessory proteins (purple). Repression of FLC transcription is crucial for the transition
from the vegetative to the reproductive state. The JmjC domain-
containing H3K27 demethylase REF6/JMJ12 promotes flowering
independently of photoperiod by repressing FLC transcription
(Noh et al., 2004). Because REF6 acts antagonistically to CLF
by removing repressive H3K27me2/3 marks (Lu et al., 2011),
binding its target genes in a sequence-specific fashion via its
C2H2 zinc-finger domains (Cui et al., 2016) and facilitating
recruitment of BRM (Li et al., 2016), the repression of FLC
by REF6 is likely to be indirect (Yang et al., 2016). ROLES OF trxG FACTORS IN FLORAL
INDUCTION AT THE SHOOT APICAL
MERISTEM SEP3 and
several other floral homeotic TFs bind their target genes prior
to detectable increases in DNA accessibility (Pajaro et al., 2014),
suggesting that they work closely with epigenetic factors to
facilitate transcription initiation during early flower development
by modulating chromatin accessibility at target loci. Finally, two methyltransferases that accelerate the floral
transition independent of photoperiod have recently been
characterized in rice. SDG708 encodes a methyltransferase that
deposits up to three methyl groups on H3K36, and promotes
flowering by catalyzing H3K36 methylation at the key flowering
time regulatory genes H3Da and RFT1, which are closely related
homologs of Arabidopsis FT, and Ehd1 (Liu et al., 2016). SDG701
encodes an H3K4 di- and tri-methyltransferase that likewise
promotes flowering by depositing H3K4me3 to elevate the
expression of H3Da and RFT1 (Liu et al., 2017). ROLES OF trxG FACTORS IN
PATTERNING THE FLORAL MERISTEM The ULT1 trxG protein induces AG transcription in the center
of the FM at stage 3 of flower formation (Alvarez-Venegas
et al., 2003; Carles and Fletcher, 2009), binding directly to the
locus to limit CLF-mediated H3K27me3 deposition and enhance
H3K4me3 deposition (Carles and Fletcher, 2009). ULT1 thus sets
the timing of the transition of the AG locus from a repressed Flowering signals induce reproductive development in plants by
reprogramming the SAM into an inflorescence meristem (IM)
that produces floral meristems (FMs) instead of leaves. A small
suite of floral homeotic transcription factor genes then specifies November 2017 | Volume 8 | Article 1925 Frontiers in Plant Science | www.frontiersin.org 5 trxG Factors in Plant Development Fletcher to an active state, helping trigger a molecular pathway that
ultimately terminates FM activity (Carles and Fletcher, 2009;
Engelhorn et al., 2014b; Cao et al., 2015; Sun and Ito, 2015). Mutations in the domesticated transposase gene ANTAGONIST
OF LIKE HETEROCHROMATIN PROTEIN1 (ALP1) enhance
ult1 FM phenotypes, and ALP1 promotes floral organ identity
gene expression in the absence of LFY (Liang et al., 2015). ALP1
antagonizes CLF function, acting genetically as a trxG factor,
and is required for the activity of PcG target genes such as AP3
and AG. Notably, the ALP1 protein complex lacks known trxG
factors but consists of core components of PRC2 and accessory
factors such as EMF1 and LHP1. ALP1 is therefore proposed
to antagonize PcG activity by blocking the interaction between
PRC2 and accessory factors that stimulate its activity (Liang et al.,
2015). Although significant progress has been made in determining
the roles of trxG factors in plant development, many gaps in
our understanding remain. It still remains to be determined
how many trxG complexes exist in plants, not to mention
their
full
composition
and
whether
that
composition
is
static or changes depending on the developmental stage
or tissue. The DNA binding proteins that recruit trxG
factors to developmental regulatory loci are only beginning
to be identified, while elucidating the chromatin signatures
of
plant
stem
cell
populations
can
provide
a
valuable
starting point for determining how tissue- and stage-specific
epigenetic states are ultimately achieved during development. Finally, much work remains to decipher how developmental
switches between trxG and PcG activities are implemented
at individual loci as well as broadly across the genome to
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genes and in atx1 mutants. Nucleic Acids Res. 33, 5199–5207. AUTHOR CONTRIBUTIONS The author confirms being the sole contributor of this work and
approved it for publication. ROLES OF trxG FACTORS IN
PATTERNING THE FLORAL MERISTEM Further
investigation in these areas will provide a more complete
picture of how plants are able to maintain and as necessary
adjust their gene expression programs during development
in response to a wealth of endogenous and environmental
cues. ACKNOWLEDGMENTS The author apologizes for references omitted due to space
limitations. This work was supported by research grants from
the USDA (CRIS 2030-21000-041-00D) and the NSF (IOS-105-
2050). CONCLUSION A variety of trxG factors exist in plants that carry out diverse
biochemical activities to promote active gene expression states:
chromatin-remodeling
ATPases,
histone
methyltransferases,
AtCOMPASS
core
components,
histone
demethylases,
as
well as DNA-binding and accessory proteins (Table 1). It is
becoming clear from recent research that members of all of
these categories of proteins play important roles in regulating
landmark post-embryonic developmental processes such as
meristem maintenance and floral induction. Moreover some trxG
factors, such as BRM and ATX1, mediate multiple developmental
processes during plant growth whereas others appear to have
more restricted roles. The coupling of a core set of reiteratively
used trxG components together with stage-, tissue- or process-
specific trxG components may provide a flexible mechanism
for tailoring the basic process of transcription activation to
discrete gene networks in response to changing endogenous and
environmental signals during the life cycle. Blilou, I., Xu, J., Wildwater, M., Willemsen, V., Paponov, I., Friml, J., et al. (2005).
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growth. Nature 449, 1008–1013. doi: 10.1038/nature06215 November 2017 | Volume 8 | Article 1925 Frontiers in Plant Science | www.frontiersin.org 7 Fletcher trxG Factors in Plant Development Proc. Natl. Acad. Sci. U.S.A. 109, 1560–1565. doi: 10.1073/pnas.111287
1109 Michaels, S. D., and Amasino, R. M. (2001). Loss of FLOWERING LOCUS C
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histone H3 K36. Nat. Cell Biol. 7, 1256–1260. doi: 10.1038/ncb1329 Schwartz, Y. B., and Pirrota, V. (2007). Polycomb silencing mechanisms and the
management of genomic programmes. Nat. Rev. Genet. 8, 9–22. doi: 10.1038/
nrg1981 Conflict of Interest Statement: The author declares that the research was
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be construed as a potential conflict of interest. Conflict of Interest Statement: The author declares that the research was
conducted in the absence of any commercial or financial relationships that could
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LOCUS C. Proc. Natl. Acad. Sci. U.S.A. 97, 3753–3758. doi: 10.1073/pnas.97.7. 3753 Copyright © 2017 Fletcher. This is an open-access article distributed under the terms
of the Creative Commons Attribution License (CC BY). The use, distribution or
reproduction in other forums is permitted, provided the original author(s) or licensor
are credited and that the original publication in this journal is cited, in accordance
with accepted academic practice. No use, distribution or reproduction is permitted
which does not comply with these terms. Smaczniak, C., Immink, R. G. H., Muino, J. M., Blanvillain, R., Busscher, M.,
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Influence of Different Concentration and Ratio of a Photoinitiator System on the Properties of Experimental Resin Composites
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Brazilian Dental Journal
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ISSN 0103-6440 ISSN 0103-6440 Brazilian Dental Journal (2017) 28(6): 726-730
http://dx.doi.org/10.1590/0103-6440201701372 1Department of Restorative
Dentistry, Dental Materials
Division, Piracicaba Dental School,
UNICAMP - Universidade Estadual
de Campinas, Piracicaba, SP, Brazil
2Department of Prosthodontics,
UNIVAG - Centro Universitário de
Várzea Grande, Cuiaba, MT, Brazil
3Dental School, UNISA - Universidade
de Santo Amaro, São Paulo, SP, Brazil
4Department of Pediatric Dentistry,
Piracicaba Dental School, UNICAMP
- Universidade Estadual de
Campinas, Piracicaba, SP, Brazil
5Restorative Dental Science
Department, Operative
Dentistry Division, College
of Dentistry, University of
Florida, Gainesville, FL, USA Correspondence: Mário Alexandre
Coelho Sinhoreti, Avenida Limeira,
901, 13414-903 Piracicaba, SP,
Brasil. Tel: +55-19-2106-5374.
e-mail: sinhoreti@unicamp.br Influence of Different Concentration
and Ratio of a Photoinitiator
System on the Properties of
Experimental Resin Composites Caio Vinícius Signorelli Grohmann1, Eveline Freitas Soares1, Eduardo José
Carvalho Souza-Junior2, William Cunha Brandt3, Regina Maria Puppin-
Rontani4, Saulo Geraldeli5, Mário Alexandre Coelho Sinhoreti1 The aim in this study was to evaluate the influence of different ratio of camphorquinone/
tertiary amine concentration on the flexural strength (FS), elastic modulus (EM), degree
of conversion (DC), yellowing (YL), water sorption (WS) and water solubility (WSL) of
experimental composites. Thus, acrylate blends were prepared with different camphorquinone
(CQ) and amine (DABE) concentrations and ratios by weight: (CQ/DABE%): 0.4/0.4% (C1),
0.4/0.8% (C2), 0.6/0.6% (C3), 0.6/1.2% (C4), 0.8/0.8% (C5), 0.8/1.6% (C6), 1.0/1.0% (C7),
1.0/2.0% (C8), 1.5/1.5% (C9), 1.5/3.0% (C10). For the FS and EM, rectangular specimens (7x2x1
mm, n=10) were photo-activated by single-peak LED for 20 s and tested at Instron (0.5 mm/
min). Then, the same specimens were evaluated by FTIR for DC measurement. For YL, disks
(5x2 mm, n=10) were prepared, light-cured for 20 s and evaluated in spectrophotometer
using the b aspect of the CIEL*a*b* system. For WS and WSL, the volume of the samples was
calculated (mm³). For WS and WSL, composites disks (5x0.5 mm, n=5) were prepared. After
desiccation, the specimens were stored in distilled water for 7 days and again desiccated,
in order to measure the WS and WSL. Data were submitted to one-way ANOVA and Tukey’s
test (5%). The groups C8, C9 and C10 showed higher DC, EM and YL means, compared to
other composites. Therefore, the FS and WS values were similar among all groups. Also,
C1, C2 and C3 presented higher WSL in 7 days, compared to other composites. In general,
higher concentrations of camphorquinone promoted higher physical-mechanical properties;
however, inducing higher yellowing effect for the experimental composites. Key Words: camphorquinone,
dental resin, curing performance. Key Words: camphorquinone,
dental resin, curing performance. Introduction material (8). According to Musanje et al. (4), higher
quantities of CQ promote a rapid generation of large
amounts of free radicals, but this may result in a reduced
ability to form an adequate polymer network, since polymers
with relatively lower molecular weight are formed. On the
other hand, in accordance with Schneider et al. (5), the high
concentration of the photoinitiator system may guarantee a
high monomer conversion and consequently higher physical
and mechanical properties of the composites. Therefore,
the intense yellow hue of CQ places practical limits on the
concentration of photoinitiators that can be used, because
of the esthetic properties, but this may not limit the degree
of polymerization and depth of cure that can be attained (9). To achieve satisfactory polymerization of the resin
composites, a proper combination of photoinitiator/co-
initiator system, which are usually camphorquinone (CQ)
and a tertiary amine, respectively (1,2) should be used. Light absorption raises CQ to an excited state that interacts
with a tertiary amine, forming a triplet exciplex that
disintegrates into free radicals. These radicals initiate the
polymerization of methacrylates, which success conducts
on obtaining a reliable bond and good polymerization (3). The concentration of CQ and amine, as well as the CQ/amine
ratio, play important roles in the polymerization efficiency
and ultimately in the physical and mechanical properties
of resin composite materials (4-6). Conversely, if the concentration of photoinitiators is
kept too low, an inadequately polymerized resin composite
may result, and the physical-mechanical properties and
durability of the restoration can be compromised (10). Also,
an incomplete photocuring process leaves the composite
with more leachable residual monomers and initiators that
can increase its biological incompatibility and jeopardize
the color stability (11,12). Therefore, it is important to find
an optimal photoinitiator and co-initiator concentration
and ratio that guarantee a satisfactory polymerization CQ is inherently yellow and can cause a color mismatch
in aged aesthetic resin-based composite restorations. This
mismatch becomes even more pronounced if bleaching
is performed in those restored teeth (7). Flexural Strength and Elastic Modulus Immediately after the DC analysis, the same bar-shaped
specimens (n=10) were used for the three-point bending
flexural test. The flexural test was performed according to
ISO 4049:2009, except for the specimen’s dimensions (7 mm
length x 2 mm width x 1 mm thickness). The specimens were
photo-activated by a LED curing unit (Radii-Cal, 900 mW/
cm2) for 20 s and dry stored in light-proof containers for
24 h at 37 °C. The three-point bending test was performed
in a universal testing machine (Instron, Canton, USA. span
between supports=5 mm) at a cross-head speed of 0.5 mm/
min. The maximum load for the specimens at fracture was
recorded and the flexural strength (FS) calculated using the
following equation: FS=3FL/(2bh2), where F is the maximum
load (N) exerted on the specimens; L the distance (mm)
between the supports; b is the width (mm) of the specimens
measured immediately prior testing; h is the height (mm) of
the specimens measured immediately prior testing. Yellowing Effect The experimental resin composites were inserted in The experimental resin composites were inserted in 727
Table 1. Concentration and ratio of the photo-initiator system of the
experimental composites
Composite
CQ wt%
DABE wt%
Composite 1 (C1)
0.4
0.4
Composite 2 (C2)
0.4
0.8
Composite 3 (C3)
0.6
0.6
Composite 4 (C4)
0.6
1.2
Composite 5 (C5)
0.8
0.8
Composite 6 (C6)
0.8
1.6
Composite 7 (C7)
1.0
1.0
Composite 8 (C8)
1.0
2.0
Composite 9 (C9)
1.5
1.5
Composite 10 (C10)
1.5
3.0 Table 1. Concentration and ratio of the photo-initiator system of the
experimental composites Concentration and ratio of photoinitiator system Concentration and ratio of photoinitiator system - Sigma-Aldrich Inc); and triethyleneglycol dimethacrylate Concentration and ratio of photo The elastic modulus was measured as the slope of
the strain x strain curve in the linear portion, using the
following equation: E=L1D310-3/4bh3d, where L1 is load (N),
d: distance (mm) between the supports, b: width (mm), h:
height (mm) and D: displacement (mm). Introduction Moreover, due
to the tendency for internal discoloration of the resin
composite, the amount of the tertiary amine may be a
concern, since these amines have double bonds, which
are capable of absorbing UV light and react with oxygen
or other aromatic groups, causing the yellowing of the Braz Dent J 28(6) 2017 aliphatic carbon-carbon double-bond absorbance peak
intensity (located at 1638 cm−1) and that of the aromatic
(C…C) (located at 1608 cm−1; reference peak) were collected. The DC (%) was calculated using the following equation: DC
(%)=100×[1−(R polymerized/R non-polymerized)], where R
represents the ratio between the absorbance peak at 1638
cm−1 and 1608 cm−1. behavior and consequently better chemical-mechanical
properties without increasing the yellowing of the composite
restorations. The purpose in this study was to determine the
optimal concentration and ratio of camphorquinone (CQ)
and tertiary amine (DABE) in relation to the degree of
conversion (DC), color stability (CS), elastic modulus (EM),
flexural strength (FS), water sorption (SP) and solubility
(SL) of microhybrid experimental composites. The tested
hypotheses were: 1) The higher the content of CQ, the
higher are the physical-mechanical properties, but also
the yellowing effect of the experimental composites; 2)
The higher the DABE ratio, the higher are the properties,
but also the yellowing effect. Materials and Methods
Resin Preparation Ten experimental resin formulations were tested in this
study. The resin matrix for all formulations consisted of a
combination of bisphenol glycidyl methacrylate – 29.0 wt%
(BisGMA – Sigma-Aldrich Inc, St Louis, MO, USA); urethane
dimethacrylate – 32.5 wt% (UDMA - Sigma-Aldrich Inc);
bisphenol ethoxylate dimethacrylate – 32.5 wt% (BisEMA
- Sigma-Aldrich Inc); and triethyleneglycol dimethacrylate
– 6.0 wt% (TEGDMA - Sigma-Aldrich Inc). Composites were
loaded at 75% by volume with silanized filler (20 wt% of
0.04 μm colloidal silica and 80 wt% of 0.7 µm Ba-Al-silicate
glass with 0.7 µm – FGM, Joinville, SC, Brazil). The inhibitor,
BHT (butylated hydroxytoluene; Sigma-Aldrich Inc), was
added to the organic matrix in a concentration of 0.1 wt%
to avoid spontaneous polymerization of the monomers. From this base-composition, ten experimental composites
were produced, varying the concentration and ratio of the
photo-initiator system (CQ - camphorquinone and DABE
- N,N-dimethyl-p-aminobenzoic acid ethylester; Sigma-
Aldrich Inc). The experimental groups are shown in Table 1. Ten experimental resin formulations were tested in this
study. The resin matrix for all formulations consisted of a
combination of bisphenol glycidyl methacrylate – 29.0 wt%
(BisGMA – Sigma-Aldrich Inc, St Louis, MO, USA); urethane
dimethacrylate – 32.5 wt% (UDMA - Sigma-Aldrich Inc);
bisphenol ethoxylate dimethacrylate – 32.5 wt% (BisEMA Degree of Conversion For the degree of conversion (DC) analysis, bar-shaped
specimens (7 mm length x 2 mm width x 1 mm thickness)
(n=10) were made in molds of silicon and photo-activated
by a LED curing unit (Radii Cal, SDI, Victoria, Australia - 900
mW/cm2), for 20 s. The energy density was standardized
at 16 J/cm2. After polymerization, the specimens were
removed from the molds and dry stored in light-proof
containers at 37 °C for 24 h. DC was measured on the
top surface of each specimen using Fourier Transformed
Infrared spectroscopy with attenuated total reflectance
(FTIR/ATR - Spectrum 100; PerkinElmer, Shelton, CA, USA). The absorption spectra of non-polymerized and
polymerized composites were obtained from the region
between 4000 and 650 cm−1 with 32 scans at 4 cm−1. The Braz Dent J 28(6) 2017 circular Teflon molds (6 mm diameter x 0.5 mm thickness)
with placement of a Mylar strip on the top and bottom
surfaces (n=10). Then, the composites were photoactivated
for 20 using a LED curing unit (SDI, 900 mW/cm2; Radii Cal)
and were dry stored in light-proof containers for 24 h at 37
°C. For the yellowing effect analysis, a spectrophotometer
CM-700d (Minolta, Corp., Ramsey, NJ, USA) was utilized to
measure the b-axis parameter of the CIELab system. This
parameter measures the yellow color of a material, with
higher b-values relating to a greater yellowing effect. no weight change) this value was recorded as m2 and the
samples were returned into the desiccators. The entire
mass reconditioning cycle was repeated and the constant
mass (two days of no weight change) was recorded as m3. The values for water sorption (WS) and solubility (WSB),
in micrograms per cubic millimeters, were calculated using
the following equations: WS=(m2–m3)/V WS=(m2–m3)/V Water Sorption and Solubility Data were analyzed by one-way ANOVA and Tukey’s
post-hoc test. Statistical significance was established at
α=0.05 for all tests. This study was performed in compliance with ISO
4049:2009 standard specifications (except for the specimen
dimensions and curing protocol) as follows. To verify the
sorption (WS) and solubility (WBS), circular samples (0.5
mm thickness x 6 mm diameter) (n=5) were photo-activated
by a LED curing unit (SDI, 900 mW/cm2; Radii Cal) for 20 s. The specimens were stored in desiccators containing silica
gel at 37 °C and weighed daily on an analytical balance (Tel
Marke; Bel Quimis, Sao Paulo, SP, Brazil) accurate to 0.0001
g, until a constant mass (m1) was obtained (i.e. three days of
no weight change). Thickness (four measurements at four
equidistant points on the circumference) and diameter of
each specimen were measured using a digital electronic
caliper (Mitutoyo Corporation, Tokyo, Japan). Mean values
were used to calculate the volume (V) of each specimen
(in mm³). Thereafter, the samples were stored in plastic
containers with 6 mL of distilled water at 37 °C for 7 days
and weighed daily after carefully wiping with absorbent
paper. When constant weight was obtained (two days of Results FS, EM, DC and yellowing effect means are shown in
Table 2. The composites C6, C7, C8, C9 and C10 showed
higher DC when compared to the other composites
(p=0.00001). For the flexural strength (FS), all composites
showed similar values (p=0.41797). In relation to the elastic
modulus (EM), the composites containing CQ/DABE in the
1.5/1.5 wt% (C9) and 1.5/3.0 wt% (C10) showed higher
values (p=0.00001). As expected, the yellowing effect
(YL) was higher for the composites having higher ratio
of phothoinitiators (C9 and C10, p=0.00001). As normal
distribution was not possible, the data for water sorption
(WS) and water solubility (WSB) were log transformed (log
x+0). After, the normalized data were submitted to one-way
ANOVA and Tukey’s test (α=0.05). The means are shown in
the Table 3. All groups showed similar WS means, after 7 Means followed by same capital letter in the same column are not
statistically different at 5%, by Tukey’s test. Concentration and ratio of photoinitiator system For the DC analysis, Alonso et al. (15), using similar
composite formulations as those in this study, found a
significant reduction of monomer conversion when a
experimental composite containing 0.5 wt% of CQ was
compared to the one containing 1.5 wt%, i.e., the higher
the initiator concentration, the higher the DC. This is
consistent with the present study in which higher CQ
concentrations resulted in higher DC. On the other hand,
Musanje et al. (4) found that composites with 1.44 wt% of
CQ, the polymer matrix would interfere badly and decline
the physical properties of the resin material. In general, the DABE ratio did not influence the
polymerization performance of the experimental
composites, except for the composites C5 and C6 in DC. Based on this, the second hypothesis was rejected, since for
the majority of the composites the DABE concentration did
not influence the selected properties. This result indicates
that the amount of CQ has a greater influence on the curing
efficiency for this resin system than the amine content
and that hydrogen abstraction with the higher amine
content does not help or interfere in forming the polymer
network (16). It might be suggested that the production
of free radicals is not limited by the concentration of
the co-initiator, but duration of curing light exposure. In
this study was used a high energy density (16 J/cm2) for
photoactivate the specimens, which may have contributed
to compensate the effect of amine content. This is in
accordance with previous investigations (24,25) where
DC and rate of polymerization were independent of co-
initiator concentration. Moreover, it has been suggested
that hydrogen abstraction necessary for polymerization
may take place without a co-initiator, possibly by direct
interaction between the initiator and resin monomer, which
contains active hydrogen ions (25). The higher DC for composites with higher concentration
of CQ/DABE may be explained by the fact that they allow
the generation of more free radicals during light application,
promoting increased monomer conversion (16-18). In this
study, the threshold level of photoinitiator concentration
was 0.8% of CQ and 1.6% of the co-initiator (C6), since this
composite reached the maximum DC with good physical
properties and a low yellowing effect. However, for the EM,
the higher the CQ concentration, the higher the EM values
for the tested composites. This finding it is also supported
by the higher DC found in these composites. C.V.S. Grohmann et al. C.V.S. Grohmann et al. 728
Table 2. Means (standard-deviation) of elastic modulus (EM), flexural
strength (FS), yellowing (YL) and degree of conversion (DC) for the
tested experimental composites
Composite
EM (GPa)
FS (MPa)
YL (b*)
DC (%)
C1
2.4 (0.5)CD
104.8 (27.5)A
13.5 (1.1)EF
56.9 (1.2)C
C2
2.1 (0.4)D
107.0 (19.8)A
12.6 (2.8)F
54.9 (3.4)C
C3
2.6 (0.3)CD
117.1 (23.1)A
16.3 (1.2)CDE
57.7 (3.1)C
C4
2.8 (0.5)BC
110.2 (32.0)A
15.7 (1.6)ED
56.9 (3.0)C
C5
2.7 (0.5)CD
121.2 (22.1)A
17.9 (0.8)BCD
61.0 (2.2)B
C6
2.6 (0.2)CD
127.4 (10.7)A
16.2 (0.3)CDE
65.2 (2.2)A
C7
2.6 (0.1)CD
122.9 (13.6)A
19.8 (0.6)B
67.7 (1.2)A
C8
2.7 (0.2)BC
122.1 (19.9)A
19.0 (1.5)BC
66.8 (1.5)A
C9
3.3 (0.4)AB
119.3 (19.3)A
24.4 (1.9)A
66.1 (1.1)A
C10
3.9 (0.2)A
125.5 (21.0)A
24.2 (0.7)A
66.7 (2.5)A
Means followed by same capital letter in the same column are not
statistically different at 5%, by Tukey’s test Table 2. Means (standard-deviation) of elastic modulus (EM), flexural
strength (FS), yellowing (YL) and degree of conversion (DC) for the
tested experimental composites Table 3. Means (standard-deviation) of water sorption (WS) and water
solubility (WSL) for the tested experimental composites
Composite
WS (µg/mm3)
WSL (µg/mm3)
C1
25.1 (6.9) A
13.6 (1.2) A
C2
16.3 (4.0) A
13.4 (1.5) A
C3
19.2 (3.0) A
8.8 (2.1) B
C4
19.8 (6.0) A
4.2 (2.8) B
C5
17.5 (5.5) A
3.5 (2.0) B
C6
12.4 (5.0) A
3.6 (1.6) B
C7
20.6 (5.3) A
9.2 (2.0) B
C8
21.5 (7.0) A
6.8 (3.4) B
C9
17.3 (4.2) A
4.0 (2.0) B
C10
16.9 (4.3) A
1.17 (0.9) C
Means followed by same capital letter in the same column are not
statistically different at 5%, by Tukey’s test. Table 3. Means (standard-deviation) of water sorption (WS) and water
solubility (WSL) for the tested experimental composites Braz Dent J 28(6) 2017 concentrations to maximize cure. However, the yellowing
effect causing by the presence of CQ may cause difficulty
in matching dental restorations, as well as result in a
tendency for internal discoloration due to the amine (1, 22). Therefore, the CQ/amine concentration should be as low as
possible to produce a satisfactory DC but without causing
esthetic problems; this is why the formulation of C6 may
be the optimal concentration of CQ/DABE in the present
study, since it presents high DC with low yellowing effect. days of immersion in distilled water (p=0.15213). C.V.S. Grohmann et al. However,
for the WSB, the composites containing the lower amount
of the photoinitiator system presented higher WSB values
after 7 days of immersion in distilled water (p=0.00001). Discussion The type and concentration of the photoinitiator
system are fundamental parameters that determine the
polymerization characteristics of a resin composite (1). Commercial resin composites are supplied with a wide
range of concentration of these molecules and their effects
on the polymerization behavior and physical-mechanical
properties have not been completely elucidated. Some
studies have evaluated CQ concentrations ranging from
0.05-3 wt% (4,5,13,14). In this study, the concentration
of CQ ranged from 0.4 to 1.5 wt% because they are the
concentrations most commonly observed in the formulation
of the current composite resins. The outcomes was shown
that this range affected the tested mechanical properties
and the yellowing effect. Thus, the first hypothesis tested
was accepted. While water sorption was similar among the composites,
the composites with higher photoinitiator concentration
resulted in less solubility. This result is consistent with
the DC outcomes. The WS may be more dependent upon
the chemical composition of the monomers, which was
equivalent in the ten composites, and thus they did not show
a difference in water uptake. The solubility may be more
related to formulation (monomers type/concentration)
and polymer network formation (monomer conversion). Composites presenting higher DC have fewer residual
monomers to be leached (23). Also, these composites would
be less degraded through hydrolytic degradation by the
water than would composites with poorer conversion (5, 24). Resumo of model resin composites. J Esthet Restor Dent 2015;27:S41-S4 O objetivo foi avaliar a influência da concentração e proporção de
canforoquinona (CQ)/amina terciária (DABE) na resistência à flexão
(RF), módulo flexural (MF), grau de conversão (GC), amarelamento (AM),
sorpção (SA) e solubilidade em água (SL) de compósitos experimentais. Blendas acrilatas foram preparadas com diferentes concentrações e
proporções de CQ/DABE em peso, como segue: (CQ/DABE%): 0,4/0,4%
(C1); 0,4/0,8% (C2); 0,6/0,6% (C3); 0,6/1,2% (C4); 0,8/0,8% (C5); 0,8/1,6%
(C6); 1,0/1,0% (C7); 1,0/2,0% (C8); 1,5/1,5% (C9); 1,5/3,0% (C10). Para RF
e MF, espécimes retangulares (7x2x1 mm, n=10) foram fotoativados com
LED de pico único (Radii Cal) por 20 s e testados em máquina Instron (0,5
mm/min). Após, o GC dos mesmos espécimes (fragmentos) foi mensurado
por FTIR. Para AM, discos de compósito (5x2 mm, n=10) foram preparados,
fotoativados por 20 s e imediatamente avaliados em espectrofotômetro,
considerando o aspecto b do sistema CIEL*a*b*. Para SA e SL, discos de
compósito (5x0,5 mm, n=5) foram preparados e seus volumes calculados
(mm³). Após desidratação, as amostras foram pesadas e armazenadas em
água destilada por 7 dias, pesadas e novamente desidratadas e pesadas,
para se calcular o SA e SL. Os dados foram submetidos a one-way ANOVA
e teste de Tukey (5%). Os grupos C8, C9 e C10 mostraram maior GC, MF e
AM, comparado aos outros grupos. No entanto, RF e SA foram similares
entre todos os grupos. Ainda, C1, C2 e C3 apresentaram maior SL em 7
dias comparado aos outros grupos. Em geral, maiores concentrações de
CQ promoveram melhores propriedades físico-mecânicas; no entanto,
levaram ao maior amarelamento dos compósitos. 10. Oliveira DC, Rocha MG, Gatti A, Correr AB, Ferracane JL, Sinhoreti
MA. Effect of different photoinitiators and reducing agents on cure
efficiency and color stability of resin-based composites using different
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of photoinitiator type on the rate of polymerization, degree of
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of dental dimethacrylate monomers: EPR studies on effects of dilution,
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polymerization and bond strength of resin composite restorations? J
Dent 2011;39:438-447. 3. Segreto DR, Naufel FS, Brandt WC, Guiraldo RD, Correr-Sobrinho L,
Sinhoreti MA. Influence of photoinitiator and light-curing source on
bond strength of experimental resin cements to dentin. Braz Dent J
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between leachability of polymerization initiator and degree of
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photoinitator camphorquinone. Dent Mater 2007;23:655-664. References Effect of photoinitiator on degree of conversion
of unfilled light cured resin. J Dent 1994;22:296–299. 8. Oliveira DC, Silva CB, Muniz BV, Volpato MC, Costa AR, Sinhoreti
MA. Effect of 4-(N,N-dimethylamino)phenethyl alcohol on degree
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composites. Braz Dent J 2014;25:538-542. Received August 2, 2017
Accepted September 11, 2017 Received August 2, 2017
Accepted September 11, 2017 730
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Effects of Psychological Capital, Coping Style and Emotional Intelligence on Self-Rated Health Status of College Students in China During COVID-19 Pandemic
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Associations of psychological capital, coping style
and emotional intelligence with self-rated health
status of college students in China during COVID-19
pandemic Xiangzhi Jing
Xinyang Vocational and Technical college
Hongling Meng
Zhengzhou Normal University
Yuping Li
Zhengzhou Normal University
Lingeng Lu
Yale University School of Public Health
yongcheng yao
(
806306944@qq.com
)
Zhengzhou Normal University
https://orci Abstract Background COVID-19 lockdown can lead to mental health problem, and the problem is heterogeneous
across individuals. In this study, we aimed to explore the association between the self-reported health
status, emotional coping style, emotional intelligence and positive psychological state of college students
during the COVID-19 pandemic in China. Methods The questionnaires of Self-rated Health Measurement Scale (SRHMS), Emotional Intelligence
Scale (EIS), Psychological Capital Questionnaire (PCQ), Simplified Coping Style Questionnaire (SCSQ)
were used for online survey. The study included 367 undergraduates. Results Undergraduate sex, sibling and birth of place were associated with either psychological capital
scores, or/and emotional intelligence. Online time per day had significantly negative associations with
self-rated health and psychological capital scores. Multivariate analysis showed that the interaction
between emotional intelligence, psychological capital and coping styles was statistically significant (β =
-0.112,p =0.045) in health status with psychological capital ranking the top (β = 0.428). Mediation
analysis showed that psychological capital and coping style modified the effect of emotional intelligence
on health status. Conclusions: Psychological capital and coping style modified the effect of emotional intelligence on
health status in college students during the COVID-19 pandemic. In this pandemic emergency, a
comprehensive intervention, such as positive coping styles with optimistic emotional perception and
understanding as well as social support, is an important measure against the pandemic-induced mental
health problem in college students in China. Research Article Keywords: college students, coping style, emotional intelligence, health status, psychological capital
Posted Date: September 9th, 2022
DOI: https://doi.org/10.21203/rs.3.rs-141892/v3
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Version of Record: A version of this preprint was published at Psychology Research and Behavior
Management on September 7th, 2022. See the published version at
https://doi.org/10.2147/PRBM.S383743. Page 1/20 Introduction An outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus
disease 2019 (COVID-19), occurred in early January 2020 in Wuhan, China, resulting in the complete
lockdown of Wuhan right before the eve of Chinese (or Lunar) New Year. To help slow the spread of
highly contagious COVID-19, most of the cities in China later on took a series of prevention and control
measures such as the restriction of traveling, party gathering and business operation, and social
distancing, isolation and contact tracing. To meet the measurement for containing COVID-19, the Ministry
of Education of China made a decision, closing all schools for the spring semester until reopen. Consequently, all students were not allowed to return to campuses for in-person class, instead stay at
home and opt for virtual online courses. Such physical isolation made individuals fewer opportunities to
engage with peers. In addition, the communications with others via internet increased the probability of
excessive online surfing, exposing individuals to a large number of complex information streams. Therefore, the long-term isolation and lockdown or closures and stressful information on COVID-19 could
result in anxiety, fear, grief and other emotional problems, consequently affecting physical, social and
mental health issues 1. Mental health issues, such as difficulty in sleeping or eating, abuse alcohol An outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus
disease 2019 (COVID-19), occurred in early January 2020 in Wuhan, China, resulting in the complete
lockdown of Wuhan right before the eve of Chinese (or Lunar) New Year. To help slow the spread of
highly contagious COVID-19, most of the cities in China later on took a series of prevention and control
measures such as the restriction of traveling, party gathering and business operation, and social
distancing, isolation and contact tracing. To meet the measurement for containing COVID-19, the Ministry
of Education of China made a decision, closing all schools for the spring semester until reopen. Consequently, all students were not allowed to return to campuses for in-person class, instead stay at
home and opt for virtual online courses. Such physical isolation made individuals fewer opportunities to
engage with peers. In addition, the communications with others via internet increased the probability of
excessive online surfing, exposing individuals to a large number of complex information streams. Introduction Therefore, the long-term isolation and lockdown or closures and stressful information on COVID-19 could
result in anxiety, fear, grief and other emotional problems, consequently affecting physical, social and
t l h
lth i
1 M
t l h
lth i
h
diffi
lt i
l
i
ti
b
l
h l Consequently, all students were not allowed to return to campuses for in-person class, instead stay at
home and opt for virtual online courses. Such physical isolation made individuals fewer opportunities to
engage with peers. In addition, the communications with others via internet increased the probability of
excessive online surfing, exposing individuals to a large number of complex information streams. Therefore, the long-term isolation and lockdown or closures and stressful information on COVID-19 could
result in anxiety, fear, grief and other emotional problems, consequently affecting physical, social and
mental health issues 1. Mental health issues, such as difficulty in sleeping or eating, abuse alcohol Page 2/20 Page 2/20 consumption or substance, worsening chronic conditions, increased due to worry and stress over the
pandemic 2-5. To alleviate this potential health problem, mental health experts provided a series of
shareable resources on how to cope with COVID-19 and how to manage stress during this difficult time,
educating individuals how to stabilize emotions with a positive attitude to fight against COVID-19-related
stress. Although college students are young with relatively less susceptible to severe COVID-19 in
comparison to the elderly (greater than 65 years old), campus closure, physical isolation (or home-
confinement) and online instruction still brought negative impacts on their mental health 6. A recent
study consisting a small sample size conducted in Poland reported that the COVID-19 outbreak led to
high stress and low general self-rated health in University students 7. As a special population, college
students’ health always can affect their parents’ and the community’s health and emotional distress. Thus, it is essential to evaluate college students’ health and assess factors influencing mental health in
the context of the COVID-19 pandemic, so that strategies could be made to improve their health and
achievements. Emotional intelligence refers to the ability of an individual to properly perceive and understand emotions,
regulate emotions through certain strategies, and use emotional knowledge to deal with difficulties 8. Introduction In
stressful situations, individuals with high emotional intelligence generally can interact with others in a
receptive and appropriate way, thereby optimally adapting themselves to surrounding environments with
motivation, perseverance, empathy and mental agility 9. In contrast, those with low emotional intelligence
are vulnerable to stress and burnout 10. During the pandemic, nurses with high emotional intelligence
have been shown to have better job performance in a study conducted in Saudi Arabia 11. Emotional
intelligence could reduce the anxiety in international students studying in China during the pandemic12. Wang and colleagues reported that professional students who had a high level of emotional intelligence
could keep mental health better during the pandemic13. Psychological capital refers to a set of resources an individual can use to help improve their performance
on the job and their success. It consists of four aspects: self-efficacy (confidence), optimism, hope and
resilience. It has been shown that psychological capital could ameliorate the negative consequences of
stress on mental health, and enhance positive psychological outcome in undergraduate students 14. High
psychological capital empowers individuals with resilience strength to cope up with adverse situations. The results from another study also suggest that increasing psychological capital has a protective effect
on the risk of burnout and traumatic stress in social workers, augmenting the sustainability of their
working conditions15. Similar results were reported in a previous study on Chinese nurses, showing that
psychological capital reduced job burnout with the help from organizational commitment16. Psychological resilience, an ability to “bounce back” from a negative emotional experience and to
confront stressful situations, is a major protective factor of burnout in nurses 17,18. Moreover, a recent
study reported that psychological support was positively associated with better mental health19, which
might alleviate psychological impact in Bangladesh university students during COVID-19 pandemic20. Psychological capital refers to a set of resources an individual can use to help improve their performance
on the job and their success. It consists of four aspects: self-efficacy (confidence), optimism, hope and
resilience. It has been shown that psychological capital could ameliorate the negative consequences of
stress on mental health, and enhance positive psychological outcome in undergraduate students 14. High
psychological capital empowers individuals with resilience strength to cope up with adverse situations. Introduction The results from another study also suggest that increasing psychological capital has a protective effect
on the risk of burnout and traumatic stress in social workers augmenting the sustainability of their Page 3/20 Page 3/20 Mediating analyses showed that psychological capital had direct and indirect effect on mental health via
social capital during COVID-19 pandemic21. When individuals confront stress or difficulties, different persons may take different coping strategies
of either task-, emotion- and avoidance-oriented coping. Previous studies have shown that a lower
burnout is accompanied with task-oriented coping, whereas an increased burnout results from emotion-
oriented coping, and the long-term avoidance-oriented coping can lead to severe mental problem 22-25. Over the period COVID-19 pandemic, individuals with poor coping styles had adverse mental health of
anxiety and depression 26,27. However, the anxiety and depression could be substantially alleviated if an
individual had a plenty of resources (psychological capital) to use, which could make the one to take an
appropriate coping strategy 28. Moreover, individuals with a strong emotional intelligence have an ability
to restrain negative emotions, low self-esteem and anxiety, and they can courageously face the challenge
of stress-related issues 29. Previous studies also showed that sex, marital status, and birth place were
also associated with job-related stress and burnout in nurses 30,31. Female students were more anxious
and stressed than males during COVID-19 pandemic in Bangladesh20. Taken together, we hypothesize that emotional intelligence, psychological capital and coping style are
associated with self-rated health of college students during the COVID-19 pandemic in China. Thus, the
purposes of this study were to investigate the associations among emotional intelligence, psychological
capital, coping style and health status in college students during the COVID-19 by conducting a cross-
sectional survey in Henan, China, so that we can provide theoretic basis to make strategies to keep
students healthy in the context of COVID-19. Participants And Methods In this study, the
Cronbach’s α coefficient of the scale was 0.890. Psychological capital: The psychological capital questionnaire (PCQ) developed by Luthans et al. was
used to comprehensively evaluate psychological capital. The Chinese version of PCQ was translated by
Li and colleagues 34. The questionnaire contains 24 items in four measure dimensions: self-efficacy,
hope, resilience and optimism. Each item uses Likert 6-level scoring method, 1 means "strongly disagree"
and 6 for "strongly agree". The higher the score is, the higher the level of positive psychology for
individuals, and the more resources they can use to improve their performance on the job and their
success. The Cronbach’s α coefficient of the scale in this study was 0.958. Coping style: The Simplified Coping Style Questionnaire (SCSQ) was used to measure the coping style of
college students35. There are 20 items in two dimensions of positive and negative coping. The score each
item is graded 0 to 3. A high score of the positive coping indicates that the individuals are more inclined
to adopt positive coping styles with efforts to solve a stressful situation/problem, and a high score of the
negative coping refers that the individuals are more inclined to adopt negative coping styles with
distancing or avoiding a stressful situation. The sum score of coping style is equal to the positive coping
score minus the negative coping score. The Cronbach’s α coefficient of the positive coping and negative
coping in this study was 0.863 and 0.741, respectively. Statistical analysis: All data analyses were performed using SPSS 18.0 software. The measurement data
showed an approximate normal distribution using the normality test with both Skew and kurtosis
methods. Online surfing time was grouped based on the cutoff of 3 and 6 h as previously described
elsewhere36. Pearson correlation analysis was performed to examine the correlation between self-rated
health, emotional intelligence, psychological capital, and coping style. ANOVA test was used to compare
the difference in numerical variables with Bonferroni correction for multiple comparisons. Multivariate
regression analysis was used to explore the effect of emotional intelligence, psychological capital, and
coping style on and their interaction in self-rated health 37. The PROCESS plugin for SPSS was used to
assess the strength of the indirect effects in mediation analyses 38. In PROCESS, total, direct and indirect
effects were calculated and tested for significance 39-41. Participants And Methods Study subjects: In May 2020, we conducted a survey on the impact of COVID-19 pandemic on health
status in college students in China using online questionnaires via the platform of “Questionnaire Star”. There are 25 universities in Zhengzhou, and one college was chosen by randomly sampling based on the
first letter of the sorted school names, each of the schools was assigned a number for selection. The
study team first contacted the class teacher for explanation of the study project. Then through WeChat
class group for each class, the questionnaire was delivered to each student in the group with the
instruction. A total number of 367 undergraduate students voluntarily participated and provided valid
questionnaires. Among the participants, 88 (24.0%) were men, and 279 (76%) women. The age ranged
from 18-24 years old, with an average of 20 years old. The basic demographics and questions include
such as sex, age, birthplace, and time length spent on internet surf per day. Informed consent was
obtained from all participants included in the study. Health status: Using the self-rated health measurement scale in Chinese version 32 (self-rated health
measurement scale, SRHMS), the health status of each participant was evaluated. The measurement
scale includes 48 items in three dimensions: physical, mental, and social health. Using 1 to 5 scoring Page 4/20 Page 4/20 method for each item, the scores of each dimension are the sum of all items in the dimension. The higher
the score is, the better the health status. The Cronbach’s α coefficient of the scale in this study was 0.939. method for each item, the scores of each dimension are the sum of all items in the dimension. The higher
the score is, the better the health status. The Cronbach’s α coefficient of the scale in this study was 0.939. Emotional Intelligence: The Emotional Intelligence Scale (EIS), which was developed by Wong and Law 33,
has 16 items in 4 measure dimensions of emotional awareness and management of one’s own and
others: self-emotional evaluation, emotional evaluation of others, emotional use and management. Using
the 5-point rating method for each item, 1 represents "strongly disagree", and 5 for "strongly agree". The
higher the score of the subject is, the higher the level of emotional intelligence is, and the higher ability to
regulate one’s own emotions is when necessary and help others to do the same. Participants And Methods The mean value for the ab (a × b) product across
the bootstrapped samples provided a point estimate of the indirect effect and 95% confidence intervals
(CI). Each analysis was based on 5000 bootstrapped samples, as suggested by Preacher 40. A p value
less than 0.05 was considered statistically significant. Page 5/20 Page 5/20 Page 5/20 Results Common-method variance (CMV) test Common-method variance (CMV) test Common-method variance (CMV) test The Harman’s single factor test was used to measure the common-method variance. The result showed
no severe common-method variance existing in this study. Twenty-two factors had eigenvalues greater
than 1, and 27.67% of the variance was explained by the first factor, which is less than the criterion of
40%42. Characteristics of self-rated health, emotional intelligence, psychological capital and coping style in
college students during COVID-19 pandemic Table 1 illustrates the associations of self-rated health, emotional intelligence, psychological capital and
coping style scores with demographic variables in college students of a university in China during the
COVID-19 pandemic period. Male students had a significantly higher score of psychological capital than
female (p < 0.05). The students who have no siblings had a significantly higher score of both emotional
intelligence and psychological capital than those having siblings (p < 0.05). There were significant
differences in emotional intelligence scores between the students from different areas (p < 0.05). Pairwise comparisons showed that the students from urban areas had a significantly higher emotional
intelligence score than those from rural areas (p < 0.05). Significant differences were also observed in
self-rated health status, and psychological capital scores between students who spent different hours per
day in internet surfing (p < 0.05). With online surfing hours per day increasing, the scores of self-rated
health and psychological capital significantly decreased. Students who spent less than 3 h online surfing
per day had significantly better health status or higher psychological capital scores than those who spent
more than 6 h (p < 0.05). Table 1 illustrates the associations of self-rated health, emotional intelligence, psychological capital and
coping style scores with demographic variables in college students of a university in China during the
COVID-19 pandemic period. Male students had a significantly higher score of psychological capital than
female (p < 0.05). The students who have no siblings had a significantly higher score of both emotional
intelligence and psychological capital than those having siblings (p < 0.05). There were significant
differences in emotional intelligence scores between the students from different areas (p < 0.05). Pairwise comparisons showed that the students from urban areas had a significantly higher emotional
intelligence score than those from rural areas (p < 0.05). Significant differences were also observed in
self-rated health status, and psychological capital scores between students who spent different hours per
day in internet surfing (p < 0.05). Common-method variance (CMV) test With online surfing hours per day increasing, the scores of self-rated
health and psychological capital significantly decreased. Students who spent less than 3 h online surfing
per day had significantly better health status or higher psychological capital scores than those who spent
more than 6 h (p < 0.05). Page 6/20 Table 1. Associations of self-rated health, emotional intelligence, psychological capital
and coping style with demographic variables in college students(mean ± SD)
Variable
N
Self-rated
health
Emotional
intelligence
Psychological
capital
Coping
style
Sex
Male
88
335.31±50.54
46.05±6.09
107.24±16.86
0.77±0.64
Female
279
335.76±42.65
44.91±4.77
102.72±15.99
0.83±0.61
t
﹣0.076
1.816
2.283﹡
﹣0.824
Having Siblings
No
61
342.56±44.09
46.70±5.18
109.21±16.13
0.90±0.69
Yes
306
334.27±44.64
44.88±5.08
102.72±16.14
0.80±0.60
t
1.326
2.558﹡
2.869﹡﹡
1.226
Birthplace
Urban
104
338.42±45.90
46.39±4.69
106.38±16.08
0.81±0.63
Suburban
41
342.34±43.23
45.02±5.94
104.44±16.59
1.00±0.65
Rural
222
333.12±44.60
44.64±5.09
102.47±16.25
0.78±0.61
F
1.021
4.216﹡
2.089
2.19
Online surfing
time
<3h
88
342.00±43.59
45.72±5.28
107.90±16.44
0.89±0.65
3~6 h
178
338.27±42.50
45.35±5.15
104.53±14.83
0.82±0.59
>6 h
101
325.50±47.72
44.43±4.92
98.95±17.53
0.74±0.65
F
3.872﹡
1.67
7.708﹡﹡
1.237
﹡P<0.05,﹡﹡P< 0.01; ﹡P<0.05,﹡﹡P< 0.01; Correlations between health status, emotional intelligence, psychological capital and coping style scores
in college students Correlations between health status, emotional intelligence, psychological capital and coping style scores
in college students The results of Pearson correlation analyses are shown in Table 2. There were significantly positive
correlations between self-rated health, emotional intelligence, psychological capital and coping style
scores in college students during the COVID-19 pandemic (p < 0.01). A moderate correlation strength was
observed between either emotional intelligence, or psychological capital or coping style, or self-rated
health, as well as between emotional intelligence and psychological capital (correlation coefficients
ranged from 0.50 to 0.65). A relatively weak positive correlation existed between coping style score and
either emotional intelligence or psychological capital (correlation coefficients 0.35 and 0.42). Page 7/20 Table 2. Pearson correlations between health status, emotional intelligence,
psychological capital and coping style in college students (N = 367)
Variable
Mean
SD
Self-rated
health
Emotional
intelligence
Psychological
capital
Coping
style
Self-rated health
335.65
44.60
1
Emotional
intelligence
45.18
5.13
0.50﹡﹡
1
Psychological
capital
103.80
16.29
0.63﹡﹡
0.65﹡﹡
1
Coping style
0.82
0.62
0.50﹡﹡
0.35﹡﹡
0.42﹡﹡
1
﹡﹡p< 0.01. Table 2. Pearson correlations between health status, emotional intelligence,
psychological capital and coping style in college students (N = 367) Table 2. Common-method variance (CMV) test Pearson correlations between health status, emotional in
psychological capital and coping style in college students (N Association of self-rated health with emotional intelligence, psychological capital and coping style To investigate the effect of emotional intelligence, psychological capital and coping style on and their
interactions in self-rated heath in college students during the period of COVID-19 pandemic, we performed
multivariate regression analyses by constructing three different models. The results are illustrated in
Table 3. All the tolerances were greater than 0.1 and VIF <10, so there is no collinearity of the variable. In
the model 1, we included the main effects of three variables with sex and having siblings as covariates
only. We found that the main effects explained 47.5% variation of self-rated health. The emotional
intelligence, psychological capital and coping style had significantly positive effects, whereas sex and
siblings were not statistically significant. The coefficient of psychological capital was the largest,
suggesting that psychological capital was the largest contributor to self-rated health in the model. In the
model 2, then we added pairwise interaction terms of emotional intelligence, psychological capital and
coping style besides the main effects. No pairwise interaction was found statistically significant. We then
added a three-way interaction term beyond the model 2 in the model 3, and we found the three-way
interaction term was statistically significant (p = 0.045). The three-way interaction independently
explained 0.6% variation of self-rated health. Page 8/20 Page 8/20 Table 3. Common-method variance (CMV) test diating effect of psychological capital and coping style on emotional
intelligence and self-rated health Note: ∗P<0.05,∗∗P<0.01,∗∗∗P<0.001; a, the effect of the independent variable on mediating variable; b, the
effect of mediating variable on self-rated health; c, the total effect of the independent variable on self-rated
health; c', the direct effect of the independent variable on self-rated health after the introduction of mediating
variable; a × b, the mediating effect of mediating variable between emotional intelligence and self-rated health To visualize the effect of emotional intelligence, psychological capital and coping style and its interaction
in self-rated health in college students during the period of COVID-19, we further constructed a figure
showing the relationship between three variables and self-rated health based on the method for a three-
way interaction as described by Dawson and Richter37 (Figure 1). With the increased emotional
intelligence, the self-rated health score increased regardless of psychological capital and coping style. At
the same level of emotional intelligence, the self-rated health status was better in college students with
high vs. low psychological capital. At the same levels of emotional intelligence and psychological capital,
the self-rated health score was better in college students who actively took positive vs. negative coping
styles. Overall, individuals with high psychological capital and coping style show the best health status
regardless of emotional intelligence. High emotional intelligence improves health status in comparison to
low one at the same level of psychological capital and coping style. Common-method variance (CMV) test Effect of emotional intelligence, psychological capital and coping style on self-
rated health in college students
Variable
b
p value
Tolerance
VIF
Model 1
Sex
0.056
0.146
0.961
1.040
Having Siblings
0.024
0.527
0.964
1.037
Emotional Intelligence
0.170
0.001
0.546
1.830
Psychological capital
0.419
<0.001
0.524
1.909
Coping style
0.250
<0.001
0.767
1.304
Adjusted R2
0.475
Model 2
Sex
0.053
0.176
0.936
1.069
Having Siblings
0.024
0.532
0.959
1.043
Emotional Intelligence
0.171
0.001
0.543
1.841
Psychological capital
0.415
<0.001
0.501
1.995
Coping style
0.253
<0.001
0.744
1.345
Emotional intelligence × psychological capital
0.053
0.335
0.473
2.114
Emotional intelligence × coping style
-0.071
0.283
0.334
2.993
Psychological capital × coping style
-0.015
0.799
0.404
2.476
Adjusted R2
0.474
ΔR2
0.003
0.517
Model 3
Sex
0.051
0.194
0.935
1.070
Having Siblings
0.028
0.476
0.957
1.045
Emotional Intelligence
0.218
<0.001
0.450
2.224
Psychological capital
0.428
<0.001
0.495
2.022
Coping style
0.289
<0.001
0.636
1.573
Emotional intelligence × psychological capital
0.068
0.217
0.464
2.154
Emotional intelligence × coping style
-0.061
0.354
0.332
3.009
Psychological capital × coping style
-0.018
0.757
0.404
2.478
Emotional intelligence × Psychological capital × coping style
-0.112
0.045
0.457
2.186
Adjusted R2
0.478
ΔR2
0.006
0.045 To further investigate the mediating effect of psychological capital and coping style on emotional
intelligence and self-rated health, we performed mediation analysis using the Bootstrap method. The
results are shown in Table 4. We found that the indirect effects from both psychological capital and
coping style were statistically significant. The contribution of psychological capital accounted for 58.8%
and coping style for 26.1%. Page 9/20 Page 9/20 Table 4. Mediating effect of psychological capital and coping style on emotional
intelligence and self-rated health
Mediating
Variable
Emotional
intelligence (a)
Self-rated
health (b)
Total
effect (c)
Direct
Effect (c’)
Indirect effect
(ab) (95%CI)
Percentage
(ab/c) %
Psychological
capital
2.05∗∗∗
1.36∗∗∗
4.74∗∗∗
1.95∗∗∗
2.79
(2.10,3.51)
58.8
Coping style
0.05∗∗∗
24.41∗∗∗
4.74∗∗∗
3.50∗∗∗
1.24
(0.86,1.66)
26.1
Note: ∗P<0.05,∗∗P<0.01,∗∗∗P<0.001; a, the effect of the independent variable on mediating variable; b, the
effect of mediating variable on self-rated health; c, the total effect of the independent variable on self-rated
health; c', the direct effect of the independent variable on self-rated health after the introduction of mediating
variable; a × b, the mediating effect of mediating variable between emotional intelligence and self-rated health. Discussion In this study, we demonstrated the associations between emotional intelligence, psychological capital,
coping style and self-rated health in college students in China during the period of COVID-19 pandemic. We found all three variables were significantly positively associated with self-rated health, and there was
a statistically significant three-way interaction. We also found that almost 60% of total effect of
emotional intelligence on health status could be explained by psychological capital, whereas coping style
accounted for 26.1%. The results suggest that college students who had high emotional intelligence,
psychological capital and coping style scores had better health conditions than their peers with low ones. Particularly, psychological capital provided more resources to help students to overcome the stress
challenge. We also found that psychological capital and coping style modified the effect of emotional
intelligence on health status. This result extends the previous findings reported by another study recently
conducted in China, in which social capital mediated the effect of psychological capital on mental health
in Chinese residents during COVID-19 pandemic21. To our knowledge, this is the first study to investigate Page 10/20 the effect of emotional intelligence, psychological capital and coping style, and their interaction on
college student health during the pandemic. Emotional intelligence is an important determinant in an individual’s life success, and affects a person's
mental health and social status. The stronger the individual's ability to regulate emotion is, the more
helpful it is for the person to relieve stress and stabilize his/her emotional state 43. A recent study
demonstrated that the emotional intelligence workshops and seminars could improve academic
engagement and performance, and reduce academic burnout in pharmacy students during the lockdown
and quarantine 44. Individuals with a positive psychological capital score showed a more determined
attitude in the face of difficulties 45, and believed that they had abilities and more resources to use to
overcome difficulties and achieve success when facing the challenges. Lebares and colleagues reported
that a high psychological resilience was significantly positively associated with a low risk of emotional
exhaustion-related burnout and low job performance 46. Similarly, Hafsa and colleagues also observed a
positive correlation between emotional intelligence and loneliness in adolescents 47. High emotional
intelligence could significantly alleviate the anxiety, maintaining mental health for student during the
pandemic12,13. Discussion Moreover, it has been shown that psychological support might mitigate the effect of
COVID-19-caused stress in university students in Bangladesh during COVID-19 pandemic20, and that
individuals with more psychological support had better mental health status19. In consistence with this
principal, our study showed that at the same level of emotional intelligence, college students with a high
psychological capital score had better self-rated health state than those with a low one. Coping style refers to the cognitive and behavioral styles that individuals take to deal with the frustrations
and difficulties when they face in daily life. The choice of coping styles, task-oriented, emotion-oriented
and avoidance-oriented, determines the consequent effect of stress events on individuals’ health 48. Individuals taking task-oriented coping will actively find a solution to solve the stress problems by such
as learning new skills or obtaining more information to manage and consequently eliminate it. Emotion-
oriented coping involves an emotional response to the perception of stress, alleviating the stressor-
associated unpleasant stressful feelings. Avoidance-oriented coping instead involves activities and
cognition to avoid the stressful situations, always accompanying with distraction and social diversion. Repeated or long-term avoidance or distancing from a stressful situation can result in a detrimental
consequence to mental health. Individuals with a positive coping style usually actively try to find a
problem solution (active coping), face the challenge, and seek social support with optimistic attitude. A
previous study demonstrated that the positive coping styles could promote individual’s mental health
with a positive attitude, and reduce psychological harm 49. For example, a negative correlation was found
between positive coping style and psychological stress in infertile women 50. A similar finding was found
in patients with either breast cancer or osteoarthritis, the coping style affected the mental health 51,52. Another study showed that undergraduate nursing students with higher personal resilience (positive
coping style) had a lower stress during COVID-19 pandemic in India53. Similarly, students who had an Page 11/20 Page 11/20 emotion-focused coping style had a higher level of anxiety during the COVID-19 pandemic in Poland54. A
two-phase longitudinal study showed that during the COVID-19 pandemic, Chinese children with active-
avoidance coping style had a low level of anxiety and depression55. Negative coping style associated
with psychological distress in Chinese college students56. Discussion A recent study conducted in a large university
of the Mid-Atlantic region in the United States showed that extraversion and avoidance coping style
moderated the effect of COVID-19 stressful life events on mental health57. Moreover, a positive
correlation was observed between positive coping style and less psychological distress in Indian
population during lockdown58. Furthermore, when an individual has more psychological capitals during
the grow-up, it is easier for him/her to take a positive coping style to find solutions with more positive
optimism when facing challenges 59, promoting the individual's health. In this study, we found that the
psychological capital was a major effect on self-rated health with a statistically significant three-way
interaction analysis. Seeking social support and taking a positive coping style are important in
maintaining a good health status when they face a stressful situation, and it is a more active strategy
even at the same level of emotional intelligence. In this study, we also found that sex, having sibling or not, birthplace and online surfing time were
associated with emotional intelligence, psychological capital and coping style. Previous studies have
shown that women had a higher emotional intelligence than men in the nursing profession 60,61. However,
we did not find a significant difference in emotional intelligence in college students. This discrepancy is
most likely duo to the difference in social experience. Undergraduate Students, particularly in China, have
much less experience in society in comparison to nurses or other professionals. Male college students
have a relatively more social relation, and more resources to use. Thus, when they face challenges and
difficulties, generally, they are more active with courage and optimism to solve the problems in
comparison to their female peers. The college students from only-one child family (no siblings) usually
had a higher emotional intelligence and psychological capital score, since they probably started earlier to
play together with peers from other families, learning mutual and social relationships through the
interaction. Similarly, the students from urban area usually had more skills to identify and manage their
own emotions and might affect others as necessary, since their family incomes were relatively higher
with more opportunities to expose themselves to different cultures in urban than in rural area. In addition,
although the internet emergency facilitates the access to obtain knowledge and information, the internet
also provides a virtual village for users, allowing online interactions behind screen but with isolating
themselves from others. Conclusion In summary, this study demonstrated the associations of emotional intelligence, psychological capital
and coping style with health status in college students in China during the period of COVID-19 pandemic
We found that interaction existed between psychological capital, coping style and emotional intelligence
in college students’ health with the psychological capital and coping style modifying the effect of
emotional intelligence on health status in college students regardless of sex and whether they had
siblings or not under the pandemic stress. The findings provide knowledge for our educational
administrators how to improve and maintain college students’ health, keeping them mentally healthy
during the period of COVID-19 pandemic. A comprehensive intervention measure targeting emotional
intelligence, coping style and psychological capital (e.g., social support) is warranted. Discussion It has been reported that dissociation positively associated with depression in
married women 62. Internet addiction made college students insufficient personal communication with
peers, thereby leading to mental health problem56. Interestingly, the significance of sex and siblings in
the Univariate analyses disappeared in the multivariate analyses, suggesting that the associations of sex
and siblings with mental health are dependent. There was a significantly positive correlation among the variables of college students’ emotional
intelligence, psychological capital, and coping styles as expected. The findings suggest that college
students with a high emotional intelligence have more resources and adopt a positive coping style, since Page 12/20 Page 12/20 they have a strong ability to perceive, evaluate, understand, express their own emotions and influence
others’ 63. When they had more psychological capitals to use, they took more positive coping styles with
optimism to face difficulties 64. Thus, a better health status was maintained. Some limitations exist in this study. This survey is a cross-sectional study, and it is difficult to make
causal inference from the association between the factors we investigated and self-rated health. Secondly, the sample size is relatively small, and the survey was conducted in one college only, bias may
exist and the participants may not well represent the whole population of college students. However, the
findings warrant further studies with a relatively large sample size. Longitudinal or intervention studies
can be carried out Some limitations exist in this study. This survey is a cross-sectional study, and it is difficult to make
causal inference from the association between the factors we investigated and self-rated health. Abbreviations SARS-CoV-2, Severe acute respiratory syndrome coronavirus 2; COVID, contagious coronavirus disease;
SRHMS, Self-rated Health Measurement Scale; EIS, Emotional Intelligence Scale; PCQ, Psychological
Capital Questionnaire; SCSQ, Simplified Coping Style Questionnaire. Ethics approval and consent to participate The procedures followed were in accordance with the ethical standards of the Committee on Zhengzhou
Normal University (ZZNU-2020-006). The questionnaire had an introductory paragraph explaining the
purpose of the survey and the name of the research center undertaking the research, and that responses
were anonymous. All procedures performed in studies involving human participants were in accordance
with the ethical standards of the institutional and/or national research committee and with the 1964
Helsinki declaration and its later amendments or comparable ethical standards. Consent for publication Consent for publication Page 13/20 Availability of data and materials The dataset supporting the conclusions of this article can be shared with the corresponding author by
email. Funding This study was supported by Henan Province Educational Science “13th Five-year Plan” Fund
(2020YB0256). All the authors listed have approved the manuscript that is enclosed. All the authors listed have approved the manuscript that is enclosed. Authors’ contributions YY and XJ designed the research, carried out data analysis, wrote the paper, and YY is the corresponding
author. HM provided guidance in study design, organized the investigation. LL, YL and XJ provided help
with the data analysis, interpretation, and write-up. YL provided help with the data collection and
interpretation. All authors read and approved the final manuscript. Acknowledgements: We thank all participants who voluntarily completed the questionnaires in this study. We also wish all students stay well during the pandemic and achieve their goals in their careers. Acknowledgements: We thank all participants who voluntarily completed the questionnaires in this study. W
l
i h
ll t d
t
t
ll d
i
th
d
i
d
hi
th i
l i
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Nursing. 2019;26(4):75-78. Figures Figure 1 Page 19/20 Interaction of emotional intelligence, psychological capital and coping style in self-rated health. Blue line
represents the individuals with high psychological capital and coping style; grey line represents those
with high psychological capital but low coping style; tangerine line represents low psychological capital
but high coping style, and orange line represents low psychological capital and coping style. Interaction of emotional intelligence, psychological capital and coping style in self-rated health. Blue line
represents the individuals with high psychological capital and coping style; grey line represents those
with high psychological capital but low coping style; tangerine line represents low psychological capital
but high coping style, and orange line represents low psychological capital and coping style. Interaction of emotional intelligence, psychological capital and coping style in self-rated health. Blue line
represents the individuals with high psychological capital and coping style; grey line represents those
with high psychological capital but low coping style; tangerine line represents low psychological capital
but high coping style, and orange line represents low psychological capital and coping style. Interaction of emotional intelligence, psychological capital and coping style in self-rated health. Blue line
represents the individuals with high psychological capital and coping style; grey line represents those
with high psychological capital but low coping style; tangerine line represents low psychological capital
but high coping style, and orange line represents low psychological capital and coping style. Page 20/20
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1. INTRODUCTION tion is not certain, effective area sampled is obtained by in-
tegrating under an estimated detection probability surface. DS
methods estimate the detection probability surface by using ob-
served distances to detections to estimate detection probability
as a function of distance from detector. CR methods have un-
til recently had no statistically rigorous method for estimating
density, but this changed with the advent of spatially explicit
capture–recapture (SECR) methods (Efford 2004; Borchers and
Efford 2008; Royle and Young 2008; Royle et al. 2013a). SECR
data do not include distances to animal locations; instead SECR
methods use the distances between detectors at which animals
are (and are not) detected to estimate a distance-based detection
probability surface. Estimating animal population density is crucial for success-
ful and efficient management and conservation of wildlife re-
sources. As a complete census is rarely feasible, this usually
requires survey sampling, most often using one of the two dom-
inant survey methods: capture–recpature (CR) or distance sam-
pling (DS; see Schwarz and Seber 1999; Borchers, Buckland,
and Zucchini 2002; Williams, Nichols, and Conroy 2002; Royle
and Dorazio 2008, for overviews of methods). In CR, a series of
detectors (e.g., traps or cameras) are deployed on multiple sam-
pling occasions. The resulting “capture history” of occasions on
which each uniquely identified animal was detected is used to
estimate the probability of detection, and hence account for un-
detected animals. DS requires only a single survey occasion and
uses the distances of detected animals from detectors to estimate
the detection probability and hence account for animals missed. As it involves a distance-based detection function, SECR is
closer to DS than is traditional CR, and in fact SECR methods
have borrowed detection function forms from DS. At the same
time, there have been developments in DS that bring it closer
to CR methods. For example, standard DS methods have been
extended to use two independent observers, generating capture
history as well as DS data—a method known as mark recap-
ture distance sampling (MRDS; Manly, McDonald, and Garner
1996; Borchers, Zucchini, and Fewster 1998). Both methods sample a subset of the area occupied by the
population of interest and both require some measure of ef-
fective area sampled to estimate animal density. A Unifying Model for Capture–Recapture and Distance
Sampling Surveys of Wildlife Populations D. L. BORCHERS, B. C. STEVENSON, D. KIDNEY, L. THOMAS, and T. A. MARQUES A fundamental problem in wildlife ecology and management is estimation of population size or density. The two dominant methods in this
area are capture–recapture (CR) and distance sampling (DS), each with its own largely separate literature. We develop a class of models that
synthesizes them. It accommodates a spectrum of models ranging from nonspatial CR models (with no information on animal locations)
through to DS and mark-recapture distance sampling (MRDS) models, in which animal locations are observed without error. Between
these lie spatially explicit capture–recapture (SECR) models that include only capture locations, and a variety of models with less location
data than are typical of DS surveys but more than are normally used on SECR surveys. In addition to unifying CR and DS models, the
class provides a means of improving inference from SECR models by adding supplementary location data, and a means of incorporating
measurement error into DS and MRDS models. We illustrate their utility by comparing inference on acoustic surveys of gibbons and frogs
using only capture locations, using estimated angles (gibbons) and combinations of received signal strength and time-of-arrival data (frogs),
and on a visual MRDS survey of whales, comparing estimates with exact and estimated distances. Supplementary materials for this article
are available online. KEY WORDS:
Abundance estimation; Acoustic survey; Closed population; Measurement error; Visual survey. Supplementary materials for this article are available online. Please go to www.tandfonline.com/r/JASA upplementary materials for this article are available online. Please go to www.tandfonline.com/r/JASA ©
David Borchers, Ben Stevenson, Darren Kidney, Len Thomas, Tiago
Marques Published with license by Taylor & Francis
Journal of the American Statistical Association
March 2015, Vol. 110, No. 509, Theory and Methods
DOI: 10.1080/01621459.2014.893884 ©
David Borchers, Ben Stevenson, Darren Kidney, Len Thomas, Tiago
Marques
This is an Open Access article distributed under the terms of the Cre-
ative Commons Attribution-Non-Commercial License (http://creativecommons.
org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distri-
bution, and reproduction in any medium, provided the original work is properly
cited. The moral rights of the named author(s) have been asserted.
D. L. Borchers (E-mail: dlb@st-andrews.ac.uk), B. C. Stevenson (E-mail:
bcs5@st-andrews.ac.uk),
D.
Kidney,
(E-mail:
dk253@st-and.ac.uk),
L.
Thomas
(E-mail:
dk253@st-and.ac.uk),
and
T.
A.
Marques
(E-mail:
tiago@mcs.st-and.ac.uk), Centre for Research into Ecological and Environ-
mental Modelling, University of St. Andrews and The Observatory, Buchanan
Gardens, Fife, KY16 9LZ, Scotland. We are grateful to the National Geographic
Society/Waitt Grants Program for a grant (W184-11) for funding the frog sur-
vey, to John Measey and Res Altwegg for conducting it, to Ben Rawson and
Conservation International for the gibbon data, and the Marine Research In-
stitute of Iceland for the minke whale data. Marques was part-funded by the
Fundacao Nacional para a Ciencia e Tecnologia, Portugal (FCT) under the
project PEst OE/MAT/UI0006/2011. This work was part-funded by EPSRC
grant EP/I000917/1. We are grateful to two anonymous referees, which sub-
stantially improved the article.
C l
i
f
f th fi
i th
ti l
b f
d
li g
( )
D. L. Borchers (E-mail: dlb@st-andrews.ac.uk), B. C. Stevenson (E-mail:
bcs5@st-andrews.ac.uk),
D.
Kidney,
(E-mail:
dk253@st-and.ac.uk),
L.
Thomas
(E-mail:
dk253@st-and.ac.uk),
and
T.
A.
Marques
(E-mail:
tiago@mcs.st-and.ac.uk), Centre for Research into Ecological and Environ-
mental Modelling, University of St. Andrews and The Observatory, Buchanan
Gardens, Fife, KY16 9LZ, Scotland. We are grateful to the National Geographic
Society/Waitt Grants Program for a grant (W184-11) for funding the frog sur-
vey, to John Measey and Res Altwegg for conducting it, to Ben Rawson and
Conservation International for the gibbon data, and the Marine Research In-
stitute of Iceland for the minke whale data. Marques was part-funded by the
Fundacao Nacional para a Ciencia e Tecnologia, Portugal (FCT) under the
project PEst OE/MAT/UI0006/2011. This work was part-funded by EPSRC
grant EP/I000917/1. We are grateful to two anonymous referees, which sub-
stantially improved the article. This is an Open Access article distributed under the terms of the Cre-
ative Commons Attribution-Non-Commercial License (http://creativecommons.
org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distri-
bution, and reproduction in any medium, provided the original work is properly
cited. The moral rights of the named author(s) have been asserted. Color versions of one or more of the figures in the article can be found online
at www.tandfonline.com/r/jasa. 3.2 Probability Model and Likelihood Gibbons are difficult to detect visually in forest but can
be detected quite easily acoustically when they make territo-
rial calls. An acoustic survey with human detectors, of north-
ern yellow-cheeked gibbon (Nomascus annamensis) was con-
ducted in northeastern Cambodia by Conservation International
in 2010. The design involved three people standing in a line
spaced approximately 500 m apart, recording estimated angles
to all gibbon groups they heard. Observers who detected a group
comprise the group’s capture history, while the estimated angles
to detected groups provide additional data on group location. Use of the additional data is shown to improve inference. Consider a survey of a region with surface area A in which K
detectors are deployed on S occasions. Following Borchers and
Efford (2008), we assume that animals are independently dis-
tributed in this region according to a nonhomogeneous Poisson
process (NHPP) with parameter vector φ and intensity D(x; φ)
at x. We denote the probability that an animal at x is detected
by at least one detector on the survey as p·(x; θ), with unknown
parameter vector θ. It follows that the locations of detected an-
imals, X = (x1, . . . , xn), are realizations of a filtered NHPP
with intensity D(x; φ)p·(x; θ) at x. We construct a probability model for the outcomes of a survey
via a product of conditional probabilities, which are developed
below. The first component of the model is the probability of
detecting n animals: P(n; φ, θ). The second is the probability
density function (pdf) of animal locations, X, conditional on
detection, which we write as fX(X; φ, θ). 2.2 Frog Survey An acoustic survey of Lightfoot’s moss frog (Arthroleptella
lightfooti) in a water seepage on Table Mountain, South Africa,
was conducted using six microphones in a roughly rectangular
arrangement. The survey is similar to the gibbon survey in that
spatial capture histories consist of the locations of detectors (mi-
crophones) at which each vocalization (frog click) was detected. The time difference of arrival (TDOA) of the same click at dif-
ferent detectors and the received SS at each detector provide
additional data on animal location. Each of the additional data
types improves inference in this case. The third component is the probability of observing the
capture histories , conditional on detections and detected
animal locations X, which we write as P( | X; θ). Here
= (ω1, . . . , ωn), where ωi is the capture history of the ith
animal. The joint pdf of n, X, and is then fn,X,(X, n, ; φ, θ) = P(n; φ, θ)fX(X; φ, θ)P( | X; θ). (1) (1) 2.3 Minke Whale Survey We now expand upon each of the terms on the RHS of this
equation, after which we add a term for (possibly noisy) obser-
vations of animal locations. As part of the 2001 North Atlantic Sightings Survey (NASS
2001; see Pike et al. 2009, for details), two independent ob-
servers surveyed the same region of sea simultaneously from an
aircraft, recording estimated distances to detected whale cues
(blows). The detectors (the observers) were at the same loca-
tion, and capture histories indicate which observer(s) detected
each cue. Having the detectors at the same location has implica-
tions for SECR analysis that we expand upon below. Additional
data on whale location are contained in the estimated distances
to detected cues, even though they are subject to measurement
error. Use of these data is shown to substantially reduce density
estimation bias. Note that our model assumes that each animal has a single x
for the survey. This does not mean that animals do not move,
just that x is the center of activity over the whole survey if they
do move. We discuss this further in Section 5. 3.2.1 Capture History Given Location. P( | X; θ). We
define an indicator variable ωiks that is 1 if animal i is detected
by detector k on occasion s and is 0 otherwise, so that the capture
history of animal i on occasion s is ωis = (ωi1s, . . . , ωiKs) and
its full capture history is ωi = (ωi1, . . . , ωiS). It is convenient
to define two indicator variables derived from ωiks, as follows:
let ωi·s = 1 if animal i was detected on occasion s and ωi·s = 0
otherwise, and ωi·· = 1 if animal i is detected at all and ωi·· = 0
otherwise. Letting B(ω, p) indicate a Bernoulli probability mass
function for ω, with parameter p, we can write P( | X; θ) as 2. MOTIVATING PROBLEMS tial capture history, and then extend this to include location
observation data. 2.1 Gibbon Survey 1. INTRODUCTION When detec- In this article, we unify DS and CR methods and in doing so
create a class of model that includes a range of models that can
be viewed as hybrids of them. Examples include MRDS sur-
veys with distance measurement error and SECR surveys that
contain additional information about animal location, such as
received acoustic signal strength (SS), precise time of acoustic
detection, or estimated bearing to detected animals. We demon-
strate the new class through a series of applications to both real
and simulated datasets. Color versions of one or more of the figures in the article can be found online
at www.tandfonline.com/r/jasa. 195 Journal of the American Statistical Association, March 2015 196 2. MOTIVATING PROBLEMS 3.1 Animal Location detaining them) and detectors that hold animals until the end
of the occasion. Appendix A contains the details for each of
these cases. It is also shown in this Appendix that in the case
of proximity detectors with a single occasion and any survey
with a single detector and multiple occasions, P( | X; θ) is
identical to the conditional likelihood of Huggins (1989). 3.2.4 Location Observation Given Capture History. f (Y |
X, ; γ ). Suppose now that in addition to observing ωiks
for animal i on occasion s, we also observe a vector yiks =
(yiks1, . . . , yiksM) containing M different kinds of data, each of
which is a noisy observation of animal location. An example
with M = 2 is an acoustic survey in which detectors are micro-
phones and SS (yiks1) and time of arrival (yiks2) of the sound at a
microphone are recorded. Writing the set of all observations yiks
as Y, we write the conditional pdf of Y given X as fY|X(Y |
X, ; γ ), where γ is a vector of parameters to be estimated. In the models we consider, the yiks’s are conditionally indepen-
dent, given X. In general, yiksm may affect detection probability,
and in this case pks(x; θ) must be replaced by pks(x, yiks; θ, γ )
in all of the above, and P(n; φ, θ), fX(X; φ, θ), P( | X; θ)
become P(n; φ, θ, γ ), fX(X; φ, θ, γ ), P( | X; φ, θ, γ ). (See
Efford, Dawson, and Borchers 2009b, and below.) So if the xi’s were observed, we could estimate abundance
using the conditional likelihood approach of Huggins (1989),
with x as the observed covariate vector. This implies that (unlike
conventional CR) estimation is possible from multiple detectors
on one occasion with proximity detectors, as recaptures within
occasion are possible. (Efford, Borchers, and Byrom 2009a, first
noted this fact.) Because animal location (x) is not observed on conventional
CR studies (only locations of capture are observed), we cannot
take the approach of Huggins (1989). But the location covari-
ate x is observed on MRDS surveys, which involve a single
occasion (S = 1) and typically two observers (K = 2), acting
as independent detectors, recording locations of detections. In
this case, we could use the approach of Huggins (1989). 3.1 Animal Location We use a generic notion of animal location, specified via
Cartesian coordinates x = (x1, x2). In DS surveys, x is the ac-
tual location of an animal at the time of the survey. If an animal
moves during the survey its location x represents the average of
its positions over the survey. In the context of trapping studies,
these locations have variously been called, “home range cen-
ters,” “centroids,” and “activity centers” (Borchers and Efford
2008; Royle and Young 2008; Royle et al. 2009a). Ideally, we
would like to observe x, but this may not be possible. Below
we derive a likelihood function that accommodates situations in
which location is observed, in which it is partially observed or
observed with error, and in which only locations of the detec-
tors are observed. We develop the likelihood for SECR surveys
without any information on animal locations other than the spa- P( | X; θ)
=
n
i=1
S
s=1 B(ωi·s, p·s(xi; θ))Pr(ωis|ωi·s = 1; θ)ωi·s
p·(xi; θ)
(2) (2) where p·s(xi; θ) = 1 −K
k=1{1 −pks(xi; θ)} is the probability
that animal i at xi is detected on occasion s, pks(xi; θ) is the
probability that animal i is detected by detector k on occasion s,
and p·(xi; θ) = 1 −S
s=1{1 −p·s(xi; θ)} is the inclusion prob-
ability for animal i, that is, the probability that it is detected
at all. P(ωis|ωi·s = 1; θ) is the probability that on occasion s
detected animal i has capture history ωis. This probability is
different for proximity detectors (which detect animals without where p·s(xi; θ) = 1 −K
k=1{1 −pks(xi; θ)} is the probability
that animal i at xi is detected on occasion s, pks(xi; θ) is the
probability that animal i is detected by detector k on occasion s,
and p·(xi; θ) = 1 −S
s=1{1 −p·s(xi; θ)} is the inclusion prob-
ability for animal i, that is, the probability that it is detected
at all. P(ωis|ωi·s = 1; θ) is the probability that on occasion s
detected animal i has capture history ωis. This probability is
different for proximity detectors (which detect animals without 197 Borchers et al.: A Unifying Model for Capture–Recapture and Distance Sampling of Wildlife Populations is a fixed number N, then n is a binomial random variable with
parameters N and p· =
R2 π(x; φ)p·(x; θ) dx. 3.3 The Likelihood Function The joint density of n, X, and Y is f (n, X, , Y; φ, θ, γ ) = P(n; φ, θ, γ )
× fXY(X, , Y | n; φ, θ, γ ),
(3) (3) × fXY(X, , Y | n; φ, θ, γ ),
(3) 3.2.2 Animal Locations, Given Detection. fX(X; φ, θ). As
noted above, MRDS methods assume an independent uniform
distribution of animals within detectable range ( Borchers,
Zucchini, and Fewster 1998). This distribution is consistent
with animals being distributed according to a homogeneous
Poisson process (HPP) in the plane. We make the more gen-
eral assumption that animals occur according to an NHPP, with
intensity D(x; φ) at x. As an animal at x is detected with prob-
ability p·(xi; θ), it follows that detected animals occur accord-
ing to a filtered NHPP with intensity D(xi; φ)p·(xi; θ). The
pdf of x given detection is obtained using Bayes’ theorem
as fx(xi; φ, θ) = D(xi; φ)p·(xi; θ)/λ(φ, θ), where λ(φ, θ) =
R2 D(x; φ)p·(x; θ) dx. Assuming independent detections, we
have fX(X; φ, θ) = n
i=1 fx(xi; φ, θ). The same fX(X; φ, θ) is
obtained if we treat the number of animals in the area as fixed at N
and assume that these animals are located independently in space
with probability density π(x; φ) = D(x; φ)/
R2 D(x; φ) dx
at x. where
fXY(X, , Y | n; φ, θ, γ )
is
the
product
of
fX
(X; φ, θ, γ ), P( | X; φ, θ, γ ) and fY|X(Y | X, ; γ ). In gen-
eral, X is not observed and this density cannot therefore be
evaluated. We obtain our likelihood by marginalizing over X in
Equation (3): L(φ, θ, γ | n, , Y) = P(n; φ, θ, γ )
×
R2 fXY(X, , Y; φ, θ, γ ) dX
(4) L(φ, θ, γ | n, , Y) = P(n; φ, θ, γ )
(4) and we estimate φ, θ, γ by maximizing this equation with re-
spect to φ, θ, γ . We obtain interval estimates using the inverse
of the negative Hessian matrix, which is obtained from numeri-
cal maximization of the likelihood. Model selection can be done
using AIC or any other likelihood-based method. 3.3.1 Estimating Animal Location. 3.1 Animal Location This
is, however, seldom done because on MRDS and other DS sur-
veys with randomized sampler locations, animal locations in the
vicinity of detectors can be treated as random variables with a
known pdf determined by design (namely a uniform distribu-
tion in the plane) and Borchers (1996) showed that using this
pdf of locations in estimation usually improves MRDS estima-
tor properties. Hence, the estimator of Huggins (1989), which
conditions on locations, is not optimal for MRDS estimation and
is generally not used for MRDS data. Instead MRDS inference
is based on likelihood functions that treat X as random. These
involve the conditional distribution of animal locations given
detection, fX(X; φ, θ), which we now consider in more detail. Following Efford, Dawson, and Borchers (2009b), we model
the expected value of the random variable ym (dropping
the iks subscript for brevity here), given x, as E(ym | x) =
μm(x) = g−1
m (β0m + β1mhmk(x)). Here gm is a link function,
βm = (β0m, β1m) is a component of γ and the “proxy function”
hmk(x) returns the component of location for which ym is a
proxy, at detector k. For example, if ym is either the observed
distance from detector to animal or the received SS, then hmk(X)
is the true distance from detector k to the animal. 4.2 Frog Survey: SECR With Arrival Times
and Signal Strength In this case, we have multivariate location data Y, comprising
the TDOA and SS of detected frog clicks. We have one occasion
(S = 1) and the arrangement of the six microphones (K = 6) is
shown in Figure 4. Here S = 1 and we model the probability of detecting animal
i with location xi in trap k on this occasion as pk1(xi; θ) =
exp{−dk(xi)2/(2θ2)}, where θ ≡θ, dk(xi) is the distance from
observer k (located at coordinates zk = (zk1, zk2)) to animal
i at xi = (xi1, xi2): dk(xi) =
(zk2 −xi2)2 + (zk1 −xi1)2. We
assume an HPP for animal locations with D(x; φ) = φ. We compare estimators using SECR methods with no location
observations, using TDOA data, using SS data, and using both. We use the same forms for pk(xi; θ), dk(xi), and D(x; φ) as
were used in the gibbon survey. Models for TDOA data and SS
are specified below, followed by analysis and simulation results
for each case. Supplementary data comprise recorded angles to animals, so
M = 1 and, dropping subscripts s and m for brevity, we let
yik denote the recorded angle between animal i and detector
k, with respect to some reference direction. The proxy func-
tion hk(xi) is the inverse tangent of (zk2 −xi2)/(zk1 −xi1)). We assume an identity link in the expectation function so that
E(ym | x) = β0 + β1hk(x), and we assume angles are estimated
without bias at all distances so that β0 = 0 and β1 = 1. A von
Mises distribution with concentration parameter γ is used to
model the angle observation error (γ ≡γ ). With independent
angle observation errors, 4.2.1 TDOA Observation. As we have only one kind of
supplementary location data (M = 1), we omit the m subscript
and we let yik denote the time of arrival of the ith clicks at
detector k. The proxy function hk(xi) is the distance function
dk(xi) (in meters) used above. We assume normal errors in time
of arrival, and constant variance σ 2
t of this error at all micro-
phones, which is consistent with randomness in time of arrival
being dominated by measurement error. 4. ANALYSES OF MOTIVATING PROBLEMS The continuum of increasingly spatially resolved spatial sam-
pling models covered in this article is illustrated in Figure 1. SECR models without location observations Y are obtained
by omitting fY|X(Y | X, ; γ ) from the model. Detection
probability of an animal at distance zero from detectors (de-
noted pks(x(k); θ), with x(k) being the location of the kth de-
tector) may be constrained to be 1 or not, depending on the
application. DS and MRDS models are obtained by defining
fY|X(Y | X, ; γ ) to be unity at Y = X and zero elsewhere. MRDS models generally have K = 2 and S = 1 and allow
pks(x(k); θ) < 1, while conventional DS models have K = 1,
S = 1 and define pks(x(k); θ) = 1. To investigate the cause of the differences we plotted esti-
mated locations of calling groups using Equation (5), and we
conducted a simulation study (with 500 simulations) in which
true parameter values were equal to those estimated using
and Y. Illustrative examples of location contours are shown in
Figure 2 and the simulated sampling distributions of the two
estimators is shown in Figure 3. The utility of angle data is apparent in Figure 2 in the form
of much tighter contours when and Y are used than for
alone. It is also apparent in Figure 3, which shows the “simple”
estimator using only to be biased (by about 15%), very much
more dispersed and with a mode far below truth (“truth” being
the density used in simulating). (Note that with three detectors
there are only seven possible capture histories and hence the
simple SECR model will estimate all animals to be at one of only
seven locations, while with the angle data, an infinite number of
locations is possible.) All the case studies below involve proximity detectors and a
single occasion (so we omit subscript s), but the methods apply
equally to multi-catch traps and multiple occasions. We do not
include any covariates or individual random effects (other than
x) in our applications for brevity and because our emphasis is
on illustration of the effects of adding supplementary data. See
Discussion for more on covariates. All analyses and plots were done with the R library
admbsecr, written by authors of this article (see online sup-
plementary material). 3.3 The Likelihood Function Given estimates ˆφ, ˆθ,
and ˆγ , animal locations can be estimated from , Y by appli-
cation of Bayes’ Theorem as follows (omitting ˆφ, ˆθ, and ˆγ for
brevity and indicating estimates by “hats” on functions): 3.2.3 Number of Detections. P(n; φ, θ). If animals are in-
dependently distributed in the plane according to an NHPP with
parameter vector φ and intensity D(x; φ) at x, and they are in-
dependently detected with probability p·(x; θ), it follows that n,
the number of detected animals, is a Poisson random variable
with rate parameter λ(φ, θ). If the number of animals in the area ˆfX|Y(X|, Y) =
ˆfY|X(Y|X, ) ˆP(|X) ˆfX(X)
R2 ˆfY|X(Y|X, ) ˆP(|X) ˆfX(X) dX
. (5) (5) Besides being of possible inherent interest, the pdf of animal
locations, ˆfX|Y(X|, Y), is useful for illustrating the effect Journal of the American Statistical Association, March 2015 198 of the location observation data Y on the precision of location
estimation, and we use it primarily for this purpose below. gibbon groups is estimated to be 0.83 groups per square kilome-
ter, with a coefficient of variation (CV) of 44%, while using
and Y it is estimated to be 0.32 with CV of 23%. The differences
arise as a consequence of the estimated detection function scale
parameter θ being much smaller when only is used ( ˆθ =
754 m; CV = 23%) than when Y is also used ( ˆθ = 1248 m; CV
= 11%). 4.1 Gibbon Survey: SECR With Estimated Angles 4.1.1 The Model. Recall that the detectors are observers
standing in a line spaced approximately 500 m apart (see
Figure 2), recording estimated angles to gibbon groups they
heard. We use SECR methods to estimate the density of calling
groups from the locations of the observers who detected the
group, both with and without the angle data. 4. ANALYSES OF MOTIVATING PROBLEMS Part of the problem is poor design: with detectors spaced only
500 m apart and scale parameter θ = 1248 the simple estimator
has no information on how detection probability varies at dis-
tances greater than 1000 m—because detections are never more
than 1000 m apart. The angle data overcome this limitation: use
of Y improves estimation. Figure 1. A continuum of increasingly spatially resolved capture–recapture models. Numbers in brackets correspond to subsections of the
article. Figure 1. A continuum of increasingly spatially resolved capture–recapture models. Numbers in brackets correspond to subsections of the
article. Figure 1. A continuum of increasingly spatially resolved capture–recapture models. Numbers in brackets correspond to subsections of the
article. incidental parameters, and to eliminate them we can base infer-
ence on the likelihood of time differences of arrival (TDOAs)
from the mean arrival time, conditional on the mean arrival time: σ 2
s , but unlike the time of arrival model, we estimate β0 and
β1 in addition to σ 2
s , so that γ = (β0, β1, σ 2
s ). In addition, be-
cause signals weaker than some specified strength c are fil-
tered out at the acoustic processing stage, detection probability
depends on received SS. We can write the probability of mi-
crophone k detecting signal i made at a distance d(xi) from
it as pk1(x, yk; θ, γ ) = 1 −Fk(c; xi, γ ), where Fk(c; xi, γ ) is
the cumulative distribution function (CDF) of a normal ran-
dom variable with mean exp{β0 + β1hk(xi)} and variance σ 2
s ,
evaluated at c. Then, fY|X(Y | X, ; γ )
∝
n+
i=1
2πσ 2
t
(1−mi)/2 exp
mi
k=1
(δk(xi) −¯δi)2
−2σ 2
t
,
(7) where n+ is the number of clicks detected on more than one
microphone, mi is the number of microphones on which the
ith of these was detected, γ ≡σ 2
t , δk(xi) = yik −E(yik | xi),
and ¯δi =
1
mi
mi
k=1 δk(xi). The same likelihood can be obtained
using a marginal approach, treating the β0s as random effects
(see online supplementary material). For this reason, and for
brevity, we do not explicitly show the conditioning on ¯δis on the
LHS of the equation. fY|X(Y | X, ; γ ) =
n
i=1
mi
k=1
Nk(yik; xi, γ )
1 −Fk(c; xi, γ ),
(8) (8) where mi is as before, the number of microphones on which
click i was detected and Nk(yik; xi, γ ) is a normal pdf with
mean exp{β0 + β1hk(xi)} and variance σ 2
s , evaluated at yik. 4.2.2 Signal Strength (SS) Observation. 4.2 Frog Survey: SECR With Arrival Times
and Signal Strength We use an identity link
so that E(yik | xi) = β0i + β1hk(xi), where β0i is the time the
ith sound was generated and β1 is the inverse of the speed of
sound in air (in meters per second). f (Y | X, ; γ ) =
n
i=1
K
k=1
exp {γ cos [yik −hk(xi)]}
2πI0 (γ )
ωik
, (6) (6) The time clicks are made is uninformative about location, as
a click made at distance d(x) at time β0 has the same expected
arrival time as one made at distance d(x) + c/β1 at time β0 −c,
for any c. The β0i’s are what (Millar 2011, pp. 188–189) called where I0( ) is the modified Bessel function of order 0. where I0( ) is the modified Bessel function of order 0. 4.1.2 Results. A total of 123 detections of 77 calls were
made. Using only capture histories (), the density of calling Borchers et al.: A Unifying Model for Capture–Recapture and Distance Sampling of Wildlife Populations
199
Figure 1. A continuum of increasingly spatially resolved capture–recapture models. Numbers in brackets correspond to subsections of the
article. Borchers et al.: A Unifying Model for Capture–Recapture and Distance Sampling of Wildlife Population 199 Borchers et al.: A Unifying Model for Capture–Recapture and Distance Sampling of Wildlife Populations 200 Journal of the American Statistical Association, March 2015 0
100
200
300
-0.01
0.01
0.03
0.05
Estimate as % deviation from Truth
pdf of Estimate
simple
angle
Figure 3. Smoothed simulated sampling distributions of estimated gibbon call density when only spatial capture history is used in estimation
(“simple”) and when capture history and observed angles are used (“angle”). The down arrow marks true (simulated) density, the horizontal axis
is percentage deviation from true density, and the up arrows are the means of the sampling distributions. 0
100
200
300
-0.01
0.01
0.03
0.05
pdf of Estimate
simple
angle Estimate as % deviation from Truth Figure 3. Smoothed simulated sampling distributions of estimated gibbon call density when only spatial capture history is used in estimation
(“simple”) and when capture history and observed angles are used (“angle”). The down arrow marks true (simulated) density, the horizontal axis
is percentage deviation from true density, and the up arrows are the means of the sampling distributions. Figure 3. Smoothed simulated sampling distributions of estimated gibbon call density when only spatial capture history is used in estimation
(“simple”) and when capture history and observed angles are used (“angle”). The down arrow marks true (simulated) density, the horizontal axis
is percentage deviation from true density, and the up arrows are the means of the sampling distributions. independent we have independent we have SECR+TDOA often differed substantially. Figure 4 shows an
example for a specific click. The average difference in received
SS for individual clicks was less than 2% of its mean value and it
may be that the distances between microphones were too small
for the contrast in received SS to be very informative about
location. The same is not true of TDOA. fY|X(Y | X, ; γ )
=
n+
i=1
2πσ 2
t
(1−mi)/2
2T √mi
exp
mi
k=1
(δk(xi) −¯δi)2
−2σ 2
t
×
n
i=1
mi
k=1
Nk(yik; xi, γ )
1 −Fk(c; xi, γ ). (9) fY|X(Y | X, ; γ )
=
n+
i=1
2πσ 2
t
(1−mi)/2
2T √mi
exp
mi
k=1
(δk(xi) −¯δi)2
−2σ 2
t
×
n
i=1
mi
k=1
Nk(yik; xi, γ )
1 −Fk(c; xi, γ ). (9) We investigate estimator properties by simulation (500
simulations),
using
the
parameter
estimates
from
the
SECR+SS+TDOA model as truth and mean sample size equal
to that observed on the survey. The ideas of this
section are taken from Efford, Dawson, and Borchers (2009b). M = 1 and we let yik denote the received SS at detector k. The proxy function hk(xi) is as above and we model the ex-
pectation as E(yik|xi) = β0 + β1hk(xi), where β0 is the mean
SS of clicks and β1 is a parameter quantifying SS loss with
propagation distance. (We also tried a log link, E(yik|xi) =
exp{β0 + β1hk(xi)}, but this was found to be inferior in terms
of AIC: AIC = 18.) As with the time of arrival model, we
assume that yik is normally distributed with constant variance, 4.2.3 TDOA and Signal Strength (SS) Observation. In
this case, M = 2 and we let yik = (yik1, yik2) where yik1
is the time of arrival and yik2 is the received SS of click
i at detector k. Both h1k(xi) and h2k(xi) are the distance
function dk(xi) and we assume the same models as above
so that E(yik|xi) = (β01 + β11h1k(xi), exp{β02 + β12h2k(xi)}),
γ = (β11, σ 2
t , β02, β12, σ 2
s ), and assuming yik1, yik2
to be −2000
0
1000
3000
5000
−2000
0
1000
x
y
−2000
−1000
0
1000
2000
−2000
−1000
0
1000
x
y
Figure 2. Example location estimates, given capture, of two different gibbons. Detectors are crosses; circled detectors are those that detected
the gibbon call. Arrows show estimated angles to detections. Dotted lines are the contours of the estimated probability of the group being
contained within the contour, given only the spatial capture history data . Dashed lines are estimated contours, given only observed angles to
detections. Solid lines are estimated contours, given capture history and angles. −2000
−1000
0
1000
2000
−2000
−1000
0
1000
x
y −2000
0
1000
3000
5000
−2000
0
1000
x
y x x Figure 2. Example location estimates, given capture, of two different gibbons. Detectors are crosses; circled detectors are those that detected
the gibbon call. Arrows show estimated angles to detections. Dotted lines are the contours of the estimated probability of the group being
contained within the contour, given only the spatial capture history data . Dashed lines are estimated contours, given only observed angles to
detections. Solid lines are estimated contours, given capture history and angles. Simulated sampling distributions
are shown in Figure 5. As expected, the addition of SS or TDOA
reduces bias and improves precision, and there is a further small
improvement in precision when both SS and TDOA data are
used: the CVs for the SECR, SECR+SS, SECR+TDOA, and
SECR+SS+TDOA models are 7.9%, 6.8%, 6.8%, and 6.1%,
respectively. (9) 4.2.4 Comparison of Estimates With and Without TDOA, SS. A total of 590 detections of 345 frog clicks were made. Using
SECR only, the click density is estimated to be 152.1 clicks per
hectare per minute, with standard error 10.6 (CV = 7.0%). When
SS is used these are reduced to 148.9 and 8.9 (CV = 6.0%), when
TDOA is used they are reduced to 134.5 and 9.5 (CV=7.1%),
and when both SS and TDOA are used, they reduce to 125.7
and 8.0 (CV = 6.4%). While both SS and TDOA reduce the
point estimate of density and its standard error, the effect of SS
on the point estimate is weaker. Investigation at the individual
click level revealed that point estimates of click locations from
the TDOA+SS model tended to agree well with those from the
simple SECR model (but were more precise), while those from 4.3 Whale Survey: MRDS With Estimated Distances Smoothed simulated sampling distributions of estimated frog click density using only spatial capture history (“simple”), using
capture history and time of arrival (“TDOA”), using capture history and signal strength (“SS”), and using capture history, time of arrival and
signal strength (“joint”). The down arrow marks true density, the horizontal axis is percentage deviation from true density, and the up arrows are
the means of the sampling distributions, expressed as percentage deviation from truth (some are almost coincident). Figure 5. Smoothed simulated sampling distributions of estimated frog click density using only spatial capture history (“simple”), using
capture history and time of arrival (“TDOA”), using capture history and signal strength (“SS”), and using capture history, time of arrival and
signal strength (“joint”). The down arrow marks true density, the horizontal axis is percentage deviation from true density, and the up arrows are
the means of the sampling distributions, expressed as percentage deviation from truth (some are almost coincident). made one pass over animals. Standard SECR methods cannot
be applied in this case because a distance-dependent detection
function cannot be estimated from detectors at a single loca-
tion. But with the addition of estimated distances to detections
(yik for observer k’s estimate of distance to cue i), estimation is
possible. (Buckland et al. 2001) and hence use a HPP for animal loca-
tions with D(x; φ) = φ. This leads to the usual cue-counting
pdf for radial distances of detected animals (see online sup-
plementary material). We found it necessary to introduce
detector-specific detection function parameters as one detec-
tor was far more efficient than the other. We use pk1(xi; θk) =
logit−1(θk2) exp{−dk(xi)2/(2θ2
k1)}, where θk = (θk1, θk2) (k =
1, 2) and θ = (θ11, θ12, θ21, θ22). The proxy function h1k(xi) is
the distance function dk(xi). Following Borchers et al. (2009),
we assume unbiased distance estimation with gamma errors,
that is, E(yik|xi) = dk(xi) and MRDS survey models treat distances as being observed with-
out error (see Borchers et al. 2009; Laake et al. 2011, for cue
counting and point transect examples); our model readily al-
lows distance measurement error to be incorporated in MRDS
inference, estimating measurement error from the pairs of
recorded distances of the two observers to recaptures, simul-
taneously with density and detection function parameters. In
this survey, measurement error is substantial, as can be seen
from Figure 6. 4.3 Whale Survey: MRDS With Estimated Distances We estimate cue density allowing probability of
detection at distance zero to be less than unity, both with and
without the assumption of no measurement error. Were we to
enforce certain detection at distance zero, we would have con-
ventional distance sampling (CDS) models with and without
measurement error. (See Borchers et al. 2010, for references to
CDS models with measurement error.) 4.3 Whale Survey: MRDS With Estimated Distances 4.3.1 The Model. We estimate the number of minke whale
cues per hectare over the sampling period from 71 detections ob-
tained on the aerial cue-counting component of the NASS 2001
survey. K = 2 as there were two detectors and S = 1 as they -10
-5
0
5
10
15
0
5
10
15
-10
-5
0
5
10
15
0
5
10
15
Figure 4. Estimated location contours given capture history and SS (left) and capture history and TDOA (right), of a click. Detectors are
crosses; circled detectors are those that detected the frog click. Dotted lines are the contours of the probability density of frog location given only
spatial capture history data . Dashed lines in the right plot are contours given only TDOA. Solid lines are contours of location given capture
history and SS (left) or capture history and TDOA (right). -10
-5
0
5
10
15
0
5
10
15 -10
-5
0
5
10
15
0
5
10
15 15 Figure 4. Estimated location contours given capture history and SS (left) and capture history and TDOA (right), of a click. Detectors are
crosses; circled detectors are those that detected the frog click. Dotted lines are the contours of the probability density of frog location given only
spatial capture history data . Dashed lines in the right plot are contours given only TDOA. Solid lines are contours of location given capture
history and SS (left) or capture history and TDOA (right). Borchers et al.: A Unifying Model for Capture–Recapture and Distance Sampling of Wildlife Populations 201 -30
-20
-10
0
10
20
0.00
0.01
0.02
0.03
Estimate as % deviation from Truth
pdf of Estimate
simple
TDOA
SS
joint
Figure 5. Smoothed simulated sampling distributions of estimated frog click density using only spatial capture history (“simple”), using
capture history and time of arrival (“TDOA”), using capture history and signal strength (“SS”), and using capture history, time of arrival and
signal strength (“joint”). The down arrow marks true density, the horizontal axis is percentage deviation from true density, and the up arrows are
the means of the sampling distributions, expressed as percentage deviation from truth (some are almost coincident). Estimate as % deviation from Truth Figure 5. fY|X(Y | X, ; γ ) fY|X(Y | X, ; γ )
=
n
i=1
mi
k=1
dk(xi)
α
α
(α)
−1
yα−1
ik
exp
−αyik
dk(xi)
, (10) (10) where yik is the radial distance measurement from observer j to
cue i. where yik is the radial distance measurement from observer j to
cue i. For the case without measurement error, we define fY|X(Y |
X, ; γ ) to be 1 if Y = X, and zero otherwise. 4.3.2 Results. When distance measurement error is accom-
modated using an SECR model with estimated distance data,
density is estimated to be 1.72 whale cues per hectare over the Following standard practice for DS surveys, we assume
an independent uniform distribution of animals in the plane -1000
-500
0
500
1000
0
200
600
1000
x
y
0
200
400
600
800
1000
1200
1400
0e+00
4e-05
8e-05
Radial distance (r)
pdf(r)
Figure 6. Estimated location contours (dotted) given capture history and recorded location (solid) of a whale detected by one of the two
detectors. Contours are such that 100α% of the density falls between the two contours marked α. The left plot shows locations in perpendicular
and forward distance space, the right curve shows it in radial distance space. Detectors are crosses. -1000
-500
0
500
1000
0
200
600
1000
x
y 0
200
400
600
800
1000
1200
1400
0e+00
4e-05
8e-05
Radial distance (r)
p
( ) pdf(r) Figure 6. Estimated location contours (dotted) given capture history and recorded location (solid) of a whale detected by one of the two
detectors. Contours are such that 100α% of the density falls between the two contours marked α. The left plot shows locations in perpendicular
and forward distance space, the right curve shows it in radial distance space. Detectors are crosses. 202 Journal of the American Statistical Association, March 2015 -50
0
50
100
0.00
0.01
0.02
0.03
Estimate as % deviation from Truth
pdf of Estimate
mrds
dist
Figure 7. Smoothed simulated sampling distributions of estimated whale cue density when capture history and exact distances are observed
(“mrds”) and when capture history and estimated distances are used (“dist”). The down arrow marks true density, the horizontal axis is percentage
deviation from true density, and the up arrows are the means of the sampling distributions. Estimate as % deviation from Truth Figure 7. 5.1 Model Extensions One topic that we have skirted, for lack of space, is how
covariate data is incorporated into the models. Covariates can
be incorporated in the density model D(x; φ) most naturally via
a log link function, in the scale parameter of detection functions
using a log link, and in the intercept parameter of detection
functions using a logit link. Borchers and Efford (2008) and
Royle et al. (2013b) contained SECR examples with a variety of
explanatory variables and the former includes individual random
effects. Marques and Buckland (2003) dealt with explanatory
variables for DS models, and Borchers, Zucchini, and Fewster
(1998) dealt with them for MRDS models. We conducted a simulation study (500 simulations) to in-
vestigate the effect of neglecting measurement error on density
estimates, using the parameters estimated from the model that
incorporates measurement error, with mean sample size of 70,
and with error CVs of 12%, 32%, and 50%. Results for the
32% case are shown in Figure 7. On the 1987 NASS survey
measurement error CV was estimated to be 32% compared to
12% on the 2001 survey—see Borchers et al. (2009). All esti-
mators were found to be positively biased but those from the
MRDS model were (in order of increasing measurement error
CVs) larger by 14%, 34%, and 68%, respectively. Biases us-
ing the SECR model with measurement error were 7.7%, 7.0%,
and 8.2%. As the model estimates six parameters from only 70
observations, we believe this to be small-sample bias. We have also not covered any detail of how NHPP or other
models that involve nonuniform animal distribution might be
implemented. Although animal distribution is typically not ho-
mogeneous in space, it is usual to assume uniform spatial distri-
bution in DS analyses (as a consequence of random placement
of detectors), but DS estimators usually use this assumption
only to estimate detection probability (estimating density con-
ditional on detection probability using design-based methods). They have been found to be relatively robust to violation of
the assumption at this level (see Buckland et al. 2001). Other
methods may not be. Johnson, Laake, and Hoef (2010) imple-
mented DS with an NHPP and Royle et al. (2013b) implemented
a Bayesian version of SECR with an NHPP, with log-linear de-
pendence on environmental covariates in both cases. We believe fY|X(Y | X, ; γ ) Smoothed simulated sampling distributions of estimated whale cue density when capture history and exact distances are observed
(“mrds”) and when capture history and estimated distances are used (“dist”). The down arrow marks true density, the horizontal axis is percentage
deviation from true density, and the up arrows are the means of the sampling distributions. duration of the survey (CV = 18%), and detection probability
at distance zero for the two detectors (pk1(0; ˆθk), k = 1, 2) to
be 1.0 (CV = 0.01%) and 0.30 (CV = 25%). When using an
MRDS estimator in which distances are assumed to be error-
free (as is the norm for such analyses), density is estimated to
be 1.61 (CV = 17%) and pk1(0; ˆθk), k = 1, 2 to be 1.0 (CV =
0.01%) and 0.30 (CV = 23%). Figure 6 shows the contours of
estimated location of a whale detected only by detector 2, when
observed distance is assumed error free and when it is estimated
with measurement error. when MRDS surveys are considered as SECR surveys, most
have the worst possible design (detectors at the same location)
and inference about density from them would be impossible
without the additional location data. In the case of DS surveys, the new class of model provides
a ready means for incorporating measurement error into in-
ference, with or without the conventional DS assumption of
certain detection at distance zero. The general model also pro-
vides a framework for incorporating into SECR surveys the
point independence (Laake 1999; Innes et al. 2002; Borchers
et al. 2006) and limiting independence (Buckland, Laake, and
Borchers 2009) methods developed in the DS literature, as a
means of reducing bias due to unmodeled heterogeneity. Formulating the MRDS survey as an SECR estimation prob-
lem with distance measurement error provides a ready means
of accommodating both measurement error and estimation of
pk1(0; θk)—something that has to date not been done in anal-
yses of DS data, with the exception of a model developed by
Hiby and Lovell (1998) which used distance interval data rather
than continuous distance measurements. 5. DISCUSSION We have shown that DS and CR are special cases of a more
general class of spatial sampling model that uses detection lo-
cations to assist in estimating detection probability, and hence
density. We have also shown that in the case of CR surveys, sup-
plementing data on locations of captures with data on animal
location (albeit noisy or incomplete) can substantially improve
inference, particularly when designs are not optimal. Indeed, 203 Borchers et al.: A Unifying Model for Capture–Recapture and Distance Sampling of Wildlife Populations imply or require that animals do not move during the survey. Nor does it require that movement between occasions on a multi
occasion survey (S > 1) be modeled; providing that either (a)
single- or multi-catch traps are used, or (b) occasions are long
enough that the distribution of points that an animal visits over
the duration of an occasion is the same as that over the duration
of the whole survey. In the former case, there is no information
on animal movement within occasions and the location is by def-
inition the center of activity across occasions. In the latter case,
the center of activity across occasions is identical to that within
occasions. If proximity detectors are used and (b) above does
not hold, then the detection functions within occasion will differ
from those across occasions (typically having shorter ranges for
shorter occasions). Models that do not allow for this are mis-
specified and may produce biased estimates. This problem can
usually be avoided by having a design with occasions that are
sufficiently long. there is a need for more flexible models that are not neces-
sarily monotonic in their dependence on explanatory variables,
and expect that these will be developed in the near future. This
could be achieved using penalized regression splines, in a sim-
ilar way to that in which Gimenez et al. (2006) used them to
model nonmonotonic dependence of survival probability in an
open-population capture–recapture model. Bayesian and frequentist versions of SECR have been devel-
oped in parallel by different authors. Bayesian inference tends to
be particularly useful in the presence of latent variables or ran-
dom effects—and animal locations are latent variables in SECR
models. However, marginalization over locations involves a sim-
ple two-dimensional integral when locations are independent,
making maximum likelihood inference straightforward. APPENDIX A: VARIETIES OF P( | X; θ) Multi-catch traps detain animals until the end of the sampling occasion
in which they are trapped (and do not fill up). Proximity detectors are
detectors that do not detain animals and therefore allow captures of the
same animal on different traps within occasions. In some proximity
detector applications, it is possible to detect the same animal more than
once at the same detector. In this case, either binary capture histories
of the sort used in the body of this article can be used or the capture
frequency of each animal at each trap on each occasion can be recorded. 5.2 Robustness and Diagnostics A final important issue that remains to be resolved for this
class of model, and indeed for many CR models of any sort, is
how to deal with uncertain recapture identification, as this can be
fraught with uncertainty when animals are not physically tagged. This general problem was addressed by Link et al. (2010) for
example, while Bonner (2013) and work referenced therein ad-
dressed the issue when there are multiple sources of individual
identification. None of these methods explicitly use location in-
formation and we expect that methods that use location data to
quantify the probability that detections are recaptures will be
useful, as they were in the MRDS analysis of Hiby and Lovell
(1998). The robustness of estimators within the class of models de-
veloped in this article to failures of assumptions is likely to be
case-specific. DS point estimators of density tend to be robust
to failure of the assumption of independent uniform animal dis-
tribution (see Buckland et al. 2001, p. 36), although interval
estimators are not. Efford, Borchers, and Byrom (2009a) found
SECR point and interval estimators with multi catch traps to be
robust to failure of assumptions of independence and uniformity
(see Table 4, p. 266), and also found density estimates to be little
affected by the form assumed for the detection function. Goodness-of-fit diagnostics are well developed for DS detec-
tion function estimators, using observed locations (see Buckland
et al. 2004, pp. 385–389). Similar diagnostics when locations are
not observed remain to be developed (for both DS and SECR es-
timators). Borchers and Efford (2008) proposed a Monte Carlo
goodness-of-fit test based on scaled deviance for the overall fit
of SECR models but this does not distinguish between lack of
fit of the animal density model and lack of fit of the detection
model. This is an area that would benefit from further research. 5. DISCUSSION In this
case, both approaches work well and it is largely a matter of per-
sonal preference which is used. Maximum likelihood estimation
has to date proved to be much faster than the MCMC methods
used for Bayesian inference, even when a random effect for un-
modeled heterogeneity in detection probability is incorporated
in the model. It seems likely that the Bayesian approach will
come into its own when there is a more complicated latent vari-
able structure—when there is dependence between latent vari-
ables, for example. In such cases, the marginalization required
for maximum likelihood inference may become infeasible. We
expect that models that do not involve independent distribution
of animal locations (as NHPPs do) will soon be developed, as
animals are often not independently distributed. A simple but
common case is when animals occur in groups; in this case,
animals within the group may not be detected independently of
one another. This can often be dealt with by treating the group as
the detection unit while simultaneously estimating mean group
size if individual animal density is of interest, but in other cases
models for spatial dependence may be required. When activity centers move between occasions, an additional
model layer for activity center movement will be required in
general. The simplest such model is probably one in which
the activity centers on each occasion are independent random
effects with mean equal to an animal’s activity center across
all occasions. But we believe that this will not be an adequate
model in many applications, because activity centers on consec-
utive occasions are likely to be correlated. If activity centers are
observed on some (but not all) occasions, the methods of Lan-
grock and King (2013) and of references therein may be useful
for modeling activity centers that were not observed, conditional
on those that were. (If animal activity centers are the same for
all occasions and some but not all are observed, the likelihood
is like Equation (4), but with integration over only those centers
that were not observed.) 5.3 Animal Movement The methods of this article assume a single location (activity
center) for each animal over the whole survey, but this does not In the case of multi catch traps, all but one of ωi1s, . . . , ωiKs are
zero and Pr(ωis|ωi·s = 1; θ) is a multinomial distribution with index Journal of the American Statistical Association, March 2015 Journal of the American Statistical Association, March 2015 204 1 and probabilities pks(xi; θ)/
k pks(xi; θ) (k = 1, . . . , K). Model-
ing pks(xi; θ) using a competing hazard formulation (see Borchers and
Efford 2008), pks(xi; θ) = rks(xi; θ)p·s(xi; θ), where rks(xi; θ) is de-
fined as hks(xi; θ)/ h·s(xi; θ), the relative hazard of detection at trap
k on occasion s for an animal at xi, hks(xi) is the detection hazard
at trap k and h·s(xi) =
k hks(xi) is the total hazard on the occasion. Hence,
k pks(xi; θ) = p·s(xi; θ) and the multinomial probabilities
are r1s(xi; θ), . . . , rKs(xi; θ). 1 and probabilities pks(xi; θ)/
k pks(xi; θ) (k = 1, . . . , K). Model-
ing pks(xi; θ) using a competing hazard formulation (see Borchers and
Efford 2008), pks(xi; θ) = rks(xi; θ)p·s(xi; θ), where rks(xi; θ) is de-
fined as hks(xi; θ)/ h·s(xi; θ), the relative hazard of detection at trap
k on occasion s for an animal at xi, hks(xi) is the detection hazard
at trap k and h·s(xi) =
k hks(xi) is the total hazard on the occasion. Hence,
k pks(xi; θ) = p·s(xi; θ) and the multinomial probabilities
are r1s(xi; θ), . . . , rKs(xi; θ). Buckland, S. T., Anderson, D. R., Burnham, K. P., Laake, J. L., Borchers, D. L., and Thomas, L. J. (2001), Introduction to Distance Sampling, Oxford:
Oxford University Press. [201,202,203] Buckland, S. T., Anderson, D. R., Burnham, K. P., Laake, J. L., Borchers, D. L.,
and Thomas, L. J. (2004), Advanced Distance Sampling, Oxford: Oxford
University Press. [203] Buckland, S. T., Laake, J. L., and Borchers, D. L. (2009), “Double-Observer
Line Transect Methods: Levels of Independence,” Biometrics, 66, 169–177. [202] Efford, M. G. (2004), “Density Estimation in Live-Trapping Studies,” Oikos,
106, 598–610. [195] In the case of proximity detectors with binary ωiks, Pr(ωis|ωi·s =
1; θ) is written as K
k=1 B(ωiks, pks(xi; θ))/p·s(xi; θ). 5.3 Animal Movement In the case of
proximity detectors with frequency data in which ωiks is the fre-
quency of detection on detector k on occasion s, Royle et al. (2009b)
proposed a Poisson model for ωi·s, such that Pr(ωis|ωi·s = 1; θ) is
K
k=1 Po(ωiks, λ0pks(xi; θ))/p·s(xi; θ), where Po(x, λ) is a Poisson dis-
tribution with parameter λ. Efford, M. G., Borchers, D. L., and Byrom, A. E. (2009a), “Density Estima-
tion by Spatially Explicit Capture-Recapture: Likelihood-Based Methods,”
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Estimated From Locations of Individuals on a Passive Detector Array,”
Ecology, 90, 2676–2682. [197,199] With binary capture histories, Equation (2) reduces to Equation (A.1)
below for proximity detectors when K = 1 and it reduces to Equation
(A.2) with either kind of detector when S = 1. Gimenez, O., Crainiceanu, C., Barbraud, C., Jenouvrier, S., and Morgan, B. J. T. (2006), “Semiparametric Regression in Capture–Recapture Modelling,”
Biometrics, 62, 691–698. [203] Hiby, L., and Lovell, P. (1998), “Using Aircraft in Tandem Formation to Estimate
Abundance of Harbour Porpoise,” Biometrics, 54, 1280–1289. [202,203] P(K=1)( | X; φ, θ) =
n
i=1
S
s=1 B(ωi1s, p1s(xi; θ))
p·(xi; θ)
(A.1)
P(S=1)( | X; φ, θ) =
n
i=1
K
k=1 B(ωik1, pk1(xi; θ))
p·(xi; θ)
. (A.2) P(K=1)( | X; φ, θ) =
n
i=1
S
s=1 B(ωi1s, p1s(xi; θ))
p·(xi; θ)
(A.1)
P(S=1)( | X; φ, θ) =
n
i=1
K
k=1 B(ωik1, pk1(xi; θ))
p·(xi; θ)
. (A.2) P(K=1)( | X; φ, θ) =
n
i=1
S
s=1 B(ωi1s, p1s(xi; θ))
p·(xi; θ)
(A.1)
P(S=1)( | X; φ, θ) =
n
i=1
K
k=1 B(ωik1, pk1(xi; θ))
p·(xi; θ)
. (A.2) Huggins, R. M. (1989), “On the Statistical Analysis of Capture Experiments,”
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Recapture-Recovery Models in the Presence of Continuous Covariates,”
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Huggins (1989). Equation (A.1) corresponds to the conventional CR
case—in which there is usually more than one trap but all traps together
are treated as a single composite trap, effectively with one location. Laake, J. L. (1999), “Distance Sampling With Independent Observers: Reduc-
ing Bias From Heterogeneity by Weakening the Conditional Independence
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D. G. Robertson, Rotterdam: Balkema, pp. 137–148. [202] Laake, J. L., Collier, B. A., Morrison, M. L., and Wilkins, R. N. (2011), “Point-
Based Mark-Recapture Distance Sampling,” Journal of Agricultural, Bio-
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UK: Wiley. [198] Bonner, S. (2013), “Response to: A New Method for Estimating Animal Abun-
dance With Two Sources of Data in Capture-Recapture Studies,” Methods
in Ecology and Evolution, 4, 585–588. [203] Pike, D. G., Paxton, C. G. M., Gunnlaugsson, Th., and Vikingsson, G. A. (2009),
“Trends in the Distribution and Abundance of Cetaceans From Aerial Sur-
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“Bayesian Inference in Camera-Trapping Studies for a Class of Spatial
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Schwarz, C. J., and Seber, G. A. F. REFERENCES (1999), “Estimating Animal Abundance:
Review III,” Statistical Science, 14, 427–456. [195] Review III,” Statistical Science, 14, 427–456. [195] Williams, B. K., Nichols, J. D., and Conroy, M. J. (2002), Analysis and Man-
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https://hal.archives-ouvertes.fr/hal-03370736/document
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English
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Short-term availability of adult-born neurons for memory encoding
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Nature communications
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cc-by
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To cite this version: Jérémy Forest, Mélissa Moreno, Matthias Cavelius, Laura Chalençon, Anne Ziessel, et al.. Short-
term availability of adult-born neurons for memory encoding. Nature Communications, 2019, 10 (1),
10.1038/s41467-019-13521-7. hal-03370736 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-03370736
https://hal.science/hal-03370736v1
Submitted on 8 Oct 2021 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
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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 ARTICLE 1 INSERM, U1028; CNRS, UMR5292; Lyon Neuroscience Research Center, Neuroplasticity and Neuropathology of Olfactory Perception Team, Lyon F-69000,
France. 2 Claude Bernard University-Lyon1 and University of Lyon, Lyon F-69000, France. 3These authors contributed equally: Jérémy Forest, Mélissa Moreno.
4These authors jointly supervised this work: Anne Didier, Nathalie Mandairon. *email: nathalie.mandairon@cnrs.fr ARTICLE ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 A F(1,106) = 9.89; p = 0.002). Enriched animals were able to dis-
criminate the two enantiomers of limonene from T1 to T3 (Paired
t-test, p < 0.05 between Hab4 and Test) but not after the longer time
periods T4 and T5 (Paired t-test, p > 0.05 between Hab4 and Test;
Supplementary Table 1, Fig. 1b). To assess the specificity of learn-
ing, discrimination of another pair of similar odorants, decanal/
dodecanone (dec/dodec), to which the animals had not been enri-
ched was also tested. No discrimination of dec and dodec was seen
after enrichment with +lim and −lim, (ANOVA group effect:
F(5,90) = 1.18, p = 0.32; trial effect: F(1,90) = 0.05; p = 0.83, p > 0.05
between Hab4 and Test) (Supplementary Table 1; Fig. 1c). NE
control animals did not discriminate any of the two odorant pairs
(Paired t-test, T1: p > 0.05 between Hab4 and Test; Supplementary
Table 1; Fig. 1b, c). A
ll animals are exposed to a changing olfactory environ-
ment requiring constant adjustments of odor perception
and memory, in order for the olfactory system to effi-
ciently guide the animal’s behavior. Adult-born neurons provided
by neuroblasts formed in the subventricular zone1–4, constantly
integrate the olfactory bulb (OB) and underlie olfactory learn-
ing5–14. However it has been proposed that, in the hippocampus,
adult neurogenesis promotes forgetting. Indeed, an increase in
hippocampal neurogenesis tends to enhance memory clearance
thereby reducing proactive interference while decreased neuro-
genesis prevents forgetting and reduces behavioral flexibility15–17. Thus, the neuronal turnover provided by adult neurogenesis
could sub-serve memory formation and regulate the transience of
the memory trace. This raises the issue of how this ‘remembering/
forgetting’ balance is regulated as a function of environmental
demand, allowing the animal to keep relevant information in
memory for optimal behavioral adaptation. However, the para-
meters determining memory persistence or forgetting such as
information relevance or the time between successive inputs and
to what extent they depend on adult-born neurons are unknown. More specifically, adult-born neurons show a critical period
during which they can be recruited by learning6 but whether they
can support successive memory traces within this critical period
and whether they suffer from memory interference are unknown. ARTICLE g
To assess adult-born neuron survival, a cohort of adult-born
neurons present in the OB at the beginning of learning was
labeled by injecting the DNA synthesis marker 5′-bromo-2′-
deoxyuridine (BrdU) 8 days before learning8,9. BrdU-positive
cells were counted in the granule cell layer for each time period
post-learning (T1 to T5). We did not assess neurogenesis in the
glomerular cell layer of the OB since we had previously found no
modulation of the rate of neurogenesis in this layer after
perceptual learning8. Analysis was performed based on learning
performance, by comparing animals who discriminated after
learning (T1, T2, and T3) to those who did not (T4 and T5) and
to their corresponding NE groups (ANOVA, F(3 32) = 4.1, p =
0.014; Fig. 1d and Supplementary Fig. 2). We observed a higher
density of BrdU-positive cells in enriched groups able to
discriminate the two odorants used for enrichment compared
to enriched groups unable to do so (T1-T2-T3 versus T4-T5,
t-test, p = 0.015, Fig. 1d) and compared to NE groups (enriched
T1-T2-T3 vs non enriched T1-T2-T3, t-test, p = 0.028, Fig. 1d
and Supplementary Fig. 2). Accordingly, the density of BrdU-
positive cells was highly correlated to the index of discrimination
(Pearson correlation: R = 0.84; p = 0.038) (Fig. 1e). The density of
BrdU-positive cells showed no decline over the time points in NE
animals (ANOVA, F(4,11) = 0.8, p = 0.5, Supplementary Fig. 2). y
y
Here, using the olfactory system in mice, we tackle the issue of
how the acquisition of new memories influences the information
already stored in the network and we identify the role of adult-
born neurons in these processes. We used perceptual olfactory
discrimination learning, which depends on newly formed neu-
rons in the OB8 to show that (1) adult-born neurons saved by
learning are present in the OB as long as the task is remembered;
(2) with a second learning task occurring soon after the first,
introducing retrograde interference, the new memory overwrites
the older one and alters survival of previously recruited adult-
born neurons unless (3) the first learned odorants are maintained
in the environment. Finally, using sequential labeling of adult-
born neurons and selective optogenetic inactivation, we showed
(4) that each successive learning is supported by a specific
population of adult-born neurons. animals (ANOVA, F(4,11) = 0.8, p = 0.5, Supplementary Fig. 2). ARTICLE To determine whether the involvement of adult-born cell
population in processing of the learned odorants relates to the
discrimination ability, we assessed the density of cells co-
expressing BrdU and Zif268 as an index of adult-born cell
activation at the different time points post learning in three
experimental groups. More precisely, we assessed the responsive-
ness of adult-born cells to +lim (odorant used for the
enrichment) in enriched and NE animals. In addition, we
assessed the response to dec (odorant not used for enrichment)
of adult-born cells in +lim/−lim enriched animals (Fig. 1f). A 2-
way ANOVA showed a group effect (F(2,40) = 33.68, p < 0.0001), a
time
effect
(F(2,40) = 14.08,
p < 0.0001)
and
an
interaction
(F(8,40) = 8.13, p < 0.0001) indicating that the differences in
BrdU/Zif268 positive cell density observed between groups
depend on the time post learning. Interestingly, the density of
Zif268 expression in adult-born neurons was significantly higher
in enriched animals in response to +limonene at T1 to T3
compared to the other groups (Tukey tests, T1 versus T4 and T5,
p = 0.0003; T2 versus T4 and T5, p = 0.006; T3 versus T4, p =
0.0005 and T3 versus T5, p = 0.0006). This result is in accordance
with the performance of discrimination (Fig. 1f). Regarding the
NE group, no time effect was observed (ANOVA, F(4,15) = 4.66,
p = 0.84). These changes were underlined not only by changes in
the density of BrdU positive cells but also by changes in
percentage of BrdU/Zif268 positive cells (Supplementary Fig. 3). Notably, a higher percentage of BrdU/Zif268 was found in
enriched animals at T1-T3 compared to T4-T5 (t-test, p =
0.00022) in response to +lim but not dec, indicating of a higher
involvement of BrdU positive cells in processing the learned Short-term availability of adult-born neurons for
memory encoding
https://doi.org/10.1038/s41467-019-13521-7
OPEN Jérémy Forest1,2,3, Mélissa Moreno1,2,3, Matthias Cavelius1,2, Laura Chalençon1,2, Anne Ziessel1,2,
Joëlle Sacquet1,2, Marion Richard1,2, Anne Didier1,2,4 & Nathalie Mandairon
1,2,4* Jérémy Forest1,2,3, Mélissa Moreno1,2,3, Matthias Cavelius1,2, Laura Chalençon1,2, Anne Ziessel1,2,
Joëlle Sacquet1,2, Marion Richard1,2, Anne Didier1,2,4 & Nathalie Mandairon
1,2,4* Adult olfactory neurogenesis provides waves of new neurons involved in memory encoding. However, how the olfactory bulb deals with neuronal renewal to ensure the persistence of
pertinent memories and the flexibility to integrate new events remains unanswered. To
address this issue, mice performed two successive olfactory discrimination learning tasks
with varying times between tasks. We show that with a short time between tasks, adult-born
neurons supporting the first learning task appear to be highly sensitive to interference. Furthermore, targeting these neurons using selective light-induced inhibition altered memory
of this first task without affecting that of the second, suggesting that neurons in their critical
period of integration may only support one memory trace. A longer period between the two
tasks allowed for an increased resilience to interference. Hence, newly formed adult-born
neurons regulate the transience or persistence of a memory as a function of information
relevance and retrograde interference. 1 INSERM, U1028; CNRS, UMR5292; Lyon Neuroscience Research Center, Neuroplasticity and Neuropathology of Olfactory Perception Team, Lyon F-69000,
France. 2 Claude Bernard University-Lyon1 and University of Lyon, Lyon F-69000, France. 3These authors contributed equally: Jérémy Forest, Mélissa Moreno. 4These authors jointly supervised this work: Anne Didier, Nathalie Mandairon. *email: nathalie.mandairon@cnrs.fr NATURE COMMUNICATIONS | (2019) 10:5609 | https://doi.org/10.1038/s41467-019-13521-7 | www.nature.com/naturecommunications 1 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 Non enriched
± lim enrichment
± lim enrichment
± lim enrichment
± lim enrichment
Test
Test
Test
Test
Test
Test
T5
T4
T3
T2
T1
± lim enrichment
0
8
17
Enrichment period
24
34
44
54
64
Days
a Non enriched
± lim enrichment
± lim enrichment
± lim enrichment
± lim enrichment
Test
Test
Test
Test
Test
Test
T5
T4
T3
T2
± lim enrichment
0
4
+lim/–lim
dec/dodec
R = 0.84, p = 0.038
3
2
Investigation time (s)
Brdu-positive cell density
(cell mm–2)
Brdu-positive cell density
(cell mm–2)
BrdU-Zif268-positive cell
density (cell mm–2)
Investigation time (s)
1
0
4
3
Hab4
Test
Non-enriched and stimulated +lim
Enriched and stimulated +lim
Enriched and stimulated dec
Hab4
Test
2
1
0
NE
70
60
50
40
30
25
20
15
10
5
0
T1
T2
T3
Discrimination index
0.6
0.1
0.2
0.3
0.4
0.5
60
50
40
30
20
10
0
T1
T2
T3
T4
T5
T4
T5
T1
T2
T3
T4
T5
NE
T1
T2
T3
T4
T5
8
17
Enrichment period
24
34
44
54
64
Days
b
c
d
e
f
Fig. 1 The presence of adult-born neurons correlates with mnesic performance. a Experimental design. b–f Behavioral results. b Discrimina
lim and -lim is assessed at different time points after the enrichment period. Non-enriched control (NE; n = 9) animals do not discriminat
−lim at T1. After enrichment, the two odorants are discriminated at T1 (n = 11), T2 (n = 5), and T3 (n = 10) but not at T4 (n = 9) and T5 (n
dodec are not discriminated at any time post-learning (NE n = 9; T1 n = 10; T2 n = 7; T3 n = 8; T4 n = 5; T5 n = 10) (for clearer representat
belonging to the Test trial of the NE group is not shown (investigation time = 6 s)). T-test, *p < 0.05. d Adult-born neuron density in the
experimental groups. Groups that discriminated +lim/−lim (T1–T3) have a higher BrdU-positive cell density compared to groups that do
(T4-T5) (n = 4/group), t-test *p < 0.05. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 c Dec and
dodec are not discriminated at any time post-learning (NE n = 9; T1 n = 10; T2 n = 7; T3 n = 8; T4 n = 5; T5 n = 10) (for clearer representation, 1 data point
belonging to the Test trial of the NE group is not shown (investigation time = 6 s)). T-test, *p < 0.05. d Adult-born neuron density in the OB in enriched
experimental groups. Groups that discriminated +lim/−lim (T1–T3) have a higher BrdU-positive cell density compared to groups that do not discriminate
(T4-T5) (n = 4/group), t-test *p < 0.05. e Positive correlation between the discrimination index and adult-born neuron density in the OB. f Density of
activated adult-born neurons (BrdU + /Zif268 + cells) in +lim/−lim enriched groups in response to +lim (n = 4/group) or dec (n = 5/group) and in control
non-enriched in response to +lim (T1 n = 2; T2 n = 4; T3 n = 2; T4 n = 3; T5 n = 4). A significant increase of double-labeled cells is observed in enriched
groups in response to learned odorant (+lim) in T1–T3 compared to T4-T5. Tukey tests, * at least p < 0.05 for comparisons between enriched animals
responding to +lim. Data are represented as data points and mean ± sem. Source data are provided as a Source Data file. Fig. 1 The presence of adult-born neurons correlates with mnesic performance. a Experimental design. b–f Behavioral results. b Discrimination between +
lim and -lim is assessed at different time points after the enrichment period. Non-enriched control (NE; n = 9) animals do not discriminate the +lim from
−lim at T1. After enrichment, the two odorants are discriminated at T1 (n = 11), T2 (n = 5), and T3 (n = 10) but not at T4 (n = 9) and T5 (n = 5). c Dec and
dodec are not discriminated at any time post-learning (NE n = 9; T1 n = 10; T2 n = 7; T3 n = 8; T4 n = 5; T5 n = 10) (for clearer representation, 1 data point
belonging to the Test trial of the NE group is not shown (investigation time = 6 s)). T-test, *p < 0.05. d Adult-born neuron density in the OB in enriched
experimental groups. Results
f Performances are linked to adult-born neurons fate. Previous
studies have reported increased survival of adult-born granule cells
after perceptual discrimination learning8. To better understand the
potential role of these neurons in long-term memory, we first asked
whether increased bulbar neurogenesis persists for as long as the
learned information is remembered. Thus, different groups of mice
underwent perceptual discrimination learning consisting in a 10-
day enrichment period with (+)limonene and (−)limonene (+lim
and −lim), odorants which are not spontaneously discriminated by
mice. Their performance in discriminating these two odorants was
then analyzed from 1 to 5 weeks post learning (T1, T2, T3, T4, and
T5, Fig. 1a) using a habituation/dishabituation test. In this test, mice
were exposed to the first odorant of the pair 4 times (Hab1 to Hab4;
inter-trial interval 5 min) followed by exposure to the second
odorant of the pair (Test, 5 min between Hab4 and Test). The time
spent by the mouse investigating the odorant was recorded for all
trials. Decreased investigation time across habituation trials reflects
habituation to the odorant used and a longer time spent investing
Test than Hab4 shows discrimination of the two odorants of the
pair. At all time-points analyzed, mice exhibited habituation
behavior (Supplementary Table 1, Supplementary Fig. 1). Regarding
discrimination, using a two-way ANOVA, we observed significant
differences between groups (NE: Non Enriched, T1, T2, T3, T4, T5:
F(5,106) = 2.39; p = 0.043) and effect of the trial (Hab4 vs Test, NATURE COMMUNICATIONS | (2019) 10:5609 | https://doi.org/10.1038/s41467-019-13521-7 | www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 e Positive correlation between the discrimination index and adult-born neuron density in the OB
activated adult-born neurons (BrdU + /Zif268 + cells) in +lim/−lim enriched groups in response to +lim (n = 4/group) or dec (n = 5/grou
non-enriched in response to +lim (T1 n = 2; T2 n = 4; T3 n = 2; T4 n = 3; T5 n = 4). A significant increase of double-labeled cells is obse
groups in response to learned odorant (+lim) in T1–T3 compared to T4-T5. Tukey tests, * at least p < 0.05 for comparisons between en
responding to +lim. Data are represented as data points and mean ± sem. Source data are provided as a Source Data file. dec/dodec
Investigation time (s)
4
3
Hab4
Test
2
1
0
NE
T1
T2
T3
T4
T5
c 4
+lim/–lim
3
2
Investigation time (s)
1
0
Hab4
Test
NE
T1
T2
T3
T4
T5
b b c Hab4 Test d Brdu-positive cell density
(cell mm–2)
70
60
50
40
30
20
10
0
T1
T2
T3
T4
T5
d R = 0.84, p = 0.038
Brdu-positive cell density
(cell mm–2)
60
50
40
30
Discrimination index
0.6
0.1
0.2
0.3
0.4
0.5
e e R = 0.84, p = 0.038 Discrimination index BrdU-Zif268-positive cell
density (cell mm–2)
Non-enriched and stimulated +lim
Enriched and stimulated +lim
Enriched and stimulated dec
25
20
15
10
5
0
T1
T2
T3
T4
T5
f BrdU-Zif268-positive cell
density (cell mm–2)
Non-enriched and stimulated +lim
Enriched and stimulated +lim
Enriched and stimulated dec
25
20
15
10
5
0
T1
T2
T3
T4
T5
f f Fig. 1 The presence of adult-born neurons correlates with mnesic performance. a Experimental design. b–f Behavioral results. b Discrimination between +
lim and -lim is assessed at different time points after the enrichment period. Non-enriched control (NE; n = 9) animals do not discriminate the +lim from
−lim at T1. After enrichment, the two odorants are discriminated at T1 (n = 11), T2 (n = 5), and T3 (n = 10) but not at T4 (n = 9) and T5 (n = 5). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 Groups that discriminated +lim/−lim (T1–T3) have a higher BrdU-positive cell density compared to groups that do not discriminate
(T4-T5) (n = 4/group), t-test *p < 0.05. e Positive correlation between the discrimination index and adult-born neuron density in the OB. f Density of
activated adult-born neurons (BrdU + /Zif268 + cells) in +lim/−lim enriched groups in response to +lim (n = 4/group) or dec (n = 5/group) and in control
non-enriched in response to +lim (T1 n = 2; T2 n = 4; T3 n = 2; T4 n = 3; T5 n = 4). A significant increase of double-labeled cells is observed in enriched
groups in response to learned odorant (+lim) in T1–T3 compared to T4-T5. Tukey tests, * at least p < 0.05 for comparisons between enriched animals
responding to +lim. Data are represented as data points and mean ± sem. Source data are provided as a Source Data file. (T4-T5) (n = 4/group), t-test *p < 0.05. e Positive correlation between the discrimination index and adult-born neuron density in the OB. f Density of
activated adult-born neurons (BrdU + /Zif268 + cells) in +lim/−lim enriched groups in response to +lim (n = 4/group) or dec (n = 5/group) and in control
non-enriched in response to +lim (T1 n = 2; T2 n = 4; T3 n = 2; T4 n = 3; T5 n = 4). A significant increase of double-labeled cells is observed in enriched
groups in response to learned odorant (+lim) in T1–T3 compared to T4-T5. Tukey tests, * at least p < 0.05 for comparisons between enriched animals
responding to +lim. Data are represented as data points and mean ± sem. Source data are provided as a Source Data file. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 3 3 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 4
3
2
1
0
30
25
20
15
10
5
0
4
0
BrdU
8
17
24
+lim/–lim
+lim/–lim
+lim/–lim
+lim/–lim
+lim/–lim
Test
Test
Test
dec/dod
dec/dod
dec/dod
dec/dod
Test
Test
34
44
54
64 Days
+lim/–lim
dec/dodec
Hab4
Test
Hab4
Stimulated with +lim
Stimulated with dec
Test
3
2
Investigation time (s)
Investigation time (s)
1
0
90
80
70
60
50
30
20
BrdU-positive cell density
(cell mm–2)
BrdU-Zif268-positive cell
density(cell mm–2)
40
10
0
T1
T1
T2′
T2′
T3′
T4′
T5′
T3′
T4′
T5′
T1
T2′
T3′
T4′
T5′
T1
T2′
T3′
T4′
T5′
T1
T2′
T3′
T4′
T5′
a
b
c
d
e
Fig. 2 Time between learning sessions has a critical impact on memory. a Experimental design. The groups differed by the interval between the two
enrichment periods: 4, 14, 24 or 34 days separating the two enrichments. b, c Behavioral results. b Discrimination between +lim/−lim. Groups T1 and T3′
discriminate. No discrimination is observed for the other groups (T2′, T4′, T5′) (T1 n = 11; T2’ n = 11; T3′ n = 11; T4′ n = 6; T5′ n = 7) t-test, **p < 0.005;
***p < 0.001. c Dec is discriminated from dodec in Group T2′ to T5′ (T1 n = 10; T2′ n = 11; T3′ n = 11; T4′ n = 10; T5′ n = 11) t-test **p < 0.005; ***p < 0.001. d Adult-born neuron density. Higher density of adult-born neurons is observed in groups that are able to discriminate +lim from −lim (T1 and T3′) (n =
6–7/group), Tukey corrected t-tests *p < 0.05; ***p < 0.001. e Density of activated adult-born neurons (BrdU+/Zif268+ cells) in response to +lim (T1 n =
4; T2′ n = 6; T3′ n = 4; T4′ n = 6; T5′ n = 6) or dec (T1 n = 4; T2′ n = 6; T3′ n = 4; T4′ n = 6; T5′ n = 6) after successive enrichments. Density of double-
labeled cells is higher in response to +lim at T1 and T3′ compared to T2′, T4′, and T5′. In response to dec, the density of BrdU/Zif268 cells is higher at T3′
compared to the other groups. T-test *p < 0.05 for enriched animals responding to +lim at T1 and T3′ different from all other groups. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 Data are represented
as data points and mean ± sem. Source data are provided as a Source Data file. 0
BrdU
8
17
24
+lim/–lim
+lim/–lim
+lim/–lim
+lim/–lim
+lim/–lim
Test
Test
Test
dec/dod
dec/dod
dec/dod
dec/dod
Test
Test
34
44
54
64 Days
T1
T2′
T3′
T4′
T5′
a 4
3
2
1
0
dec/dodec
Hab4
Test
Investigation time (s)
T1
T2′
T3′
T4′
T5′
c 4
+lim/–lim
Hab4
Test
3
2
Investigation time (s)
1
0
T1
T2′
T3′
T4′
T5′
b b c 90
80
70
60
50
30
20
BrdU-positive cell density
(cell mm–2)
40
10
0
T1
T2′
T3′
T4′
T5′
d d 30
25
20
15
10
5
0
Stimulated with +lim
Stimulated with dec
BrdU-Zif268-positive cell
density(cell mm–2)
T1
T2′
T3′
T4′
T5′
e e e Fig. 2 Time between learning sessions has a critical impact on memory. a Experimental design. The groups differed by the interval between the two
enrichment periods: 4, 14, 24 or 34 days separating the two enrichments. b, c Behavioral results. b Discrimination between +lim/−lim. Groups T1 and T3′
discriminate. No discrimination is observed for the other groups (T2′, T4′, T5′) (T1 n = 11; T2’ n = 11; T3′ n = 11; T4′ n = 6; T5′ n = 7) t-test, **p < 0.005;
***p < 0.001. c Dec is discriminated from dodec in Group T2′ to T5′ (T1 n = 10; T2′ n = 11; T3′ n = 11; T4′ n = 10; T5′ n = 11) t-test **p < 0.005; ***p < 0.001. d Adult-born neuron density. Higher density of adult-born neurons is observed in groups that are able to discriminate +lim from −lim (T1 and T3′) (n =
6–7/group), Tukey corrected t-tests *p < 0.05; ***p < 0.001. e Density of activated adult-born neurons (BrdU+/Zif268+ cells) in response to +lim (T1 n =
4; T2′ n = 6; T3′ n = 4; T4′ n = 6; T5′ n = 6) or dec (T1 n = 4; T2′ n = 6; T3′ n = 4; T4′ n = 6; T5′ n = 6) after successive enrichments. Density of double-
labeled cells is higher in response to +lim at T1 and T3′ compared to T2′, T4′, and T5′. In response to dec, the density of BrdU/Zif268 cells is higher at T3′
compared to the other groups. ARTICLE 3b, c) and that the
second enrichment with dec/dodec when performed 4 days after
the first one erased the ability to discriminate +lim/−lim (group
2, Fig. 3b, c, Supplementary Table 3). Interestingly, if we
maintained the enrichment with +lim/−lim during the second
enrichment
period
(group
3),
the
animals
were
able
to
discriminate both odorant pairs (Fig. 3b, c, Supplementary
Table 3). This prevention of forgetting can be due to network
reactivation or relearning. Finally, when the retention of the task
was tested at T3′, mice were, as previously, able to discriminate
both odorant pairs (group 4, Fig. 3b, c, Supplementary Table 3). e
o y ete t o
(Supp e
e ta y
ab e
,
g. b, c). BrdU-positive cell density was then assessed as in the first
experiment. BrdU-positive cell density differed between groups
(ANOVA, group effect, F(4,26) = 16.1; p < 0.0001) (Fig. 2d). Post
hoc Tukey corrected t-tests were then performed and showed that
when the second enrichment was performed 4 days after the first
one (T2′), the density of labeled adult-born cells was significantly
decreased compared to T1 (Tukey corrected t-test p = 0.025). This
was accompanied by impaired +lim/−lim discrimination suggest-
ing that, at 4 days between two learning, new memory formation
altered adult-born neuron survival and already stored informa-
tion. However, when the second enrichment was performed
14 days after the first (T3′), both pairs of odorants were
discriminated and the density of adult-born cells remained high
(Tukey corrected t-test, T1 versus T3′: p = 0.2). Finally, when the
second enrichment occurred even later (T4′ or T5′), BrdU density
decreased significantly (Tukey corrected t-test, T3′ versus T4′: p <
0.001; T5′ versus T3′: p < 0.001) to a level comparable to T2′
(Tukey corrected t-test, T4′ versus T2′: p = 0.59; T5′ versus T2′:
p = 0.99) which is in accordance with the first experiment
(Fig. 2d). To study the involvement of adult-born neurons in
processing the learned information, we assessed the density of
adult-born neurons responding to +lim or to dec (group effect) at
the different delays between enrichments (Fig. 2e). Using two-way
ANOVA, we found a group effect (F(1,42) = 8.61, p = 0.0054), a
time
effect
(F(1,42) = 42.53,
p < 0.0001)
and
an
interaction
(F(1,42) = 13.08, p < 0.0001). ARTICLE More precisely, we labeled a first pool of 8-day-old adult-
born cells in the OB at the beginning of the first enrichment (with
+lim/−lim) with CldU and a second pool of 8-day-old adult-
born cells in the OB at the beginning of the second enrichment
(with dec/dodec) but not present in the OB at the beginning of
the first enrichment (Fig. 3a, d) with IdU. The densities of CldU-
and IdU-positive cells were assessed in the OB at T2. Overlapping
between CldU and IdU was never observed. Results showed
differences between experimental groups for both markers
(ANOVA, CldU: F(3,16) = 19.06, p < 0.0001; IdU: F(3,24) = 7.8,
p = 0.0008). Post hoc Tukey corrected t-tests revealed that the
level of CldU-positive cells was lower in group 2 compared to the
other groups, in accordance with +lim/−lim discrimination
performance (group 2 versus group 1: p = 0.056, group 2 versus
group 3: p = 0.0029, group 2 versus group 4: p < 0.001; Fig. 3e). Interestingly,
the
density
of
CldU-positive
cells
was
not
significantly higher in group 3 compared to group 1 (p = 0.48)
suggesting that the second enrichment recruited a new pool of
adult-born neurons. Supporting this hypothesis, all groups that
were submitted to dec/dodec enrichment after the +lim/−lim
enrichment, including group 2, showed a higher level of IdU-
positive cells than group 1 (respectively p = 0.03, p = 0.017 and p
< 0.001) (Fig. 3f). This strongly suggests that the second learning
recruited a new population of adult-born neurons younger than
that recruited during the first learning. We learned from this experiment that reactivating the network
of the first learned odorant pair during the second task prevented In all groups, mice exhibited habituation behavior (Supple-
mentary Table 3, Supplementary Fig. 8). Using two-way ANOVA,
we observed significant effects of trial and group (trial: F(1,164) =
16.88; p < 0.0001, group: F(3,164) = 12.55; p < 0.0001) (Fig. 3b) for
+lim/−lim discrimination and a trial effect and a group effect
(trial: F(1,164) = 31.33; p < 0.0001, group: F(3,164) = 3.14; p = 0.026)
(Fig. 3c) for dec/dodec discrimination. As in the first experiment
(Fig. 1a), we observed that +lim/−lim enrichment improved
discrimination between these two odorants when tested at T2’
(group 1, see Supplementary Table 3, Fig. NATURE COMMUNICATIONS | (2019) 10:5609 | https://doi.org/10.1038/s41467-019-13521-7 | www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 ARTICLE would avoid this overwriting of the memory. To do this we
maintained exposure to the first pair of odorants during enrich-
ment with the second pair (Fig. 3a). in this case) (Supplementary Fig. 6). When the time between the
two enrichments increased (14 days, T3′), the animals were able
to discriminate the two odorants of each pair (Supplementary
Table 2, Fig. 2b, c) suggesting that in this experimental config-
uration the formation of a new memory did not impact the
memory already stored. At longer times between enrichments (24
and 34 days, respectively T4′ and T5′), +lim and −lim were no
longer discriminated while dec and dodec were, which is in
accordance with the first experiment reporting the duration of
memory retention (Supplementary Table 2, Fig. 2b, c). ment with the second pair (Fig. 3a). In all groups, mice exhibited habituation behavior (Supple-
mentary Table 3, Supplementary Fig. 8). Using two-way ANOVA,
we observed significant effects of trial and group (trial: F(1,164) =
16.88; p < 0.0001, group: F(3,164) = 12.55; p < 0.0001) (Fig. 3b) for
+lim/−lim discrimination and a trial effect and a group effect
(trial: F(1,164) = 31.33; p < 0.0001, group: F(3,164) = 3.14; p = 0.026)
(Fig. 3c) for dec/dodec discrimination. As in the first experiment
(Fig. 1a), we observed that +lim/−lim enrichment improved
discrimination between these two odorants when tested at T2’
(group 1, see Supplementary Table 3, Fig. 3b, c) and that the
second enrichment with dec/dodec when performed 4 days after
the first one erased the ability to discriminate +lim/−lim (group
2, Fig. 3b, c, Supplementary Table 3). Interestingly, if we
maintained the enrichment with +lim/−lim during the second
enrichment
period
(group
3),
the
animals
were
able
to
discriminate both odorant pairs (Fig. 3b, c, Supplementary
Table 3). This prevention of forgetting can be due to network
reactivation or relearning. Finally, when the retention of the task
was tested at T3′, mice were, as previously, able to discriminate
both odorant pairs (group 4, Fig. 3b, c, Supplementary Table 3). To understand the dynamics of learning-dependent survival of
adult-born neurons underlying these effects, we tagged two adult-
born neuron populations differing in age using two analogues of
thymidine, ChlorodeoxyUridine (CldU) and IododeoxyUridine
(IdU). ARTICLE In enriched animals in response to
+lim, the density of BrdU/Zif268 cells is higher at T1 and T3’
compared to T2′, T4′, and T5′ (Time effect F(4,21) = 73.09, p <
0.0001; Tukey corrected t-test p < 0.05) which is in accordance
with the performance of +lim versus −lim discrimination. In
enriched animals in response to dec, the density of BrdU/Zif268
cells is higher at T3′ compared to the other groups (Time effect
F(4,21) = 12.08, p < 0.0001; Tukey corrected t-test p < 0.05). These
changes in density relied at least partly on changes in percentage
of BrdU/Zif268 double-labeled cells (Supplementary Fig. 7). For
the T2′, the decrease in the BrdU-Zif268 density is due more to a
decrease in BrdU-positive cell density than to a decrease in the
percentage of BrdU/Zif268 double-labeled cells. However, overall,
the percentage of BrdU/Zif268-positive cells was higher in groups
that discriminate +lim from −lim (T1 and T3′) than in groups
that do not discriminate (T2′, T4′, and T5′) (p = 0.007). p
g
p
g
pp
y
To understand the dynamics of learning-dependent survival of
adult-born neurons underlying these effects, we tagged two adult-
born neuron populations differing in age using two analogues of
thymidine, ChlorodeoxyUridine (CldU) and IododeoxyUridine
(IdU). More precisely, we labeled a first pool of 8-day-old adult-
born cells in the OB at the beginning of the first enrichment (with
+lim/−lim) with CldU and a second pool of 8-day-old adult-
born cells in the OB at the beginning of the second enrichment
(with dec/dodec) but not present in the OB at the beginning of
the first enrichment (Fig. 3a, d) with IdU. The densities of CldU-
and IdU-positive cells were assessed in the OB at T2. Overlapping
between CldU and IdU was never observed. Results showed
differences between experimental groups for both markers
(ANOVA, CldU: F(3,16) = 19.06, p < 0.0001; IdU: F(3,24) = 7.8,
p = 0.0008). Post hoc Tukey corrected t-tests revealed that the
level of CldU-positive cells was lower in group 2 compared to the
other groups, in accordance with +lim/−lim discrimination
performance (group 2 versus group 1: p = 0.056, group 2 versus
group 3: p = 0.0029, group 2 versus group 4: p < 0.001; Fig. 3e). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 T-test *p < 0.05 for enriched animals responding to +lim at T1 and T3′ different from all other groups. Data are represented
as data points and mean ± sem. Source data are provided as a Source Data file. odorant in groups showing the ability to discriminate +lim
from −lim. discrimination, for +lim/−lim, using two-way ANOVA, we
observed a group effect (T1,T2′,T3′,T4′,T5′, F(4,94) = 4.63; p =
0.002) and a trial effect (F(1,94) = 19.99; p < 0.0001). For dec/
dodec, there was no group effect (F(4,108) = 2.19; p = 0.08) but a
trial effect (F(1,108) = 45.08; p < 0.0001) and interaction (F(4,108) =
5.49; p = 0.0005). When the second enrichment was performed
4 days after the first one (T2’), animals were only able to dis-
criminate the second odorant pair (Supplementary Table 2,
Fig. 2b, c) suggesting that the acquisition of the second task
erased the memory of the first one. This result was confirmed
using another strain of mice (129 mice, Supplementary Fig. 5). As
an additional control, we switched the training order, the animals
were enriched first with dec/dodec and then with +lim/−lim. Result was similar to that previously obtained: animals were only
able to discriminate the second learned odorant pair (+lim/−lim In summary, perceptual memory showed a natural memory
decay between T3 and T4. This memory alteration is accom-
panied by a decrease in the density of adult-born neurons and in
the percentage of adult-born neurons responding to the learned
odorants. Time between learning sessions affects stored memory. In total
4, 14, 24 or 34 days after a first enrichment with +lim/−lim, mice
were enriched with a new odorant pair, dec/dodec, tested for
discrimination of the two pairs of odorants and sacrificed at
the same times as in the previous experiment (groups T2′ to
T5′) (Fig. 2a). All groups exhibited habituation behavior (Sup-
plementary
Table
2,
Supplementary
Fig. 4). Regarding NATURE COMMUNICATIONS | (2019) 10:5609 | https://doi.org/10.1038/s41467-019-13521-7 | www.nature.com/naturecommunications 4 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 8
0
8
13 17
21
23 30 31
34
40
44
Days
Group4
Group3
Group2
Group1
T2′
T3′
+lim/–lim
+lim/–lim
No odor
Test
Test
Test
Test
dec/dod
dec/dod
dec/dod
+lim/–lim
7
6
5
Investigation time (s)
Investigation time (s)
4
3
2
1
0
Group 1
Group 2
Group 3
Group 4
Group 1
Group 2
Group 3
Group 4
8
a
b
+lim/–lim
dec/dodec
7
6
5
Hab4
Test
Hab4
Test
4
3
2
1
0
+lim/–lim
+lim/–lim
c a 8
7
6
5
Investigation time (s)
4
3
2
1
0
Group 1
Group 2
Group 3
Group 4
b
+lim/–lim Investigation time (s)
Group 1
Group 2
Group 3
Group 4
8
dec/dodec
7
6
5
4
Hab4
Test
4
3
2
1
0
c Group 1
Group 2
Group 3
Group 4
Group 1
Group 2
Group 3
Group 4
80
70
60
50
40
CldU-positive cell density
(cell/mm2)
IdU-positive cell density
(cell/mm2)
30
20
10
0
+lim/–lim
discrimination
dec/dodec
discrimination
–
–
+
+
+
+
+
+
80
70
60
50
40
30
20
10
0
d
e
f
CldU/IdU/DAPI
10 μm d
CldU/IdU/DAPI
10 μm d d +lim/–lim
discrimination Fig. 3 Reactivation of learned information prevented cell death and maintained memory. a Experimental design. b, c Behavioral results. b +lim and -lim
are discriminated in groups 1 (n = 18), 3 (n = 20) and 4 (n = 10) but not in group 2 (n = 18). c Dec/dodec are discriminated in Group 2 (n = 18), 3 (n = 20)
and 4 (n = 10) but not in group 1 (n = 18). d Example of CldU/DAPI and IdU/DAPI double labeling. e. Density of CldU-positive cells. A lower density is
observed in group 2 compared to groups 1, 3, and 4 and a higher density in group 4 compared to groups 1, 2, and 3 (n = 5/group). f Density of IdU-positive
cells is higher in groups 2 (n = 8), 3 (n = 7) and 4 (n = 5) compared to group 1 (n = 8). Tukey corrected t-tests *p ≤0.05, ***p < 0.001. Data are
represented as data points and mean ± sem. Source data are provided as a Source Data file. but not of dec/dodec (Fig. 4c, Supplementary Table 4). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 d lt b
h
t f
di ti
t
i
W
b t
t
f d
/d d
(Fi
4
S
l
t
T bl 4) A i
8
0
8
13 17
21
23 30 31
34
40
44
Days
Group4
Group3
Group2
Group1
T2′
T3′
+lim/–lim
+lim/–lim
No odor
Test
Test
Test
Test
dec/dod
dec/dod
dec/dod
+lim/–lim
7
6
5
Investigation time (s)
Investigation time (s)
4
3
2
1
0
Group 1
Group 2
Group 3
Group 4
Group 1
Group 2
Group 3
Group 4
Group 1
Group 2
Group 3
Group 4
Group 1
Group 2
Group 3
Group 4
8
a
b
+lim/–lim
dec/dodec
7
6
5
Hab4
Test
Hab4
Test
4
3
2
80
70
60
50
40
CldU-positive cell density
(cell/mm2)
IdU-positive cell density
(cell/mm2)
30
20
10
0
+lim/–lim
discrimination
dec/dodec
discrimination
–
–
+
+
+
+
+
+
80
70
60
50
40
30
20
10
0
1
0
+lim/–lim
+lim/–lim
c
d
e
f
CldU/IdU/DAPI
10 μm
ctivation of learned information prevented cell death and maintained memory. a Experimental design. b, c Behavioral results. b +lim and
minated in groups 1 (n = 18), 3 (n = 20) and 4 (n = 10) but not in group 2 (n = 18). c Dec/dodec are discriminated in Group 2 (n = 18), 3 (n =
= 10) but not in group 1 (n = 18). d Example of CldU/DAPI and IdU/DAPI double labeling. e. Density of CldU-positive cells. A lower dens
n group 2 compared to groups 1, 3, and 4 and a higher density in group 4 compared to groups 1, 2, and 3 (n = 5/group). f Density of IdU-po
her in groups 2 (n = 8), 3 (n = 7) and 4 (n = 5) compared to group 1 (n = 8). Tukey corrected t-tests *p ≤0.05, ***p < 0.001. Data are
d as data points and mean ± sem. Source data are provided as a Source Data file. ARTICLE Interestingly,
the
density
of
CldU-positive
cells
was
not
significantly higher in group 3 compared to group 1 (p = 0.48)
suggesting that the second enrichment recruited a new pool of
adult-born neurons. Supporting this hypothesis, all groups that
were submitted to dec/dodec enrichment after the +lim/−lim
enrichment, including group 2, showed a higher level of IdU-
positive cells than group 1 (respectively p = 0.03, p = 0.017 and p
< 0.001) (Fig. 3f). This strongly suggests that the second learning
recruited a new population of adult-born neurons younger than
that recruited during the first learning. In summary, the time between the two enrichments is critical
to the upkeep of the memory already stored and associated adult-
born neurons survival and functional involvement. These data
further suggest that the survival of at least some of these adult-
born neurons is not assured as, for one week after learning, they
go through a fragile state, which is vulnerable to a new learning
challenge. g
g
We learned from this experiment that reactivating the network
of the first learned odorant pair during the second task prevented
memory erasure and the disappearance of adult-born cells. Thus,
for a short time between two successive learning tasks, adult-born
neurons can be maintained in the network providing that the
relevant sensory inputs remain in the environment. However,
despite the presence of these adult-born neurons still in their
critical period of integration, a distinct pool of younger adult-born
neurons was recruited by the second task. It remains the question
of whether the second learning relies on the last recruited adult-
born neuron population (IdU-positive cells) or on both adult-born
neuron populations (IdU- and CldU-positive cells). In other
words, are adult-born neurons allocated to a memory trace able to
underlie or contribute to subsequent learning? Sensory reactivation prevents cell and memory loss. With a
short period between the two learning sessions (T2′), we found
that the new memory altered previously stored information and
induced apoptosis of adult-born neurons. Since cell survival is
known to be input-dependent, we tested whether reactivating the
network that responded to the odorants used for the enrichment 5 5 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 Animals
infected with a control virus and submitted to light stimulation
behaved as group 3 of the previous experiment, suggesting no
direct effect of the light. To test that light-triggered inhibition of
adult-born neurons in halorhodopsin animals had no deleterious
effect on spontaneous odor discrimination, we assessed the dis-
crimination of a pair of dissimilar odorants (+lim/+carvone) and
found that it was not impaired by light (Fig. 4d, Supplementary
Table 4). We then analyzed the level of viral transfection in
control and halorhodopsin groups and found no difference of
EYFP-positive cell density (t-test, p = 0.5, Fig. 4e). We also ver-
ified the effectiveness of light-mediated inhibition of adult-born
granule cells by assessing the expression of Zif268-positive cells in Distinct adult-born neuron cohorts for distinct memories. We
used the same learning paradigm as previously (group 3; Figs. 3a
and 4a) in which mice underwent a first enrichment period with
+lim/−lim and 4 days later a second one with dec/dodec in the
presence of +lim/−lim to avoid memory loss. Eight days before
the first enrichment period, adult-born neuron progenitors were
transduced with halorhodopsin-expressing or control lentiviruses
coupled with EYFP expression and animals were implanted with
optical fibers in the OB. Light stimulation was used to block
adult-born cell activity during the test trial of the habituation/
dishabituation task. Results showed that light-triggered inhibition
of neurons born 8 days before the first learning task altered the
discrimination of +lim/−lim (Fig. 4b, Supplementary Table 4) Distinct adult-born neuron cohorts for distinct memories. We
used the same learning paradigm as previously (group 3; Figs. 3a
and 4a) in which mice underwent a first enrichment period with
+lim/−lim and 4 days later a second one with dec/dodec in the
presence of +lim/−lim to avoid memory loss. Eight days before
the first enrichment period, adult-born neuron progenitors were
transduced with halorhodopsin-expressing or control lentiviruses
coupled with EYFP expression and animals were implanted with
optical fibers in the OB. Light stimulation was used to block
adult-born cell activity during the test trial of the habituation/
dishabituation task. Results showed that light-triggered inhibition
of neurons born 8 days before the first learning task altered the
discrimination of +lim/−lim (Fig. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 4b, Supplementary Table 4) NATURE COMMUNICATIONS | (2019) 10:5609 | https://doi.org/10.1038/s41467-019-13521-7 | www.nature.com/naturecommunications 6 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 7
b
a
c
e
f
d
0
Halor
8
17
21
30
36
Optogenetic
inhibition
Test
Days
T2′
6
Investigation time (s)
Investigation time (s)
Investigation time (s)
5
4
3
1
2
0
200
60
40
20
0
100
EYFP-positive cell density
(cell mm–2)
EYFP-Zif268-positive
cells (%)
0
Control
Halo
Control
Halo
Hab1
Hab2
Hab3
Hab4
Test
Hab1
Hab2
Hab3
Hab4
Test
Hab1
Hab2
Hab3
Hab4
Test
7
11
dec/dodec
dec/dod
+lim/–lim
+lim/–lim
+lim/–lim
+lim/+carvone
Cont
Halo
10
9
8
7
6
5
4
3
2
1
0
6
5
4
ns
3
1
2
0
Fig. 4 Optogenetic inhibition revealed that distinct adult-born neuron cohorts underlined distinct memories. a Experimental design. Adult-born neurons
aged 8 days at the beginning of the first enrichment were transduced with a lentivirus expressing halorhodopsin (Halo) or with an empty virus (Cont). Light
stimulation was performed during the test trial of the habituation/dishabituation task. b–d Behavioral results. b The learned discrimination of +lim/−lim is
abolished by light stimulation in the Halo group (n = 13), but not in the Control group (n = 12). c Discrimination between dec/dodec is not altered by light
stimulation (Cont n = 13; Halo n = 14). d Discrimination between a dissimilar odorant pair +lim/ +carvone is not altered by light-triggered inhibition of
adult-born neurons (Cont n = 9; Halo n = 14). t-tests *p < 0.05, **p < 0.01, ***p < 0.001 (for difference between Hab4 and Test in b–d). e The density of
EYFP-positive transduced cells in the OB is similar between Control (n = 9) and Halo (n = 7) animals. f Percentage of EYFP/Zif268-positive cells after
light-triggered inhibition is decreased in Halo (n = 7) compared to Control (n = 8). t-test *p < 0.05 (for difference between Halo and Control groups in f). Data are represented as data points and mean ± sem. Source data are provided as a Source Data file. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 It
is worth noting that in the short time condition, the number of
adult-born cells is reduced but a substantial fraction of adult-born
neurons (>20%) still respond to the learned odorant even though born neurons present in the OB after the first task are prematurely
killed and memory is lost. This conclusion is based on the loss of
BrdU-positive neurons and is consistent with previous reports
showing that blocking adult-born cell death prevented memory
erasure19. In addition to training-dependent changes in BrdU-
positive cell density, the functional involvement of these cells in
response to the learned odorant increased with performance for
neurons aged 8 days at the beginning of learning. We further show
that these two learning tasks, despite they are spaced by a short 4-
day time period, induced two distinct waves of adult-born neuron
survival and that optogenetically inactivating the first cohort
altered the first memory, without affecting the second learning. This further reinforces the idea that the 20-day old BrdU-tagged
neurons exited the time window during which they are most
crucial to enrichment-induced improvement in discrimination. It
is worth noting that in the short time condition, the number of
adult-born cells is reduced but a substantial fraction of adult-born
neurons (>20%) still respond to the learned odorant even though Hence, silencing a cohort of adult-born neurons aged between
8 and 18 days at the time of learning leads to memory erasure
demonstrating
the
crucial
role
of
adult-born
neurons
in
perceptual memory. However, this manipulation did not affect
the memory of a new discrimination task learned shortly
afterwards, suggesting that these adult-born neurons are allocated
to only one memory trace. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 Light
stimulation was performed during the test trial of the habituation/dishabituation task. b–d Behavioral results. b The learned discrimination of +lim/−lim is
abolished by light stimulation in the Halo group (n = 13), but not in the Control group (n = 12). c Discrimination between dec/dodec is not altered by light
stimulation (Cont n = 13; Halo n = 14). d Discrimination between a dissimilar odorant pair +lim/ +carvone is not altered by light-triggered inhibition of
adult-born neurons (Cont n = 9; Halo n = 14). t-tests *p < 0.05, **p < 0.01, ***p < 0.001 (for difference between Hab4 and Test in b–d). e The density of
EYFP-positive transduced cells in the OB is similar between Control (n = 9) and Halo (n = 7) animals. f Percentage of EYFP/Zif268-positive cells after
light-triggered inhibition is decreased in Halo (n = 7) compared to Control (n = 8). t-test *p < 0.05 (for difference between Halo and Control groups in f). Data are represented as data points and mean ± sem. Source data are provided as a Source Data file. EYFP-positive neurons after stimulation with light. The expres-
sion of Zif268 was significantly lower in the halorhodospin
compared to the control group (t-test, p = 0.03, Fig. 4f). born neurons present in the OB after the first task are prematurely
killed and memory is lost. This conclusion is based on the loss of
BrdU-positive neurons and is consistent with previous reports
showing that blocking adult-born cell death prevented memory
erasure19. In addition to training-dependent changes in BrdU-
positive cell density, the functional involvement of these cells in
response to the learned odorant increased with performance for
neurons aged 8 days at the beginning of learning. We further show
that these two learning tasks, despite they are spaced by a short 4-
day time period, induced two distinct waves of adult-born neuron
survival and that optogenetically inactivating the first cohort
altered the first memory, without affecting the second learning. This further reinforces the idea that the 20-day old BrdU-tagged
neurons exited the time window during which they are most
crucial to enrichment-induced improvement in discrimination. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 7
b
a
c
d
0
Halor
8
17
21
30
36
Optogenetic
inhibition
Test
Days
T2′
6
Investigation time (s)
Investigation time (s)
Investigation time (s)
5
4
3
1
2
0
Hab1
Hab2
Hab3
Hab4
Test
Hab1
Hab2
Hab3
Hab4
Test
Hab1
Hab2
Hab3
Hab4
Test
7
11
dec/dodec
dec/dod
+lim/–lim
+lim/–lim
+lim/–lim
+lim/+carvone
Cont
Halo
10
9
8
7
6
5
4
3
2
1
0
6
5
4
ns
3
1
2
0 a
0
8
17
21
30
36
Optogenetic
inhibition
Test
Days
T2′
dec/dod
+lim/–lim
+lim/–lim a d d b e
f
200
60
40
20
0
100
EYFP-positive cell density
(cell mm–2)
EYFP-Zif268-positive
cells (%)
0
Control
Halo
Control
Halo f Fig. 4 Optogenetic inhibition revealed that distinct adult-born neuron cohorts underlined distinct memories. a Exper Fig. 4 Optogenetic inhibition revealed that distinct adult-born neuron cohorts underlined distinct memories. a Experimental design. Adult-born neurons
aged 8 days at the beginning of the first enrichment were transduced with a lentivirus expressing halorhodopsin (Halo) or with an empty virus (Cont). Light
stimulation was performed during the test trial of the habituation/dishabituation task. b–d Behavioral results. b The learned discrimination of +lim/−lim is
abolished by light stimulation in the Halo group (n = 13), but not in the Control group (n = 12). c Discrimination between dec/dodec is not altered by light
stimulation (Cont n = 13; Halo n = 14). d Discrimination between a dissimilar odorant pair +lim/ +carvone is not altered by light-triggered inhibition of
adult-born neurons (Cont n = 9; Halo n = 14). t-tests *p < 0.05, **p < 0.01, ***p < 0.001 (for difference between Hab4 and Test in b–d). e The density of
EYFP-positive transduced cells in the OB is similar between Control (n = 9) and Halo (n = 7) animals. f Percentage of EYFP/Zif268-positive cells after
light-triggered inhibition is decreased in Halo (n = 7) compared to Control (n = 8). t-test *p < 0.05 (for difference between Halo and Control groups in f). Data are represented as data points and mean ± sem. Source data are provided as a Source Data file. Fig. 4 Optogenetic inhibition revealed that distinct adult-born neuron cohorts underlined distinct memories. a Experimental design. Adult-born neurons
aged 8 days at the beginning of the first enrichment were transduced with a lentivirus expressing halorhodopsin (Halo) or with an empty virus (Cont). NATURE COMMUNICATIONS | (2019) 10:5609 | https://doi.org/10.1038/s41467-019-13521-7 | www.nature.com/naturecommunications Discussion
h For the olfactory enrichment, 100 μL of pure odorant
were placed in two separate tea balls of the home cages for one hour daily over
10 days. For the multiple enrichment (experiment 3, group 3), the two pairs of
odorants were presented with an interval of 1 h (SI Methods). We assessed the spontaneous discrimination between two pairs of similar
odorants: +lim/−lim and dec/dodec using olfactory habituation/dishabituation. A
test session consisted of four 50-s odor presentations of a first odorant (Hab) at
5 min intervals, followed by one 50-s presentation of the second odorant of the pair
(Test). Investigation was defined as active sniffing within 1 cm of the tea ball (SI
Methods)8,24,25. Exploration assessments were done blind with regard to the
experimental group. Perceptual learning. This implicit olfactory learning consisted in passive exposure
to odorants (enrichment). For the olfactory enrichment, 100 μL of pure odorant
were placed in two separate tea balls of the home cages for one hour daily over
10 days. For the multiple enrichment (experiment 3, group 3), the two pairs of
odorants were presented with an interval of 1 h (SI Methods). We assessed the spontaneous discrimination between two pairs of similar
odorants: +lim/−lim and dec/dodec using olfactory habituation/dishabituation. A
test session consisted of four 50-s odor presentations of a first odorant (Hab) at
5 min intervals, followed by one 50-s presentation of the second odorant of the pair
(Test). Investigation was defined as active sniffing within 1 cm of the tea ball (SI
Methods)8,24,25. Exploration assessments were done blind with regard to the
experimental group. Data analysis. Data analysis was performed using R software (CRAN). Normality
was assessed using the Kolmogorov-Smirnov test. Global two-way ANOVAs were
performed to evaluate changes in discrimination abilities between groups. Then
intra-group one-way RM-ANOVAs and unilateral paired t-tests were performed to
determine whether the mice exhibited habituation (trial effect) as well as dis-
crimination (by comparing Hab4 and Test). Discrimination was indicated by a
significant increase in investigation time during the test trial. Discrimination index
was calculated as [1-(Hab4/Test)]26 (SI Methods). Sample sizes were determined
based on previous reports8,14,27. Animal assignation to the various experimental
groups was randomized. Adult-born cell density assessment. Bromodeoxyuridine (BrdU) (Sigma-
Aldrich) was injected 8 days before the enrichment period (50 mg kg-1 in saline, 3x
at 2-h intervals; i.p.)14,22. Much of what we experience is ultimately forgotten, but
memories for some events persist. Discussion
h The OB is continuously receiving waves of adult-born neurons
and so contains adult-born neurons of different ages and levels of
maturation18. When learning happens successively, which neu-
rons take part in underlying behavioral changes? When the time
between the two learning tasks is short (4 days), part of the adult- 7 7 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 discrimination is lost. This state differs from that observed for
longer between-task times where both density of adult-born
neurons and their responsiveness to the learned odorants are
strongly reduced. This suggests only a partial withdrawal of the
remaining adult-born neurons from processing the first learned
odorants in the short time condition, sufficient however to prevent
discrimination. Nevertheless, we conclude that cohorts of neurons
of different ages, but still within their critical period, are required
to allow the encoding of successive learning experiences. Impor-
tantly, when the time between tasks is longer (14 days), no cell loss
occurred and performances were maintained. Altogether, these
data provide evidence for an unexpectedly early and sharp func-
tional transition within the critical period. The critical period of
adult-born neurons which, based on their vulnerability to cell
death, morphological development and synaptic current devel-
opment and plasticity, has been reported to last from 30 days to
60 days post neuronal birth6,10,20–23. Within this critical period,
our data suggest that availability of adult-born neurons for
encoding new experience lasts no longer than 20 days. tested on their spontaneous discrimination between +lim and −lim and also between
another pair of perceptually similar odorants (decanal (dec) and dodecanone
(dodec)). Discrimination was assessed using an olfactory habituation/dishabituation
task. Animals were sacrificed 24, 34, 44, 54 or 64 days after BrdU injections (Fig. 1a). Regarding the second experiment, eight days after BrdU injection, a different set
of mice was similarly enriched with +lim and −lim but this was followed by a
second 10-day enrichment period with dec and dodec either 4, 14, 24, 34 or 44 days
after the first. At the end of both enrichments, mice were tested on their
spontaneous discrimination between the two odorants of each of the pairs using an
olfactory habituation/dishabituation task and animals were sacrificed 24, 34, 44, 54
and 64 days post BrdU injections (Fig. 2a) In the third experiment, we used two other DNA markers (analogues of BrdU), 5′-
chloro-2′-deoxyuridine (CldU) and 5′-iodo-2′-deoxyuridine (IdU), to label two
different populations of adult-born cells. Discussion
h Here, we report that modula-
tion of the OB circuitry, as a function of the environment and
dependent on the fate of maturing adult-born neurons, is
responsible for the balance between the transience or persistence
of memory. This is made possible thanks to distinct neuronal
populations encoding temporally distinct experiences. 24, 34, 44, 54 and 64 days post BrdU injection, five mice were taken randomly
from each experimental group for sacrifice and BrdU, IdU and CldU
immunohistochemistries were carried on as described in SI Methods14,22. 24, 34, 44, 54 and 64 days post BrdU injection, five mice were taken randomly
from each experimental group for sacrifice and BrdU, IdU and CldU
immunohistochemistries were carried on as described in SI Methods14,22. The method used for BrdU-, IdU- and CldU-positive cell counting is described
in SI Methods14,28. The mean positive cell density of each array was calculated and
averaged within each experimental group. Between-groups comparisons of the
mean cell density were performed by ANOVA followed by post hoc t-tests with
Tukey corrections. Unilateral t-tests were performed for comparisons between two
groups. The level of significance was set to 0.05. The method used for BrdU-, IdU- and CldU-positive cell counting is described
in SI Methods14,28. The mean positive cell density of each array was calculated and
averaged within each experimental group. Between-groups comparisons of the
mean cell density were performed by ANOVA followed by post hoc t-tests with
Tukey corrections. Unilateral t-tests were performed for comparisons between two
groups. The level of significance was set to 0.05. Discussion
h CldU was injected 8 days before the first
enrichment (again with +lim and -lim) while IdU was injected 8 days before the
second enrichment period. The second enrichment period varied among groups: no
enrichment (group 1); dec and dodec (group 2 and group 4), +lim and −lim plus dec
and dodec (group 3). The time between the two enrichments was either 4 or 14 days. As previously, discrimination was assessed using a habituation/dishabituation task
and animals were sacrificed 34 or 44 days post-CldU injection (Fig. 3a). y
j
g
In the fourth experiment using group 3 experimental configuration from the
previous experiment, we performed targeted lentiviral-induced halorhodospin
channel expression (NpHR3.0) in the subventricular zone, and used optogenetics
to specifically inhibit the population of adult-born neurons arriving in the OB at
the beginning of the +lim/−lim enrichment (Fig. 4a). g
p
g
y
When previously learned information is reintroduced into the
environment, we observed no premature cell death and the second
learning did no longer erase the first one. To maintain the first
learned discrimination, the network could either use adult-born
neurons already integrated into this network or recruit a new cohort
of adult-born neurons. Optogenetic inactivation of the 8-day old
cohort of adult-born neurons present in the OB at the beginning of
the first training impaired memory of the first task. This strongly
suggests that performance relies on adult-born neurons recruited in
the initial task suggesting that the memory is recalled rather than
re-encoded. Hence, reactivated memories are not over-written and
forgotten. On the contrary, neurons encoding obsolete information
(i.e. not present in the environment) could be sentenced to death
after about 45 days unless another learning task occurs soon after,
causing interference and leading to earlier neuronal death. Our data
thus suggest that adult-born neurons are sensitive to interference
only during their first 3 weeks and that if further learning occurs
when adult-born neurons present in the OB are more than 21 days
old, they are resistant to death and memory persists. On the other
hand, a second learning task causes neurons aged less than 21 days
old to die and erases the encoded memory. This difference could be
due to the state of synaptic integration of the adult-born neurons20
which are more or less sensitive to competition from later waves of
adult-born neurons. Perceptual learning. This implicit olfactory learning consisted in passive exposure
to odorants (enrichment). Additional information y
6. Mouret, A. et al. Learning and survival of newly generated neurons: when time
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019-13521-7. 7. Lazarini, F. et al. Cellular and behavioral effects of cranial irradiation of the
subventricular zone in adult mice. PLoS ONE 4, e7017 (2009). Correspondence and requests for materials should be addressed to N.M. 8. Moreno, M. M. et al. Olfactory perceptual learning requires adult
neurogenesis. Proc. Natl Acad. Sci. USA 106, 17980–17985 (2009). Peer review information Nature Communications thanks the anonymous reviewers for
their contribution to the peer review of this work. Peer reviewer reports are available. Peer review information Nature Communications thanks the anonymous reviewers for
their contribution to the peer review of this work. Peer reviewer reports are available. g
9. Moreno, M. M. et al. Action of the noradrenergic system on adult-born cells is
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neurogenesis promotes synaptic plasticity in the olfactory bulb. Nat. Neurosci. 12, 728–730 (2009). Reprints and permission information is available at http://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. 11. Sultan, S. et al. Learning-dependent neurogenesis in the olfactory bulb
determines long-term olfactory memory. FASEB J. 24, 2355–2363 (2010). 12. Sultan, S., Lefort, J. M., Sacquet, J., Mandairon, N. & Didier, A. Acquisition of
an olfactory associative task triggers a regionalized down-regulation of adult
born neuron cell death. Front. Neurosci. 5, 52 (2011). 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. ARTICLE ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13521-7 19. Sultan, S., Rey, N., Sacquet, J., Mandairon, N. & Didier, A. Newborn neurons
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in the OB, with the following coordinates respective to bregma: antero-posterior
+4.6 mm, medio-lateral ±0.75 mm, dorso-ventral −2 mm. Mice were injected with a
ketoprophen solution (2 mg kg−1) after the surgery as well as during the following
days and allowed to recover with food and water ad libidum. 20. Kelsch, W., Lin, C.-W., Mosley, C. P. & Lois, C. A critical period for activity-
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patterns mimicking the average light stimulating pattern during the test trial (0.75 s
light ON, 5 s light OFF for 1 h). After brain sectioning (see above), EYFP and
Zif268 double immunohistochemistry was performed: incubation with rabbit
Zif268 antibody (1:1.000, Santa Cruz, ref: Sc-189), chicken GFP antibody (1:1.000,
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cellular markers of olfactory aging and their response to enrichment. Neurobiol. Aging 33, 626.e9–626.e23 (2012). Data availability y
The data associated to all main figures are provided as a Source Data file. The data associated to all main figures are provided as a Source Data file. 27. Forest, J. et al. Role of adult-born versus preexisting neurons born at p0 in
olfactory perception in a complex olfactory environment in mice. Cereb. Cortex https://doi.org/10.1093/cercor/bhz105 (2019). Author contributions 2. Lois, C. & Alvarez-Buylla, A. Long-distance neuronal migration in the adult
mammalian brain. Science 264, 1145–1148 (1994). J.F., M.M., A.D., and N.M. designed the research. J.F., M.M., A.Z, M.C., J.S., and L.C. performed the research. J.F., M.M., M.R., A.D., and N.M. wrote the paper. 3. Ming, G. & Song, H. Adult neurogenesis in the mammalian central nervous
system. Annu. Rev. Neurosci. 28, 223–250 (2005). Acknowledgements This work was supported by CNRS, Inserm, Lyon 1 University and Ecole Normale Supér-
ieure de Lyon. We would like to thank C. Benetollo from the Neurogenetic and Optogenetic
Platform of the CRNL for lentiviral production and G. Froment, D. Nègre and C. Costa from
the lentivector production facility /SFR BioSciences de Lyon (UMS3444/US8). This work was supported by CNRS, Inserm, Lyon 1 University and Ecole Normale Supér-
ieure de Lyon. We would like to thank C. Benetollo from the Neurogenetic and Optogenetic
Platform of the CRNL for lentiviral production and G. Froment, D. Nègre and C. Costa from
the lentivector production facility /SFR BioSciences de Lyon (UMS3444/US8). The authors declare no competing interests. 5. Alonso, M. et al. Olfactory discrimination learning increases the survival of
adult-born neurons in the olfactory bulb. J. Neurosci. 26, 10508–10513 (2006). adult-born neurons in the olfactory bulb. J. Neurosci. 26, 10508–10513 (2006). 6. Mouret, A. et al. Learning and survival of newly generated neurons: when time
matters. J. Neurosci. 28, 11511–11516 (2008). Methods
Mi
Ad l Mice. Adult C57BL/6J mice (8 weeks old, male, Charles River, L’arbresles, France)
and 129 mice (8 weeks old, male, Charles River, L’arbresles, France) were used in
this study. They were housed in standard laboratory cages with water and food ad
libitum and were kept on a 12-hr light/dark cycle at a constant temperature of
22 °C. All behavioral training was conducted in the afternoon (12:00–18:00). Experiments were done following procedures in accordance with the European
Community Council Directive of 22 September 2010 (2010/63/UE) and approved
by the National Ethics Committee (Agreement DR2013-48(vM)). Every effort was
made to minimize animal suffering. BrdU/Zif268 experiment. To investigate immediate early gene expression in
response to odorant exposure, mice were presented with a tea ball containing 100 µl
of pure +lim or dec for 1 h, 1 h before sacrifice. A rabbit anti-Zif268 (1:1000, Santa
Cruz Biotechnology) and a rat anti-BrdU (1:100, Harlan Sera-Lab) were used. The
appropriate secondary antibodies, coupled to Alexa Fluor 633 and 488 (Invitrogen)
were used for revelation of the different markers. BrdU/Zif268 density was cal-
culated by combining the mean percentage of BrdU/Zif268 positive cells per group
to individual density of BrdU positive cells. All cell counts were conducted blind
with regard to the experimental group. Different animals were used in different experimental groups. Histological data
were obtained from a sample of animals used for behavioral tasks. Optogenetics in freely behaving mice. Hundred and fifty nanoliters of pLenti-
hSyn-eNpHR3.0-EYFP lentivirus (9.22 × 106IU ml−1) or 300 nL of control pLenti-
hSyn-EYFP (1.1 × 106 IU ml−1, expressing only the reporter gene EYFP29) injections
were done bilaterally in the subventricular zone, with the following coordinates
respective to bregma: antero-posterior + 1 mm, medio-lateral ± 1 mm, dorso-ventral
−2.3 mm and at a rate of 150 nL min−1. Just after virus infusions, mice were Experimental designs. Regarding the first experiment, mice were injected with a
DNA marker, BrdU, in order to label a cohort of adult-born neurons. Eight days later,
they began the enrichment procedure with (+)limonene (+lim) and (−)limonene
(−lim) for one hour daily over 10 days. At the end of the enrichment, mice were NATURE COMMUNICATIONS | (2019) 10:5609 | https://doi.org/10.1038/s41467-019-13521-7 | www.nature.com/naturecommunications 8 Competing interests 4. Ming, G. & Song, H. Adult neurogenesis in the mammalian brain: significant
answers and significant questions. Neuron 70, 687–702 (2011). p
g
The authors declare no competing interests. Additional information If material is not included in the
article’s Creative Commons license and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this license, visit http://creativecommons.org/
licenses/by/4.0/. 13. Grelat, A. et al. Adult-born neurons boost odor-reward association. Proc. Natl
Acad. Sci. USA 115, 2514–2519 (2018). 14. Mandairon, N. et al. Opposite regulation of inhibition by adult-born
granule cells during implicit versus explicit olfactory learning. eLife 7, e34976
(2018). 15. Akers, K. G. et al. Hippocampal neurogenesis regulates forgetting during
adulthood and infancy. Science 344, 598–602 (2014). y
16. Frankland, P. W. & Josselyn, S. A. Hippocampal neurogenesis and memory
clearance. Neuropsychopharmacology 41, 382–383 (2016). 17. Richards, B. A. & Frankland, P. W. The persistence and transience of memory. Neuron 94, 1071–1084 (2017). 18. Carleton, A. et al. Making scents of olfactory neurogenesis. J. Physiol.-Paris 96,
115–122 (2002). © The Author(s) 2019 9 NATURE COMMUNICATIONS | (2019) 10:5609 | https://doi.org/10.1038/s41467-019-13521-7 | www.nature.com/naturecommunicatio
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Serine/Threonine Kinase Inhibitor XL418
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Definitions
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Serine/Threonine Kinase Inhibitor XL418 National Cancer Institute National Cancer Institute Qeios ID: 3TS65E · https://doi.org/10.32388/3TS65E Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Source National Cancer Institute. Serine/Threonine Kinase Inhibitor XL418. NCI Thesaurus. Code
C68963. A selective, orally active small molecule, targeting protein kinase B (PKB or AKT) and
ribosomal protein S6 Kinase (p70S6K), with potential antineoplastic activity. XL418 inhibits
the activities of PKB and p70S6K, both acting downstream of phosphoinosotide-3 kinase
(PI3K). These kinases are often upregulated in a variety of cancers. Inhibition of PKB by
this agent will induce apoptosis, while inhibition of p70S6K will result in the inhibition of
translation within tumor cells. Qeios ID: 3TS65E · https://doi.org/10.32388/3TS65E 1/1
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https://openalex.org/W2605618870
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https://aip.scitation.org/doi/pdf/10.1063/1.4981800
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Topography evolution of germanium thin films synthesized by pulsed laser deposition
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AIP advances
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RESEARCH ARTICLE | APRIL 17 2017
Topography evolution of germanium thin films synthesized
by pulsed laser deposition RESEARCH ARTICLE | APRIL 17 2017 P. Schumacher; S. G. Mayr; B. Rauschenbach AIP Advances 7, 045115 (2017)
https://doi.org/10.1063/1.4981800 Articles You May Be Interested In Initial perturbation matters: Implications of geometry-dependent universal Kardar–Parisi–Zhang statistics
for spatiotemporal chaos for spatiotemporal chaos Chaos (November 2021) Effects of grains’ features in surface roughness scaling Variational approach to KPZ: Fluctuation theorems and large deviation function for entropy production
Chaos (July 2020) 24 October 2024 05:20:36 AIP ADVANCES 7, 045115 (2017) Topography evolution of germanium thin films synthesized
by pulsed laser deposition P. Schumacher,1,a S. G. Mayr,1,2 and B. Rauschenbach1,2
1Leibniz-Instiut f¨ur Oberfl¨achenmodifizierung (IOM), Permoserstr. 15, 04318 Leipzig, Germany
2Fakult¨at f¨ur Physik und Geowissenschaften, Universit¨at Leipzig, 04103 Leipzig, Germany
(Received 1 February 2017; accepted 9 April 2017; published online 17 April 2017) P. Schumacher,1,a S. G. Mayr,1,2 and B. Rauschenbach1,2
1Leibniz-Instiut f¨ur Oberfl¨achenmodifizierung (IOM), Permoserstr. 15, 04318 Leipzig, Germany
2Fakult¨at f¨ur Physik und Geowissenschaften, Universit¨at Leipzig, 04103 Leipzig, Germany
(Received 1 February 2017; accepted 9 April 2017; published online 17 April 2017) (Received 1 February 2017; accepted 9 April 2017; published online 17 April 2017) Germanium thin films were deposited by Pulsed Laser Deposition (PLD) onto
single crystal Ge (100) and Si (100) substrates with a native oxide film on
the surface. The topography of the surface was investigated by Atomic Force
Microscopy (AFM) to evaluate the scaling behavior of the surface roughness of
amorphous and polycrystalline Ge films grown on substrates with different rough-
nesses. Roughness evolution was interpreted within the framework of stochastic
rate equations for thin film growth. Here the Kardar-Parisi-Zhang equation was
used to describe the smoothening process. Additionally, a roughening regime was
observed in which 3-dimensional growth occurred. Diffusion of the deposited Ge
adatoms controlled the growth of the amorphous Ge thin films. The growth of poly-
crystalline thin Ge films was dominated by diffusion processes only in the initial
stage of the growth. © 2017 Author(s). All article content, except where other-
wise noted, is licensed under a Creative Commons Attribution (CC BY) license
(http://creativecommons.org/licenses/by/4.0/). [http://dx.doi.org/10.1063/1.4981800] 24 October 2024 05:20:36 aElectronic mail: philipp.schumacher@iom-leipzig.de I. INTRODUCTION Since the first report of Pulsed Laser Deposition (PLD) in 1965,1 it has become an established
technique for coating surfaces and producing thin films. This technique has several advantages with
respect to molecular beam epitaxy, among which are the high versatility, applicability and possi-
bility to perform stoichiometric transfer, even with complicated compounds.2,3 For applications of
the PLD technique, it is important to understand the physical principles that govern the growth
process. Recent studies performed with Ge have yielded results about the roughness evolution,
parameter dependence of the epitaxial breakdown and coarsening of mound-like structures on the
surface.4–6 PLD has also been used to grow Ge nanostructures that have potential importance in device
fabrication.7 This work contributes to the understanding of basic, physical mechanisms in thin film growth
by PLD by taking into account the crystalline phase, film thickness, as well as substrate material for
the growth of Ge as an exemplary material. Particularly, diffusion rates of adatoms in PLD can be
increased and thereby change the surface topography significantly in comparison to other deposition
techniques.8 Consequently, the question arises, for which conditions diffusion processes dominate the
growth. With this objective in mind, the scaling behavior of the surface roughness was investigated. By applying the power spectral density (PSD) function to the surface topography as obtained by an
Atomic Force Microscope (AFM), physical mechanisms that govern the growth processes on certain
length scales can be identified. The basic feasibility of such an approach has been demonstrated
within different scopes previously.9,10 In this context, the following continuum models that describe
the surface evolution were considered. In a simplest case, random deposition is assumed, which
means that particles are deposited on random positions and remain there. I. INTRODUCTION The growth exponent β,
which characterizes the scaling behavior of the roughness over the time, can be calculated as β=0.5
for random deposition.11 If surface diffusion is a dominant factor (in addition to random deposition) aElectronic mail: philipp.schumacher@iom-leipzig.de © Author(s) 2017 7, 045115-1 7, 045115-1 7, 045115-1 2158-3226/2017/7(4)/045115/7 045115-2 Schumacher, Mayr, and Rauschenbach AIP Advances 7, 045115 (2017) AIP Advances 7, 045115 (2017) the Wolf-Villain equation12 that combines Mullins-Herring curvature-induced surface diffusion13,14
with stochastic noise has been demonstrated to yield results in good accordance with experimental
data: the Wolf-Villain equation12 that combines Mullins-Herring curvature-induced surface diffusion13,14
with stochastic noise has been demonstrated to yield results in good accordance with experimental
data: ∂h(x, y, t)
∂t
= -K ∇4h (x, y, t) + η(x, y, t). (1) (1) Here, h(x, y, t) is the height of the surface at position (x, y) for time t. K is a temperature dependent
constant and η(x, y, t) a noise term. For the first term, diffusion of particles on the surface is assumed
to occur according to the local chemical potential, dependent on the local curvature of the surface. The second term is a noise term that takes into account the randomness of the process. For this process
the growth exponent is β=0.25, assuming a two-dimensional system.15 The roughness exponent α
characterizes the scaling behavior of the roughness, depending on the length scale. For processes
dominated by the Mullins-Herring equation, the roughness exponent is α=1 for two dimensions.15
Another growth mechanism is represented by the Kardar-Parisi-Zhang equation16 Here, h(x, y, t) is the height of the surface at position (x, y) for time t. K is a temperature dependent
constant and η(x, y, t) a noise term. For the first term, diffusion of particles on the surface is assumed
to occur according to the local chemical potential, dependent on the local curvature of the surface. The second term is a noise term that takes into account the randomness of the process. For this process
the growth exponent is β=0.25, assuming a two-dimensional system.15 The roughness exponent α
characterizes the scaling behavior of the roughness, depending on the length scale. I. INTRODUCTION For processes
dominated by the Mullins-Herring equation, the roughness exponent is α=1 for two dimensions.15
Another growth mechanism is represented by the Kardar-Parisi-Zhang equation16 ∂h(x, y, t)
∂t
= v∇2h (x, y, t) + λ
2
∇h(x, y, t)2 + η (x, y, t) ,
(2) (2) (2) where h(x, y, t) is again the height of the surface at the position (x, y) and for time t. λ and ν are
constants. The first term reflects a deposition-desorption phenomenon. Depending on the chemical
potential of the vapor and surface, which depends on the local surface curvature, either desorption
from ordepositionontothesurfacedominates.Thesecondtermreflectstheexistenceoflateralgrowth,
while the third term takes into account noise since random deposition is assumed. Growth which is
governed by this equation shows a scaling behavior characterized by a roughness exponent α=0.39
and a growth exponent β=0.25.17 These exponents are numerical results of the Kardar-Parisi-Zhang
equation. 24 October 2024 05:20:36 If three-dimensional growth occurs, the coarsening exponent γ describes the relationship between
the film thickness and separation of mound-structures on the surface. For Ge films produced by PLD,
Shin et al. experimentally found a coarsening exponent γ=0.40±0.05.4 II. EXPERIMENTAL CONDITIONS In the present investigations, Ge films were deposited from a high purity Ge target by means of
PLD. As substrates, Si (100) and Ge (100) were used. The native oxide layers on the substrates were
not removed. A KrF-excimer laser with a pulse length of 20 ns and wavelength of 248 nm was used
for PLD. The pulse frequency was 10 Hz and the pulse energy was fixed at 220 mJ, with a focal spot
size of roughly 0.05 cm2 that resulted in a fluence of 4.4 J/cm2. For all depositions, the background
pressure was <8×108 mbar. The average deposition rate was on the order of 0.001 nm per pulse. The
thickness of the Ge films was controlled via the number of laser pulses and monitored by a quartz
crystal microbalance, which was calibrated regularly by means of X-ray reflectometry. The substrates
could be heated using a resistive heater and the temperature was corrected using a thermocouple. The
topography of the films was investigated by an AFM in tapping mode with a scan size of 2x2 µm2. The Si tips had a nominal radius of 7 nm. From the statistical point of view, the height fluctuations
of the surface could be described by the root mean square roughness (RMS) Rq, which is given by Rq =
r
1
NM
XN
i=1
XM
j=1 [h(xi, yj) −¯h]2,
(3) (3) where h(xi, yj) is the surface height at a given point (xi, yj), N and M are the number of points in
x- and in y-direction, respectively and ¯h =
1
NM
PN
i=1
PM
j=1 h(xi, yj) is the average height of the surface. The RMS value of each film was measured on a square area of 500 x 500 nm2. During film growth,
different points on the surface are not independent and the height at each point is related to the height
at other points nearby. The information about these correlations and about characteristic distances of
structures is provided by the height-height correlation function H
δx, δy, t
=
D
[h
x + δx, y + δy, t
−h(x, y, t)]2E
,
(4) (4) Schumacher, Mayr, and Rauschenbach AIP Advances 7, 045115 (2017) AIP Advances 7, 045115 (2017) 045115-3 where δx and δy are distances in x- and in y-direction respectively. The angular brackets denote
averaging over the plane. II. EXPERIMENTAL CONDITIONS Additionally, the PSD function was calculated in order to analyze the height
profiles in reciprocal space. The scaling behavior of this function is related to the aforementioned
roughness exponents.18 It is therefore possible to deduce the physical principles governing the growth
of the material. By performing the discrete two dimensional fast Fourier transformation (FFT) of the
height profile of the AFM images, the dominating spatial frequencies on the surface and amplitude
of the roughness can be determined. For the discrete case, a square image of N x N points, the
two-dimensional fast Fourier transformation is given by FFT
fx, fy
= 1
N2
XN
i=1
XN
j=1 h
xi, yj
exp
"
−2iπ
N
xifx + yjfy
#
,
(5) (5) where f x and f y are the spatial frequency coordinates along the x-axis and y-axis, respectively. By calculation of the magnitude square of FFT(f x, f y), the two-dimensional power spectral density
function PSD(f x, f y) can be obtained.19 The structures on the surface were expected to be isotropic, so
the angular averaged PSD(f ) was used to evaluate the experimental roughness data. The averaging was
performed over all spatial frequencies with constant magnitude f =
q
f 2x + f 2y . For the PSD function,
an expression can be derived from which the contributing processes can be deduced10 PSD (f , t) = D(f )1 −e−2t P4
i=1 aif i
P4
i=1 aif i
(6) (6) where D(f ) is the strength of the white noise, t is the time and ai ≥0 are constants whose relations
contain information about the growth process and the dominating mechanisms. 24 October 2024 05:20:36 III. RESULTS AND DISCUSSION Epitaxial growth of Ge on Si (100) by physical vapor deposition has been previously observed
(see e.g. Ref. 20). However, in this study, epitaxial growth was not expected due to the presence of
native oxide on the substrates and the comparably low temperatures during the deposition process. FIG. 1. (a,b) AFM images of polycrystalline Ge films deposited on Ge at 175 ◦C. The RMS value and the thickness d are
given below each image. (c) The roughness evolution of polycrystalline Ge films deposited on Ge at 175 ◦C. (d) The evolution
of the roughness in the roughening regime with a rescaled film thickness (see text), shown with a power law fit. The same data
is also plotted with respect to the number of laser pulses, indicated by circular data points. FIG. 1. (a,b) AFM images of polycrystalline Ge films deposited on Ge at 175 ◦C. The RMS value and the thickness d are
given below each image. (c) The roughness evolution of polycrystalline Ge films deposited on Ge at 175 ◦C. (d) The evolution
of the roughness in the roughening regime with a rescaled film thickness (see text), shown with a power law fit. The same data
is also plotted with respect to the number of laser pulses, indicated by circular data points. 045115-4
Schumacher, Mayr, and Rauschenbach AIP Advances 7, 045115 (2017) FIG. 2. The mound separation determined by the height-height correlation function with respect to the film thickness of
polycrystalline Ge films deposited at 175 ◦C. IG. 2. The mound separation determined by the height-height correlation function with respect to the film thickness of
olycrystalline Ge films deposited at 175 ◦C. The evolution of the surface topography was studied by variation of both the substrate material and
substrate temperature. The Ge deposition was either carried out at room temperature to produce
amorphous films or at (175±30) ◦C to produce polycrystalline films. The evolution of the surface topography was studied by variation of both the substrate material and
substrate temperature. The Ge deposition was either carried out at room temperature to produce
amorphous films or at (175±30) ◦C to produce polycrystalline films. In Figs. 1 (a) and (b), AFM images of polycrystalline Ge films deposited on Ge substrates
and with two different film thicknesses are compared. Additionally, Fig. 1 presents the roughness
evolution with respect to the film thickness. III. RESULTS AND DISCUSSION The AFM images and roughness evolution revealed that
the deposited Ge film was not initially closed. The Ge film was smoothened until it closed above
a critical layer thickness of approximately 45 nm. Consequently, the roughness rose with the layer
thickness, which was caused by a coarsening process during the deposition. In agreement to the
study of Shin et al.,4 the formation of irregularly shaped mounds was observed. During the pulsed
laser ablation process, the deposition rate was not constant; therefore, the film thickness was not a
time-equivalent quantity. For this reason, the film thickness d was rescaled in order to determine
the growth exponent. The rescaled and time-equivalent film thickness was calculated according to
d′ = (¯r/r) (d −45nm). Here, d is divided by the time-averaged deposition rate r of each sample and
multiplied by a constant ¯r. This rescaling generated the film thickness that would exist if all the
samples were deposited at a constant deposition rate of ¯r. The result of this rescaling is depicted
in Fig. 1 (d). The growth exponent β could be determined for the roughening regime above a film 24 October 2024 05:20:36 FIG. 3. The roughness evolution of Ge films on Si substrates at two different temperatures. The filled points represent the
data for the polycrystalline films (deposited at 175 ◦C), while the hollow points represent the data for the amorphous films
(deposited at room temperature). The circular points (red) show the roughness evolution with respect to the number of laser
pulses, and the square points (black) show the roughness evolution with respect to the film thickness. FIG. 3. The roughness evolution of Ge films on Si substrates at two different temperatures. The filled points represent the
data for the polycrystalline films (deposited at 175 ◦C), while the hollow points represent the data for the amorphous films
(deposited at room temperature). The circular points (red) show the roughness evolution with respect to the number of laser
pulses, and the square points (black) show the roughness evolution with respect to the film thickness. 045115-5
Schumacher, Mayr, and Rauschenbach AIP Advances 7, 045115 (2017) FIG. 4. The PSD function calculated from AFM measurements of Ge films with different thicknesses on various substrates:
(a) amorphous films, deposited on Si at room temperature, (b) polycrystalline films, deposited on Si at 175 ◦C and (c)
polycrystalline films, deposited on Ge at 175 ◦C. thickness of 45 nm. III. RESULTS AND DISCUSSION It was calculated from the slope of the log-log representation of the roughness as FIG. 4. The PSD function calculated from AFM measurements of Ge films with different thicknesses on various substrates:
(a) amorphous films, deposited on Si at room temperature, (b) polycrystalline films, deposited on Si at 175 ◦C and (c)
polycrystalline films, deposited on Ge at 175 ◦C. thickness of 45 nm. It was calculated from the slope of the log-log representation of the roughness as
a function of thickness. By fitting the roughness data, β=0.45±0.17 was obtained. Compared to the
aforementioned growth exponents, random deposition gave the most adequate representation of this thickness of 45 nm. It was calculated from the slope of the log-log representation of the roughness as
a function of thickness. By fitting the roughness data, β=0.45±0.17 was obtained. Compared to the
aforementioned growth exponents, random deposition gave the most adequate representation of this Schumacher, Mayr, and Rauschenbach AIP Advances 7, 045115 (2017) 045115-6 roughening regime. Nevertheless, the values for growth dominated by diffusion and growth along the
local surface were within the margins of error. The roughening regime was analyzed by means of the height-height correlation function (see
eq. 4). The separation of mounds was found by determining the distance of the first maximum to
the center peak in the height-height correlation. After rescaling,7 it could be shown, that the mound
separation increased with the film thickness (see Fig. 2). The coarsening exponent was determined to
be 0.48±0.29. This corresponded to a high coarsening rate, which was also found by Shin et al.4 In that
context, the high coarsening exponent was justified by defect-mediated filling of gaps and increased
effective corner-diffusion, which was confirmed by the observation of irregularly shaped mounds
(Fig. 1 (b)). The roughness evolution of Ge films deposited on Si substrates at room temperature and
at 175 ◦C is depicted in Fig. 3. For small film thicknesses the RMS roughness increased slightly up to
0.2 nm corresponding to a polycrystalline film thickness of about 10 nm. After that, a smoothening
regime occurred. For large film thicknesses (>50 nm) the roughness was rather constant. For all films, only small differences between the roughness evolution of the amorphous and
polycrystalline films were observed. In Fig. 4, the evolution of the PSD function for different crystalline phases of the film and different
substrate materials are presented. III. RESULTS AND DISCUSSION For the deposition of amorphous Ge on Si, the fits revealed that a4
(see eq. 6) was large compared to the other constants, while a3 was not negligible either. Mullins-
Herring diffusion was therefore identified as the presumably dominating process. On small length
scales, surface diffusion was important for the evolution of the surface. From the PSD curves at
intermediate spatial frequencies (f ≈0.03 nm-1), the evolution of the topography could be described
by the Kardar-Parisi-Zhang equation. This behavior was similar for all the film thicknesses shown
here. For the deposition of polycrystalline Ge on Si, the fits revealed a comparably high a4-constant
for small film thickness (4 nm), while a4=0 and a3 dominated for medium film thickness (8 nm and 30
nm). In the beginning of the growth, surface diffusion as described by Mullins and Herring governed
the process. After a certain film thickness, surface diffusion was suppressed and the quadratic term
of the Kardar-Parisi-Zhang equation became more prominent. Traditionally the interpretation of this
term has been desorption, however due to the relatively small deposition temperatures, desorption
was assumed to be negligible here. Different, more complex mechanisms might also account for such
a functional dependency. For high film thickness (112 nm), ‘bumps’ and irregularities in the PSD
function appeared due to the formation of mound structures with characteristic separations. 24 October 2024 05:20:36 pp
p
For the deposition of polycrystalline Ge on Ge, the fits revealed domination of surface diffusion
up to a film thickness of 57 nm. In these cases the PSD curves show an f −4-behavior for large f
and an f 1-behavior for smaller f. Traditionally the f 1-dependency has been interpreted as viscous
flow.21 It is to be noted however, that noise might have also contributed here. It is not possible to
distinguish between noise and the real f 1-dependency. For larger film thicknesses, again ‘bumps’
evolved, leading to relatively large deviations between the fits and measured data. The ‘bumps’
moved to lower spatial frequencies from film thicknesses of 74 nm to 96 nm. This shift indicated,
that the average separation of mound-structures increased, as was also shown above by means of the
height-height correlation. Kinetic Monte-Carlo simulations of PLD of Si on Si have already been performed.22 For thermal
deposition, the occurrence of mound-like structures and existence of a second maximum in the
height-height correlation were observed. III. RESULTS AND DISCUSSION Additionally the roughness exponent was determined to be
α≈0.95±0.1 for a deposition temperature of 400 ◦C. Comparable roughness exponents were found
in this study at lower temperatures depending on the exact deposition conditions. IV. SUMMARY For the deposition of polycrystalline Ge on Ge at 175 ◦C the roughness of the films initially
decreased. This regime was dominated by a Mullins-Herring diffusion process. Beginning at a certain
film thickness (approx. 45 nm), a roughening occurred. This roughening was accompanied by the
occurrence of mound-structures, which were observed in the height-height correlation function as
well as in the PSD spectra. These changes were also accompanied by a comparably high coarsening
rate. 045115-7 Schumacher, Mayr, and Rauschenbach AIP Advances 7, 045115 (2017) AIP Advances 7, 045115 (2017) For the deposition of amorphous Ge on Si at room temperature, the surfaces were smoothened
after reaching film thicknesses of approx. 10 nm. Here, Mullins-Herring diffusion was found to
be the governing mechanism on small length scales, independent of the film thickness. For the
deposition of polycrystalline Ge at 175 ◦C, the surfaces were also smoothened after reaching film
thicknesses of several nm. However, the Mullins-Herring diffusion was identified as present only
for small film thickness (4 nm). For larger film thicknesses, surface diffusion was suppressed and
the Kardar-Parisi-Zhang equation was a suitable description of the process already on small length
scales. ACKNOWLEDGMENTS We would like to thank Dr. Erik Thelander for the technical assistance and for several useful
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11 J. T. Drotar, Y.-P. Zhao, T.-M. ACKNOWLEDGMENTS Lu, and G.-C. Wang, Phys. Rev. B 62, 2118 (2000). 12 11 J. T. Drotar, Y.-P. Zhao, T.-M. Lu, and G.-C. Wang, Phys. Rev. B 62, 2118 (2000). 12 1 J. T. Drotar, Y.-P. Zhao, T.-M. Lu, and G.-C. Wang, Phys. Re 12 D. E. Wolf and J. Villain, Europhys. Lett. 13, 389 (1990). 12 D. E. Wolf and J. Villain, Europhys. Lett. 13, 389 (1990). 24 October 2024 05:20:36 13 W. Mullins, J. Appl. Phys. 28, 333 (1957). 13 W. Mullins, J. Appl. Phys. 28, 333 (1957). 14 14 C. Herring, J. Appl. Phys. 21, 301 (1950). 14 C. Herring, J. Appl. Phys. 21, 301 (1950). 5 15 A. L. Barabasi and H. E. Stanley, Fractal Concepts of Surface Growth (Cambridge University Press, 1995). 16 5 A. L. Barabasi and H. E. Stanley, Fractal Concepts of Surfac
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18 W. M. Tong and R. S. Williams, Annu. Rev. Phys. Chem. 45, 401 (1994). 19 18 W. M. Tong and R. S. Williams, Annu. Rev. Phys. Chem. 45, 40 19 Y. Zhao, G.-C. Wang, and T.-M. Lu, Characterization of amorphous and Crystalline Rough Surfaces: Principles and
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Applications (Academic Press, San Diego, 2001). Applications (Academic Press, San Diego, 2001). 20 A. N. Larsen, Mat. Sci. Semicon. Proc. 9, 454 (2006). 21 20 A. N. Larsen, Mat. Sci. Semicon. Proc. 9, 454 (2006). 21 21 C. C. Umbach, R. H. Headrick, and K.-C. Chang, Phys. Rev. Lett. 87, 246104 (2001). 22 21 C. C. Umbach, R. H. Headrick, and K.-C. Chang, Phys. Rev. Lett. 87, 246104 (2001). 22 22 S. G. Mayr, M. Moske, K. Samwer, M. E. Taylor, and H. A. Atwater, Appl. Phys. Lett. 75, 4091 (1999). ACKNOWLEDGMENTS 22 S. G. Mayr, M. Moske, K. Samwer, M. E. Taylor, and H. A. Atwater, Appl. Phys. Lett. 75, 4091 (1999).
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Agile logistics challenges of petroleum product distribution in Nigeria
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Agile logistics challenges of petroleum product distribution in Nigeria national Journal of Science and Research Archive, 2024, 11(02), 531–542 Publication history: Received on 04 February 2024; revised on 12 March 2024; accepted on 15 March 2024 Article DOI: https://doi.org/10.30574/ijsra.2024.11.2.0436 Abstract Agile distribution of petroleum product is a strategy towards uninterrupted supply of quality products at affordable
prices. Industry and economic productivity as well as transportation in Nigeria is largely driven by petroleum energy
due to low investments in other sources of energy, yet the petroleum energy supply and distribution is marred by
lingering history of scarcity, high prices, and distribution bottlenecks to end users all over the nation. The study through
a descriptive survey following literature view, primary and secondary data collection sought to determine at granular
level the limiting factors of agile petroleum product distribution in Nigeria. Petroleum supply, consumption and pricing
are significant aspects of the study survey. Study findings shows limiting factors of distribution agility cut across pricing,
supply, technology, infrastructure, accessibility and lead time, storage, management, and Human Resource issues. Significantly lack of local refining capacity and poor distribution network are notorious for supply limitations and rising
price of products. Addressing the challenges would require strong political will, driven by genuine commitment and
focused attention towards economic growth and improvement in the welfare of the citizens. Keywords: Petroleum; Distribution; Agility; Supply Keywords: Petroleum; Distribution; Agility; Supply Copyright © 2024 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons * Corresponding author: Goodluck Tochukwu Njoku Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4 2.1. Distribution Enterprise Task A significant aspect of the enterprise logistics function is distribution. Specifically referring to the delivery of the finished
products to the customer. Schewe and Smith (1980) define distribution as the physical movement of products to the
ultimate consumers. It consists of order processing, warehousing, and transportation. Distribution Straka (2017)
identify is responsible for storage and transportation of goods to consumers as well as related information,
management, and control activities. It ensures the most appropriate way, selection, and analysis of transport, which is
most suitable for transfer of products manufactured by enterprises to achieve failure-free performance of the market. The distribution network comprise of specific nodes which include; factories where products are manufactured or
assembled; a depot or deposit relating to standard type of warehouse for storing of merchandise (high level of
inventory); distribution centres for order processing and fulfillment (lower level of inventory) and also for receiving
returning items from clients, transit points built for cross docking activities, which consist in reassembling cargo units
based on deliveries scheduled (only moving merchandise). 2. Literature review Energy is key to economic productivity and industrial growth and is central to the operation of any modern economy. Energy is one of the most important inputs for economic development and from a physical viewpoint, the use of energy
drives economic productivity, consumption, and growth. Many production and consumption activities involve energy
as a basic input (Zahid, 2008). In Nigeria the petroleum products and the derivatives constitute the main source of
energy for production, manufacturing, distribution and general logistics of goods, passengers, and services. A logistics
system which meets the need for timely availability, quality, and affordable products is necessary to drive economic
productivity and growth. The components of petroleum and its by-products traded include the following: Premium
Motor Spirit (PMS), Automotive Gas Oil (AGO), Household Kerosene (HHK), Dual Purpose Kerosene (DPK), Aviation
Turbine Kerosene (ATK), Low Pour Fuel Oil (LPFO), Liquefied Petroleum Gas (LPG), Bitumen, Lubricants and Base Oil. Fuels are extracted from crude petroleum and pass through various processing units by thermal cracking in the
refineries. International Journal of Science and Research Archive, 2024, 11(02), 531–542 reflecting rapid growth in the number of automobiles, industries, households, intensified rural-urban migration,
economic and political developments. The bulk of products consumption has been the Premium Motor Spirit (PMS) or
petrol, Automotive Gas Oil (AGO) or diesel, dual purpose kerosene and bitumen/asphalt. Together, they account for
more than 60.0 per cent of the total consumption of petroleum products. The petroleum product distribution in Nigeria
is challenged by short deliveries, high lead time, increased cost of distribution, scarcity of products and high pump
prices. Nigeria is an emerging and fast developing market and the demand for petroleum product continues to icrease. As such, the need for sufficient and uninterrupted supplies is a major task for national development and economic
growth. A sustainable economic development cannot be successfully achieved without an appreciable vibrant supply
chain which involves the even distribution of petroleum products to keep alive economic activities such as
manufacturing, production distribution and general logistics involving the movement of materials and human resources
from one place to the other. In this regard, the study seeks to:
Examine the limiting factors to agile distribution of petroleum products in Nigeria.
Evaluate petroleum supply and consumption.
Evaluate petroleum supply and
And analyse product pricing. 1. Introduction The petroleum industry is involved in a global supply-chain that includes domestic and international transportation,
ordering and inventory visibility and control, materials handling, and information technology. Logistics is a supply chain
function that plays an essential role to ensure a safe, timely and cost-effective delivery. Oil and gas logistics includes
typically transportation of crude oil from the production sites to refineries as well as transportation and distribution of
oil products to markets and customers. The demand for oil and gas has increased in tandem with the economy of the
world as it continued to prosper. From the reference point of 1960, Organization of the Petroleum Exporting Countries
(OPEC) projects that oil demand would increase up to five times by 2035 (OPEC, 2014). The demand for petroleum product is affected by myriad of challenges. Alongside the need to replace depleted wells,
old plant, and equipment, and to accommodate ever higher standards and tighter regulations is the need to adopt
logistics strategies that ensure consumers continue to receive products in a timely and orderly manner (OPEC, 2014). An effective and efficient logistics process is an industry key for reducing lead times and costs, increase the company's
profits and in managing supply chains; demand management, efficient distribution of petroleum products among
customers, better transportation scheduling, warehouse management, and quality and timeliness of information
(Lisitsa, Levina and Lepekhin, 2019). In Nigeria, the consumption of petroleum products Onwioduokit and Adenuga (2000) recount has continually been on
the increase with most of the demand for products fulfilled over time by importation due to the poor condition of the
refineries. Starting from the 1980s the demand for petroleum products they acknowledge have increased tremendously * Corresponding author: Goodluck Tochukwu Njoku International Journal of Science and Research Archive, 2024, 11(02), 531–542 The agile supply chain is a more recent concept which focuses on leveraging responsiveness to customer demand. Here,
logistics plays a crucial role in aligning material flow across the supply network with customer demand, and in ensuring
execution in a flexible and customized manner (Harrison & Hoek, 2008). The origins of the agility paradigm in logistics
and supply chain can be traced back to the theory of lean enterprises, which comprises concepts and methods which
aim to minimize waste and consequently maximize efficiency and cost-effectiveness as the company uses fewer
resources (capital, financial, human, organisational) and less time to achieve the same goal (Sajdak, 2015). Agile logistics strategy of petroleum product distribution represents a distribution process which ensures the
availability right products to the right customer at the right place, in the right condition and right quantity at the right
time, and right (lowest possible) costs. Logistics is the task of managing two key supply chain flows; material flow of the
physical goods from suppliers through the distribution centers, stores and flow of Information; demand data from the
end-customer back to purchasing and to suppliers, and supply data from suppliers to the retailer, so that material flow
can be accurately planned and controlled (Harrison & Hoek, 2008). The agile strategy ensures the adequacy of place
(moving refined products to places where there is a demand for them) and time (maintaining the right stocks levels)
and proper distribution of the products through procedural alignment, end-to-end accountability, social proximity, and
deep integration of suppliers. 2.2. Agility of Petroleum Product Distribution The petroleum industry is frequently classified into three main sections: upstream, midstream, and downstream
operation. Upstream activities refer to the exploration and production of crude oil. This includes searching for potential
underground or underwater oil and gas fields, drilling of exploratory wells, and subsequently operating the wells that
recover and bring the petroleum crude oil and/or raw natural gas to the surface. Midstream activities refer to the
refining, transportation (by pipelines, rail, tankers), storage and wholesale marketing of crude or refined petroleum
products. Downstream activities refer to the transportation and marketing of end-user products, e.g. petrol, diesel, and
liquefied petroleum gas (LPG). The agility of petroleum product distribution is a strategy to building an agile supply chain of the same. Agility
Tiefenbacher (2020) denotes is a strategy to thrive, improve performance and be competitive in VUCA environments,
that is environments that are volatile, uncertain, chaotic, and ambiguous characterized by being unpredictable and
thereby necessitating adaptive, flexible approaches and interventions to muster them. Logistics and Supply chains are
one of the same ultimately concerned with delivering the right thing to the right place at the right time (LaBotz, 2022). 532 International Journal of Science and Research Archive, 2024, 11(02), 531–542 2.4.1. Lack Local Refinery Capacity 2.4.1. Lack Local Refinery Capacity Nigeria is blessed with the abundance of petroleum resource and natural gas, but the country is seriously challenged by
local refining capacity which meet the soaring energy need. World Energy 2023 report according to Akintayo (2023)
reveal local refineries output of Nigeria has dropped to near zero output in 10 years. According to data the statistical
review, output from the nation’s four refineries fell from 92,000 barrels per day in 2012 to just 6,000 barrels per day in
2022 representing a 92 per cent drop in production capacity. Similarly, the Organisation of the Petroleum Exporting
Countries’ Annual Statistical Bulletin (2023), also revealed the country’s crude oil refining capacity fell from 33,000 bpd
in 2018 to 6,000 bpd in 2022, representing about 81 per cent drop in production output. The drop in refining capacity
has made the country to rely solely on the importation of refined petroleum products to meet the local energy
consumption needs. NBS report spanning from 1997 up to 2014 presented in table 1 below shows a continued decline
in capacity utilization of the refineries and year on year growth in crude oil refining percentage respectively. Adeosun
and Oluleye (2017) acknowledge Nigeria despite having a nameplate refining capacity, 445,000 bpd which exceeds
demand has over the last four decades consistently struggled to keep its refineries functioning optimally and is ranked
as the 3rd highest importer of petroleum products in Africa, importing over 80% of products consumed. 2.3. Petroleum Distribution Chain in Nigeria The actors in the Nigerian industry consist of both private and public organizations. The public actors are the
government agents and functionaries such as the Nigerian National Petroleum Corporation (NNPC) and its subsidiaries,
the Department of Petroleum Resources (DPR), the petroleum products pricing regulatory authority (PPPRA), the
Pipeline and Product Marketing Company (PPMC) among others. The private segment consists of both indigenous and
foreign actors. The PPMC is charged with the task of large quantities supply, distribution, and marketing of petroleum
products in Nigeria. There exists network of four thousand (4,000) kilometers of pipelines which are interconnected
with twenty-one (21) highly dispersed depots. The products may be obtained from the four local refineries or in the event of a supply short-fall from off-shore
refineries by way of imports. Imported refined products are received at PPMC depots at Atlas Cove. From here, products
are pumped to nearby depots at Mosimi in Shagamu, from which products are pumped into various other depots
through the pipelines; Booster pump stations are provided along the route and between two adjoining depots. This
arrangement is necessary to boost the flow of products in the pipelines along the routes. Movement of products from
the depots is the responsibility of major oil marketers. Source: NNPC/PPMC Bulletin, 2010
Figure 1 Chanel of Petroleum Product Distribution in Nigeria Source: NNPC/PPMC Bulletin, 2010 Source: NNPC/PPMC Bulletin, 2010 Figure 1 Chanel of Petroleum Product Distribution in Nigeria 533 International Journal of Science and Research Archive, 2024, 11(02), 531–542 International Journal of Science and Research Archive, 2024, 11(02), 531–542 2.4.1. Lack Local Refinery Capacity Table 1 Yearly Domestic Crude Oil Refining 1997-2014
Year
Crude
oil
received
Crude
oil
processed
CAPACITY UTILIZATION
(%)
Year on Year Growth in Crude Oil
Refining %
1997
80,473,488.91
78,001,995.34
140.8
1998
52,422,070.13
54,304,185.98
93.16
-30.38
1999
65,979,134.80
66,409,253.60
116.18
22.29
2000
36,189,139.39
36,282,789.51
58.64
-45.36
2001
82,578,546.57
81,512,099.57
140.41
124.66
2002
78,160,576.55
79,579,031.26
142.65
-2.37
2003
42,754,815.49
44,811,925.54
72.11
-43.69
2004
38,019,906.64
38,026,965.93
66.14
-15.14
2005
72,360,780.00
70,637,019.00
129.44
85.76
2006
42,471,739.00
43,445,397.00
62.45
-38.49
2007
18,191,136.45
19,059,670.28
24.87
-56.13
2008
45,533,304.84
39,264,519.46
75.92
106.01
2009
19,392,571.25
17,745,656.61
78
-54.80
2010
33,633,907.94
34,871,693.84
167.87
96.51
2011
40,405,605.00
39,408,108.00
61.95
13.01
2012
33,595,260.00
33,628,602.00
68.95
-14.67
2013
36,193,237.20
35,233,125.59
74.5
4.77
2014
25,839,373.09
23,360,372.27
44.42
-33.70
Source: National Bureau of Statistics e-Library, 2014 Table 1 Yearly Domestic Crude Oil Refining 1997-2014 The scarcity of petroleum products and the rising cost of the petroleum products is an indication that refined product
importation is not sustainable for agile distribution of petroleum products. Okereke, Emodi and Diemuodeke, (2022)
lament the shortage of refining capacity at existing oil refineries is the main driver of Nigeria’s fuel crisis, which hampers
the socio-economic development of the country, placing a high subsidy burden on the government and has long made
Nigeria dependent on imported petroleum products. Similarly, Ehinomen and Adeleke (2012) decry the low-capacity
utilization of government owned refineries and petrochemical plants in Kaduna, Warri and Port Harcourt and
inadequate crude oil allocation to the refineries for domestic consumption as well as long turnaround time for the 534 International Journal of Science and Research Archive, 2024, 11(02), 531–542 maintenance of the refineries poses a lot of challenge to NNPC and result into petroleum product shortage and eventual
importation of the products a situation that puts a drain on the scarce foreign exchange of the nation. An attempt by the government at the removal of subsidy without addressing the issues of local refining capacity has
compounded the issue of product scarcity and high prices with PMS and AGO pricing reaching 600 NGN and 1300 NGN
respectively, the highest prices ever recorded in the history refined product distribution in Nigeria. 2.4.1. Lack Local Refinery Capacity Because industrial
production activity and business operation in Nigeria largely depend on petroleum energy following the epileptic
electric power supply and lack of technology to harness the solar power, the increase in the price of the petroleum
products have resulted to hyperinflation which has affected the cost of production, local cost of transportation and
prices of consumer commodities. Sales and business struggle due to high cost of doing business, low demand, and
patronage while many shut down and workers lose their jobs. Source: Author’s Analysis
Figure 2 Capacity Utilization and Year on Year % Growth in Oil Refining Figure 2 Capacity Utilization and Year on Year % Growth in Oil Refining Figure 2 above presents a graphical analysis of the capacity utilization and year on year percentage growth in crude
refining with reference table 1. The line graph shows a staggered progression in the refinery capacity utilization and
year on year growth in oil refining for the years referenced depicting a very hopeless situation for the industry in
Nigeria. There has not been a serious approach in addressing the issues of local refinery capacity in the country since
time and the challenges have compounded till date. With growth in population and increasing demand for development
which put pressure on energy supply to drive industrial and economic productivity, the reliance on importation of
refined products has become highly unsustainable. A significant aspect of agile product distribution is delivering the
products available to the end users at affordable cost. This cannot be realized by dependance on importation to feed the
local market. No doubt pump price of petroleum products in Nigeria is due to importation is determined by world crude
prices, and inflation in the import countries. Interruption in production due political instability, social unrest and
natural disaster in the import countries would translate to scarcity of product in Nigeria. 2.4.4. Dependency on Road Haulage The supply of refined petroleum products to end users is faced with many challenges. Efficient transportation of
petroleum product requires a modal integration of shipping via vessels, pipeline, road trucking and rail tankers. While
the pipeline is acknowledged the most efficient single mode due to the benefit of large volume delivery at high speed
and constant flow resulting to cheaper cost relative to other modes, zero accidents and limited environmental concerns
the case is different in Nigeria where on the contrary road haulage is the dominant mode of petroleum distribution. This
is because the pipeline mode is underdeveloped and fraught by the combined force of vandalism by oil bunkering,
industrial sabotage, militancy, poor maintenance, and neglect. According to Bataiya, (2018) 98% petroleum products
that include fuel components like premium motor spirit, domestic pure kerosene, and automotive gas oil’s lifting from
supply sources to all parts of the country is done by road which of itself is in gross disrepair and affecting the distribution
of the products. 70% of the federal road which constitute a 70% of the vehicular freight movement is overstretched
with 40% and 27% in poor and fair condition respectively requiring rehabilitation and maintenance work and a 3%
unpaved. 78% and 87% of state and local government roads respectively are in poor condition. The road transport
research acknowledge is not efficient for petroleum distribution over long distances due to lower speed, interrupted
flow, limited capacity leading to high cost of transportation per unit volume. Nwoloziri et al. (2021) acknowledge the
inefficiency of railways and pipeline transportation heavily contribute to total dependency on road transportation in
the distribution of petroleum products to various depots and stations located all over the country. Ehinomen and
Adeleke (2012) recounts the despair, neglect and repeated vandalization of the state-run petroleum products pipelines
and oil movement infrastructure nationwide, coupled with frequent accidents of haulage trucks on the nations heavily
used highways pose complex problems managers, and operators in the oil industry. Road haulage of petroleum products
is marred by road traffic accidents, theft, and robbery incidence, bad and poor road networks as well as various
hindrances such as delays at police, customs check points and many other bottlenecks (Thomas et al., 2014, Nwachukwu
& Chike, 2015, Bataiya, 2018, Nwoloziri et al., 2021, Ehinomen & Adeleke, 2012). International Journal of Science and Research Archive, 2024, 11(02), 531–542 Nigeria recorded over over 16,083 cases of “pipeline tampering” and vandalism amounting to a loss of 174.57 billion
(or approximately $484 million) and between 2016 and 2017, a total of 992 cases of pipeline vandalism were recorded
across pipelines and depot lines, amounting to a loss of 167 billion (approximately $464 million). Nigeria Extractive
Industries Transparency Initiative (NEITI) records between 2017 and 2021 (in 5 years) Nigeria recorded a total of 7,143
cases of pipeline vandalism. The country recorded in 11 years (2009-2020) a loss of 619.7 million barrels of oil valued
at $46.16 billion to theft, pipeline tampering and vandalism. This is higher than the size of the country’s foreign reserves. In addition, Nigeria lost 4.2 billion litres of petroleum products from refineries, valued at $1.84 billion at the rate of
1440,000 barrels per day, from 2009 to 2018 (Aduloju, 2023). 2.4.3. Corruption and Mismanagement Unscrupulous and corrupt practices of government agencies, oil companies and marketers as well as mismanagement
of petroleum revenue largely fraught the agility of petroleum product distribution in Nigeria. Subsidy of petroleum
products leads to a situation where the pump price of petroleum product is higher in neighbouring countries, Benin,
Cameroon, and Chad. The aim of the petroleum subsidy was undermined following large-scale smuggling of petroleum
meant for Nigerian consumption and subsidized by the government to the neighbouring countries where they are sold
at higher prices making superfluous profits short-changing the government and causing scarcity in Nigeria (Terkimbi,
2015 & Ayanruoh, 2023). Oshunkeye, (2012) alleges the oil subsidy is bedeviled by criminal tendencies of oil importers
and sharp practices in the distribution of import allocation, approval of subsidy payments and actual release of subsidy
cheques. While the significance of petroleum resources as a substantial source of economic advantages became
increasingly obvious, susceptibility of the resource regions to military attacks, regional pressure, and oil-related
problems, such as oil spills, crude oil theft, illegal refining of crude oil, accidents involving oil trucks, pipeline vandalism,
and explosions will continued to exacerbate and requiring strategic management innovations by government to bring
under control (Francis et al., 2022). Mismanagement, lack of proper guidelines and standards for the design,
construction, and operation of major oil pipelines result in corrosion and aging pipelines, ruptures, operational error,
and mechanical failure which lead to leaks and explosion that disrupt product flow (Akinpelu 2021, Achebe, Nneke, &
Anisiji, 2012,). 2.4.2. Insecurity, Terrorism and Acts of Sabotage Agile supply and distribution of petroleum products will remain a mirage in the presence of insecurity and safety
concerns which disrupt operation and investment in oil production, refining and distribution. Industrial sabotage, crude
oil theft, illegal refining operations, pipeline vandalism and piracy present significant challenges which adversely impact
onshore oil and gas production and fraught delivery of products to the market. The disruptive activities of the various
militant group and instability in the Niger Delta region is acknowledged a major threat to the security of the oil & gas
industry. Oturugbum (2021) acknowledge pipeline vandalism and militancy represents a means which the inhabitants
of the oil producing areas adopt in pressing their demands to the government and oil companies. Unfortunately, this is
due to the prevalence of corruption, poverty, and governments unfulfilled promises, among others. The Petroleum
Production Act and other legislation which criminalize the act of tampering and vandalism of crude oil and natural gas
pipelines with the death penalty and life imprisonment have not deterred the commission of relevant offences as the
challenge continues to escalate. Adeosun and Oluleye (2017) lament Nigeria lost over NGN 50 billion to pipeline
vandalism between January and April 2016. Okumagba (2019) reports within a space of 10 years between 2006 – 2016, 535 International Journal of Science and Research Archive, 2024, 11(02), 531–542 International Journal of Science and Research Archive, 2024, 11(02), 531–542 3.1. Limiting factors of Agile Distribution The qualitative data is coded with the capability of the SPSS and analysed for
data results and discussions. The perceived limiting factors of distribution agility cut across pricing, supply, technology,
infrastructure, accessibility and lead time, storage, management, and Human Resource (HR) issues. The questionnaire instrument follows a five-point likert scale ranged 1 to 5 where 1 = strongly disagree, 2 = agree, 3 =
undecided, 4 = agree, 5 = strongly agree. The qualitative data is coded with the capability of the SPSS and analysed for
data results and discussions. The perceived limiting factors of distribution agility cut across pricing, supply, technology,
infrastructure, accessibility and lead time, storage, management, and Human Resource (HR) issues. The questionnaire instrument follows a five-point likert scale ranged 1 to 5 where 1 = strongly disagree, 2 = agree, 3 =
undecided, 4 = agree, 5 = strongly agree. The qualitative data is coded with the capability of the SPSS and analysed for
data results and discussions. The perceived limiting factors of distribution agility cut across pricing, supply, technology,
infrastructure, accessibility and lead time, storage, management, and Human Resource (HR) issues. The questionnaire instrument follows a five-point likert scale ranged 1 to 5 where 1 = strongly disagree, 2 = agree, 3 =
undecided, 4 = agree, 5 = strongly agree. The qualitative data is coded with the capability of the SPSS and analysed for
data results and discussions. The perceived limiting factors of distribution agility cut across pricing, supply, technology,
infrastructure, accessibility and lead time, storage, management, and Human Resource (HR) issues. Table 3 Descriptive Statistics of limiting factors to agile distribution Table 3 Descriptive Statistics of limiting factors to agile distribution
N
Minimum
Maximum
Mean
Std. 3.1. Limiting factors of Agile Distribution At a very granular level, the study sought to survey the factors which limit the agile distribution of refined petroleum
products to end users in Nigeria. Distribution agility based on the review of literature hinges on continuous product
availability at affordable prices without quality compromise. Research observation and findings shows the distribution
of refined petroleum products in Nigeria is fraught with the challenges of scarcity, product adulteration, and high prices. And efforts by the government to address this have not yielded the desired results. Perceived factors which limit
distribution agility were presented to selected staff of six major independent petroleum marketers and NNPC retail as
presented in Table 2 below. 30 questionnaires were distributed to the executive staff of each of the study organizations
drawn from sales, marketing, admin, HR, imports, finance, business analyst, logistics and transport units via one-on-one
interview making a total of 210 questionnaires. 187 questionnaires, which represent 89% of the total questionnaires
distributed, were returned valid for the research. Table 2 Instrument Analysis
Company
Questionnaires Distributed
Returned and Valid
% Returned and Valid
Total Energies
30
28
13
OVH Energy (Oando)
30
27
13
MRS
30
26
12
Conoil Plc
30
27
13
Ardova Plc
30
27
13
11 Plc (Mobil)
30
28
13
NNPC Retail
30
24
11
Total Energies
210
187
89
Source: Authors Field Survey Table 2 Instrument Analysis y
Company
Questionnaires Distributed
Returned and Valid
% Returned and Valid
Total Energies
30
28
13
OVH Energy (Oando)
30
27
13
MRS
30
26
12
Conoil Plc
30
27
13
Ardova Plc
30
27
13
11 Plc (Mobil)
30
28
13
NNPC Retail
30
24
11
Total Energies
210
187
89
Source: Authors Field Survey
The questionnaire instrument follows a five-point likert scale ranged 1 to 5 where 1 = strongly disagree, 2 = agree, 3 =
undecided, 4 = agree, 5 = strongly agree. The qualitative data is coded with the capability of the SPSS and analysed for
data results and discussions. The perceived limiting factors of distribution agility cut across pricing, supply, technology,
infrastructure, accessibility and lead time, storage, management, and Human Resource (HR) issues. The questionnaire instrument follows a five-point likert scale ranged 1 to 5 where 1 = strongly disagree, 2 = agree, 3 =
undecided, 4 = agree, 5 = strongly agree. 3. Study Framework and Data The agile logistics of petroleum product distribution is a concept representing a distribution strategy which ensures
uninterrupted supply of right products to the right customer at the right place, in the right condition and right quantity
at the right time, and right (lowest possible) costs. The study follows a descriptive survey design based on quantitative
and qualitative approach relying on primary and secondary data. While Primary data is gotten by the distribution 536 International Journal of Science and Research Archive, 2024, 11(02), 531–542 structures questionnaire, secondary data is ensued from e-library of National Bureau of Statistics (NBS) and literatures
reviews. 3.1. Limiting factors of Agile Distribution Deviation
Fluctuation in crude prices and scarce foreign exchange
(F1)
187
2.00
5.00
4.4813
0.76441
Insecurity, fire incidence, theft, and acts of terrorism
((F2)
187
1.00
5.00
4.2193
0.82960
Lack of technology and integrated ICT system (F3)
187
3.00
5.00
4.4652
0.67411
Lack of local refinery capacity (F4)
187
3.00
5.00
4.5134
0.71360
Access limitations to retail delivery locations (F5)
187
3.00
5.00
4.3422
0.74088
Long delivery cycle, transit time and high cost (F6)
187
2.00
5.00
4.4118
0.69288
Lack of integrated management system and process (F7)
187
3.00
5.00
4.3636
0.76648
Poor transport infrastructure and networks (F8)
187
3.00
5.00
4.4332
0.64722
Expertise limitations (F9)
187
2.00
5.00
4.1551
0.76377
Valid N (listwise)
187
Source: Author’s SPSS Data Computation Output Source: Author’s SPSS Data Computation Output 537 International Journal of Science and Research Archive, 2024, 11(02), 531–542 Table 3 above presents the analysis of the response of the respondents as per their level of agreement or disagreement
to the perceived limiting factors of petroleum distribution agility in Nigeria. The sampled factors on a five-point likert scale shows a mean response ranged 4 – 4.5 higher than the likert mean value
3 and standard deviation values less the likert scale mean value indicating the respondents largely agree the sampled
variables are limiting factors to petroleum product distribution in Nigeria. The sample factor of lack of local refinery
capacity has the highest mean response above 4.5 indicating the respondents are very highly concerned about the
limitations of product importation to meet local consumption demand. The data is further subjected to K-S Test for more
information about the sample and to test the significance of the sample mean to the liket mean value and that the data
result is not by random chance. Table 4 One-Sample Kolmogorov-Smirnov Test
F1
F2
F3
F4
F5
F6
F7
F8
F9
N
187
187
187
187
187
187
187
187
187
Normal
Parametersa,b
Mean
4.4813
4.2193
4.4652
4.5134
4.3422
4.4118
4.3636
4.4332
4.1551
Std. Deviation
0.76441 0.82960 0.67411 0.71360 0.74088 0.69288 0.76648 0.64722 .76377
Most
Extreme
Differences
Absolute
0.377
0.276
0.353
0.383
0.315
0.326
0.337
0.328
.240
Positive
0.249
0.173
0.214
0.242
0.187
0.200
0.203
0.230
.206
Negative
-0.377
-0.276
-0.353
-0.383
-0.315
-0.326
-0.337
-0.328
-.240
Test Statistic
.377
0.276
0.353
0.383
0.315
0.326
0.337
0.328
0.240
Asymp. Sig. (2-tailed)
.000c
0.000c
0.000c
0.000c
0.000c
0.000c
0.000c
0.000c
0.000c
a. Test distribution is Normal. b. 3.1. Limiting factors of Agile Distribution Calculated from data. c. Lilliefors Significance Correction. Table 4 One-Sample Kolmogorov-Smirnov Test The K-S Test is presented in table 4. The variables F1-F9 represent the perceived limiting factors of agile product
distribution presented in table 9. The K-S Test mean values and standard deviation as in descriptive statistics of table 9
are greater and less than the liker mean values respectively. The P value < 0.05, and negative and positive upper and
lower bound mean interval for the data parameters are indication that the mean of the data parameters is significantly
different from the likert mean value 3. This validates the descriptive result of table 3 and leads to accept that the sample
variables are limiting factors to agile distribution of petroleum products. 3.2. Supply Consumption Analysis 3.2. Supply Consumption Analysis
Table 5 Product Supply and Consumption
Product
Category
2017
2018
2019
PMS
Import
17,313,512,018
20,147,419,127
20,892,934,107
Consumption
18,335,739,889
19,467,446,039
20,584,964,536
Local Refinery Output
1,022,227,871
-679,973,088
-307,969,571
5.58%
-3.49%
-1.50%
AGO
Import
4,277,630,139
4,236,496,957
5,148,860,318
Consumption
4,748,102,354
4,583,290,750
5,158,861,275
Local Refinery Output
470,472,215
346,793,793
10,000,957
9.91%
7.57%
0.19% 538 International Journal of Science and Research Archive, 2024, 11(02), 531–542 International Journal of Science and Research Archive, 2024, 11(02), 531–542 J
,
,
(
),
HHK
Import
340,333,700
537,605,965
128,110,313
Consumption
944,390,851
619,859,750
270,215,589
Local Refinery Output
604,057,151
82,253,785
142,105,276
63.96%
13.27%
52.59%
Source: NBS E-library Two sources of product supply import and local refinery are identified. Data is sourced from the NBS e-library and
products considered are Premium Motor Spirit (PMS), Automated Gass Oil (AGO) and Household Kerosene (HHK). Data
parameter of product category examined are import volume, local consumption, and local refinery output in liters for
the period of 2017 – 2019 as available on the NBS e-library data base. With regard to table 5 above, data analysis shows
PMS import has increased steadily from 17.3 billion liters in 2017 to 20.8 billion liters in 2019 representing a 17.6%
increase. The consumption volume also experienced a steady increase form 18.3 billion liters in 2017 to 20.5 billion
liters in 2019 representing a 11% increase. The local refinery capacity decreased from 1 billion liters in 2017 to 307
million liters in 2019 representing a 70% decline in output. The local refinery contribution to PMS consumption nose-
dived from 5.58% in 2017 to 0% in 2018 and 2019. There is a gap of 679 and 307 million liters of PMS in the 2018 and
2019 respectively which infers smuggled volume PMS. This is also validated by the recorded supply excess against local
consumption for the same years. The imports and local consumption of AGO increased from 4 billion liters in 2017 to 5
billion liters in 2019 representing an increase of 25% respectively. Local refining capacity decreased from 470 million
in 2017 to 10 million liters in 2019 which is a 97% decline. Local refinery contribution to total supply decrease from
9.9% in 2017 to 0% in 2019. HHK imports decreased from 340 million litres in 2017 to 128 million litres in 2019
representing a 62% decline. Consumption decreased from 944 million litres in 2017 to 270 million litres in 2019 which
is a 73.4% decline in consumption. 4. Study Findings The agility of petroleum product distribution is a strategy towards uninterrupted supply of refined petroleum products
to consumers at affordable prices and best quality. Research observation and finding shows the petroleum supply and
distribution in Nigeria have had history of challenges ranging from product scarcity, inconsistent pricing, and quality
compromise. At the root of the challenge is festering issues of corruption, mismanagement petroleum resource and
infrastructure, lack of local refinery capacity, dependency on importation, poor distribution networks and technology
constraints. Lack of local refinery capacity is fuel by corruption, sabotage, and lack of political will. There is no identified
radical approach by the government to resuscitate the hitherto moribund refineries and to build new ones. The local
refineries currently operate at near zero out levels. Corrupt gains of oil subsidy and importation has engineered the
sabotage of any government effort at building local refinery capacity. Insecurity, pipeline vandalism, theft, and terrorism which disrupt supply are acts of sabotage and militancy that the
government must address. Product importation is the reason for increasing cost of petroleum products following rising
foreign exchange, devaluation of Naira, rising global oil prices and inflation in the country of import. The product
importation itself places foreign exchange burden on the Naira leading to further devaluation of the currency. Also
supply disruption in the exporting countries will translate to scarcity in the country of import. Storage capacity
constraints, access limitations to retail delivery locations, poor transport infrastructure and networks are transport and
distribution challenges which lead to long delivery cycle, high transit time and delivery cost per unit volume. Furthermore, expertise limitations, lack of integrated management system and technology driven process have led to
poorly coordinated distribution system. 3.3. Product Pricing The price of the products has increased over the years with YOY percentage
increase nearing a 200% for PMS in the year 2023. This is occasioned by the sudden removal of subsidy on the product
without plans of local supply. The AGO and HHK reached highest price increase of 167% and 96% respectively in the
year 2022. This is also following the removal of subsidy on the products. Table 6 above presents the yearly average price of AGO, PMS and HHK for the period of 2017 to 2023 alongside with
the Year on Year (YOY) percentage increase. The price of the products has increased over the years with YOY percentage
increase nearing a 200% for PMS in the year 2023. This is occasioned by the sudden removal of subsidy on the product
without plans of local supply. The AGO and HHK reached highest price increase of 167% and 96% respectively in the
year 2022. This is also following the removal of subsidy on the products. 3.2. Supply Consumption Analysis Contribution of local refinery to consumption decreased from 63% to 52%. Figure 3 Product Supply and Consumption Analysis Figure 3 Product Supply and Consumption Analysis Figure 3 Product Supply and Consumption Analysis Figure 3 shows local consumption for the years under review increasingly depends on foreign supply of products. Local
supply capacity for all product group have declined over the years with PMS reaching negative levels in 2018 and 2019. This explains the reason for low levels of agility in product supply and distribution. There is a limit to which a country
will effectively meet her local consumption requirements depending solely on importation. Figure 3 shows local consumption for the years under review increasingly depends on foreign supply of products. Local
supply capacity for all product group have declined over the years with PMS reaching negative levels in 2018 and 2019. This explains the reason for low levels of agility in product supply and distribution. There is a limit to which a country
will effectively meet her local consumption requirements depending solely on importation. Dependence on imported refined products is notorious for the high cost of products in the hands of the consumers
following rising cost of dollar, cost of production in the exporting countries port clearance and landing costs. It also
means Nigeria will not be in control of its supply. Any challenge in production, supply shortage, political unrest or
economic concern in the exporting countries will translate to supply shortages in Nigeria. The supply country would 539 International Journal of Science and Research Archive, 2024, 11(02), 531–542 prioritize local consumption and cut short export in the event of production constraints. This will translate to severe
shortage to Nigeria, scarcity of product and hike in price as few distributors with products would hoard them to sell at
exploitive prices. prioritize local consumption and cut short export in the event of production constraints. This will translate to severe
shortage to Nigeria, scarcity of product and hike in price as few distributors with products would hoard them to sell at
exploitive prices. 3.3. Product Pricing The study surveys the pump price of AGO, PMS and HHK for the period of 2017 to 2023 to examine how the price of the
products have fared over the years. I significant aspect of product agility is continual availability of best quality at
affordable prices. But it is unfortunate that the price of the refined petroleum products has continued to increase over
the years becoming unaffordable and making life difficult for the people following the attendant hyperinflation bearing
in mind that industrial and economic productivity in the country is driven largely by the petroleum energy. Table 6 Petroleum Pump Price for the period of 2017 to 2023
Product
2017
2018
2019
2020
2021
2022
2023
AGO
Yearly Average
212.73
210.79
227.19
223.69
248.95
664.81
1,152.00
YOY Difference
0.00
-1%
8%
-2%
11%
167%
73%
PMS
Yearly Average
146.40
154.82
145.45
147.77
166.47
181.01
483.02
YOY Difference
0.00
6%
-6%
2%
13%
9%
167%
HHK
Yearly Average
297.57
287.70
315.35
340.31
394.02
771.78
1,211.65
YOY Difference
0.00
-3%
10%
8%
16%
96%
57%
Source: NBS E-Library Table 6 Petroleum Pump Price for the period of 2017 to 2023
Product
2017
2018
2019
2020
2021
2022
2023
AGO
Yearly Average
212.73
210.79
227.19
223.69
248.95
664.81
1,152.00
YOY Difference
0.00
-1%
8%
-2%
11%
167%
73%
PMS
Yearly Average
146.40
154.82
145.45
147.77
166.47
181.01
483.02
YOY Difference
0.00
6%
-6%
2%
13%
9%
167%
HHK
Yearly Average
297.57
287.70
315.35
340.31
394.02
771.78
1,211.65
YOY Difference
0.00
-3%
10%
8%
16%
96%
57%
Source: NBS E-Library
Table 6 above presents the yearly average price of AGO, PMS and HHK for the period of 2017 to 2023 alongside with
the Year on Year (YOY) percentage increase. The price of the products has increased over the years with YOY percentage
increase nearing a 200% for PMS in the year 2023. This is occasioned by the sudden removal of subsidy on the product
without plans of local supply. The AGO and HHK reached highest price increase of 167% and 96% respectively in the
year 2022. This is also following the removal of subsidy on the products. Table 6 Petroleum Pump Price for the period of 2017 to 2023 Table 6 above presents the yearly average price of AGO, PMS and HHK for the period of 2017 to 2023 alongside with
the Year on Year (YOY) percentage increase. References [1]
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Adeosun,
O. and
Oluleye,
A. (2017). Nigerian’s
refining
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Akintayo, O. (2023, Dec. 23). Domestic refining output drops by 92%. The Punch Nigeria. [4]
Bataiya, K. I. (2018, May 10th). Road infrastructure in Nigeria: Impact on petroleum product distribution [4th
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kassim-ibrahim-bataiya-national-president-nigerian-association-of-road-transport-owners-narto-deliv/ [5]
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traditional legal approach. International Energy Law Review, 37(7),181-189. [6]
Okumagba, E. O. (2019). Oil and gas pipeline "vandalism" in nigeria: analysing alternative options beyond the
traditional legal approach. International Energy Law Review, 37(7),181-189. [7]
Straka, M. (2017). The position of distribution logistics in the logistic system of an enterprise. [7]
Straka, M. (2017). The position of distribution logistics in the logistic system of an enterprise. [8]
Thompson, O. O., Martina, F. and James J. W. (2014). Road haulage constraints in the transportation of petroleum
products in northern Nigeria. Journal of Environmental Science, Toxicology and Food Technology, 8(3) 1-8. [8]
Thompson, O. O., Martina, F. and James J. W. (2014). Road haulage constraints in the transportation of petroleum
products in northern Nigeria. Journal of Environmental Science, Toxicology and Food Technology, 8(3) 1-8. [9]
Zahid, A. (2008). Energy–gdp relationship: a causal analysis for the five countries of South Asia. Journal of Applied
Econometrics and International Development, 8 (1). [9]
Zahid, A. (2008). Energy–gdp relationship: a causal analysis for the five countries of South Asia. Journal of Applied
Econometrics and International Development, 8 (1). [10] Oshunkeye, S. (2012, Jan. 2015). At Last, People’s Power. Sun Nigeria. [10] Oshunkeye, S. (2012, Jan. 2015). At Last, People’s Power. Sun Nigeria. [11] Ayanruoh, F. (2023, June 1st) Fuel Subsidy Removal is Good for Nigeria Vanguard, Thursda [12] Johnson, F. I., Laing, R., Bjeirmi, B & Leon, M. (2022). Examining the causes and impacts of pipeline disasters in
Nigeria. AIMS Environmental Science, 9(5): 636–657 [13] Nwoloziri, C. N, Dakoru, E and Nwabueze, E. and Elias, Eleyi (2021). Risk assessment of long-distance road
haulage of petroleum products in Nigeria. International Journal of Science and Research Archive, 2024, 11(02), 531–542 Savings from removal of petroleum subsidy as implemented by the current
government should be invested in local refinery capacity, revitalization of the pipeline for efficient distribution and
development of other sources of energy especially electricity supply to reduce over dependence on petroleum energy. International Journal of Science and Research Archive, 2024, 11(02), 531–542 and other sources of energy. Over time, the demand for petroleum products has soared in the following rising
population, increasing demand for economic productivity, general logistics and distribution, as well as quest for
improved standard of living. While the consumption demand for petroleum product has continued to increase, the
issues of scarcity, high prices and corrupt practices which fraught the distribution of petroleum in Nigeria has
compounded and successive governments have not shown a political will in addressing them. Significantly lack of local
refinery capacity and poor distribution network the study identifies are notorious for supply limitations and rising price
of products. Nigeria is blessed with abundance of natural petroleum resource yet depends on importation of refined
petroleum products to satisfy her local consumption demand. The reliance on road haulage in distribution is inefficient
and possess significant bottleneck and risk to product supply. To ensure the availability of products which meet local
demand at affordable prices and even distribution to all regions, the government is tasked to show genuine commitment
in addressing the issues identified. Savings from removal of petroleum subsidy as implemented by the current
government should be invested in local refinery capacity, revitalization of the pipeline for efficient distribution and
development of other sources of energy especially electricity supply to reduce over dependence on petroleum energy. and other sources of energy. Over time, the demand for petroleum products has soared in the following rising
population, increasing demand for economic productivity, general logistics and distribution, as well as quest for
improved standard of living. While the consumption demand for petroleum product has continued to increase, the
issues of scarcity, high prices and corrupt practices which fraught the distribution of petroleum in Nigeria has
compounded and successive governments have not shown a political will in addressing them. Significantly lack of local
refinery capacity and poor distribution network the study identifies are notorious for supply limitations and rising price
of products. Nigeria is blessed with abundance of natural petroleum resource yet depends on importation of refined
petroleum products to satisfy her local consumption demand. The reliance on road haulage in distribution is inefficient
and possess significant bottleneck and risk to product supply. To ensure the availability of products which meet local
demand at affordable prices and even distribution to all regions, the government is tasked to show genuine commitment
in addressing the issues identified. Disclosure of conflict of interest The author declares that there is no conflict of interest in this manuscript. 5. Conclusion More than 70% of energy requirement in Nigerian for transportation, domestic consumption, economic and industrial
productivity depend on petroleum and its derivatives. This is chiefly because of the underdevelopment of electric power 540 References International Research Journal of Modernization in Engineering
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the Niger delta area of Nigeria Proceeding of the International Multiconference of Engineers and Computer
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superb-profits-and-sustainability-0-0-0-0 [22] van Hoek R., Harrison A. and Christopher M. (2001). Measuring agile capabilities in the supply chain. International Journal of Operations and Production Management, 21(1–2) 126–47 [23] Harrison, A. and Hoek (2008). Logistics Management and Strategy; competing through the supply chain, 3rd
edition. Pearson Education Limited Edinburgh, Gate Harlow Essex CM20 2JE, England. 542
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Clinical Activity and Tolerability of SL-401 (Tagraxofusp): Recombinant Diphtheria Toxin and Interleukin-3 in Hematologic Malignancies
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Biomedicines
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biomedicines
Review
Clinical Activity and Tolerability of SL-401
(Tagraxofusp): Recombinant Diphtheria Toxin and
Interleukin-3 in Hematologic Malignancies
Omar Alkharabsheh *
and Arthur E. Frankel
Division of Medical Oncology, Mitchell Cancer Institute, University of South Alabama, Mobile, AL 36604, USA;
afrankel@health.southalabama.edu
* Correspondence: oalkharabsheh@health.southalabama.edu; Tel.: +1-251-665-8000
Received: 20 November 2018; Accepted: 3 January 2019; Published: 5 January 2019
Abstract: Overcoming the leukemia stem cell resistance to intensive chemotherapy has been an
area of extensive research over the last two decades. Advances and greater understanding of the
molecular biology of leukemia stem cells are in rapid progress. Targeted therapies are currently being
used in clinical practice with reasonable response rates, but a cure is being achieved in only a small
percentage of patients, most likely due to tumor mutational heterogeneity. A genetically engineered
diphtheria toxin fused with interleukin-3 (SL-401 or tagraxofusp) has shown robust activity in blastic
plasmacytoid dendritic cell neoplasm and promising response rates in different myeloid malignancies,
including eradication of minimal residual disease. Multiple clinical trials are being conducted using
this drug and the preliminary results are encouraging. This article reviews the clinical trials for
SL-401, its mechanism of action, clinical activity, and the adverse event profile.
Keywords: SL-401 (tagraxofusp); diphtheria immunotoxin; adverse events; Myeloid neoplasms
1. Introduction
The current treatment approach for myeloid malignancies is heavily dependent on intensive
chemotherapy regimens that follow the old strategy of “one size fits all”. Novel targeted agents are
emerging in clinical practice and clinical trials. Data on their efficacy and tolerability are promising,
although no treatment has produced a cure thus far [1]. Intensive chemotherapy treatments are
designed to induce hypocellular marrow and allow hematopoietic stem cells to recover and resume
normal production of blood cell components. The durability and duration of remission by this
kind of therapy can vary and will depend primarily on the mutational signature of the disease and
epigenetic aberration. In addition, the planned consolidation strategy, whether it is allogeneic stem
cell transplantation or further consolidative chemotherapy, has an effect on leukemia-free survival
and how deeply we can eradicate leukemic stem cells. New agents that target actionable mutations,
such as FLT3 and IDH, have been approved for clinical practice [2,3]. However, they have not had
a significant enough effect on overall survival rates to replace chemotherapy, partly because of the
diversity of cytogenetic abnormalities and the clonal evolution of new mutations. Nevertheless, it is
important to assess the performance status and comorbidities of these treatments before exploring
which treatment approach is the most appropriate in terms of toxicity and survival. Unfortunately,
even with the most intensive treatment, the risk of relapse is high in the first two years, especially in
patients with adverse cytogenetic profiles [4]. Extensive time and effort were spent in clinical trials
optimizing the sequence of well-known acute leukemia chemotherapy protocols. There is a need to
revolutionize the treatment of acute leukemia.
Unlike traditional chemotherapy, the specificity of the target on the tumor cells is the focus
of the new era in cancer treatment. Various examples have been investigated in clinical trials, and
Biomedicines 2019, 7, 6; doi:10.3390/biomedicines7010006
www.mdpi.com/journal/biomedicines
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many agents have been approved and are available in clinical practice. These include, for example,
tyrosine kinase inhibitors to target the FLT3 mutation, monoclonal antibodies, bispecific antibodies,
and chimeric antigen receptor T cell therapies. The ideal target, for example, in acute myeloid and
lymphoid leukemia, is highly expressed in leukemic blasts, with minimal or no expression on normal
hematopoietic cells, to induce deeper remission. Targets that have been studied in acute myeloid
leukemia include CD33, CD135, FLT3, CXCR4, and vascular endothelial growth factor [5]. This article
focuses on the diphtheria toxin-interleukin 3 fusion protein as a targeted therapy to leukemic blasts.
Hematopoiesis is a complex process with multiple factors, undertaken to avoid the production
of clones and leukemic stem cells. Interleukin 3 (IL-3) is one of the growth factors and cytokines
that participate in this process at the level of granulocytic and monocytic lineage [6]. Expression of
the interleukin 3 receptor (IL-3R) starts at the CD34+ hematopoietic cell and is maintained during
all stages of development for granulocytes and monocyte precursors. Multiple investigators have
targeted this receptor for the treatment of different types of myeloid leukemia [7]. Most recently,
experiments have used chimeric antigen receptor T cell technology to target CD123 in Hodgkin
lymphoma in an effort to overcome the immunosuppressed microenvironment and allow the patient’s
T lymphocytes to attack tumor cells [8]. The structure of IL-3R consists of an α subunit, which is the
site of ligand attachment and represents the specificity of the receptor, and a β subunit, which is shared
with the granulocyte-macrophage colony-stimulating factor and is important for signal transduction,
internalization of the ligand–receptor complexes, and activation through phosphorylation of the Ras
pathway [9].
The development of monoclonal antibodies and cytokines was a major advance in cancer
treatment because of their ability to deliver cancer therapies, such as cytotoxic drugs, isotopes, and
toxins. Toxins are powerful pro-apoptosis agents, and they achieve their cellular effect by various
pathways. One of them is by shutting down protein synthesis [10]. After removal of the natural
binding domain of the toxins, they are conjugated to a monoclonal antibody or cytokine as their
ligand [11]. One toxin that has been used in cancer therapy is the diphtheria toxin, which is an exotoxin
secreted by Corynebacterium diphtheriae [12]. This toxin inhibits protein synthesis in the host, which
is the underlying pathophysiology of diphtheria infection [8]. Previously, the diphtheria toxin was
engineered to target interleukin-2 and was approved by the Food and Drug Administration (FDA)
in 2002 for the treatment of cutaneous T cell lymphoma [13]. However, because of the side effects,
marketing of this drug was discontinued by the manufacturer.
Diphtheria toxin consists of three domains and several linkers elements: The C terminus binding
domain, the N terminus catalytic domain, and the translocation domain [14]. IL-3 is a cytokine that
promotes the differentiation of hematopoietic stem cells into various myeloid cells [12]. IL-3R is
composed of two subunits (α and β). The α subunit (CD123) directly binds to IL-3, whereas the β
subunit (CDw131) functions as the signaling subunit (see Figure 1) [10]. The idea is to remove the
diphtheria toxin binding domain and replace it with IL-3 to target leukemic stem cells that express
CD123 and CDw131 by inhibiting intracellular protein synthesis and therefore inducing cell death.
This combination (diphtheria toxin and IL-3) is named SL-401 (tagraxofusp) and has reached
phase II clinical trials, with robust activity in the blastic plasmacytoid dendritic cell neoplasm (BPDCN)
and other hematological malignancies [15–20]. Subsequently, the FDA approved this drug in December
2018 for adult and pediatric BPDCN. In this review, we report the preclinical data and describe the
clinical trials for SL-401 and its activity in various myeloid malignancies.
Biomedicines 2019, 7, 6
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Figure
Structure
andand
Mechanism
of Action
(provided
by Stemline
Therapeutics,
Inc., New
Figure1.1.SL-401
SL-401
Structure
Mechanism
of Action
(provided
by Stemline
Therapeutics,
Inc.,
New NY
York,
NYUSA).
10022,
USA). Abbreviations:
IL-3, 3;
interleukin
3; IL-3R,
interleukin
3 receptor;
York,
10022,
Abbreviations:
IL-3, interleukin
IL-3R, interleukin
3 receptor;
DT, diphtheria
DT, diphtheria
toxin;
EF2,
elongation
factor ribonucleic
2; mRNA,acid;
messenger
ribonucleic
acid; ADP,
toxin;
EF2, elongation
factor
2; mRNA,
messenger
ADP, adenosine
diphosphate.
adenosine diphosphate.
This combination (diphtheria toxin and IL-3) is named SL-401 (tagraxofusp) and has reached
2. Preclinical Data and Experiments
phase II clinical trials, with robust activity in the blastic plasmacytoid dendritic cell neoplasm
(BPDCN)
and other
hematological
malignancies
[15–20].
Subsequently,
theGM-CSF
FDA approved
drug
Previously,
investigators
fused
the truncated
diphtheria
toxin with
using athis
His-Met
in
December
2018
for
adult
and
pediatric
BPDCN.
In
this
review,
we
report
the
preclinical
data
anda
linker. However, due to the toxicity profile and the hepatic side effects, they decided to explore
describe
clinical
trials[21].
for SL-401
andof
itsIL-3R
activity
in various
myeloid
malignancies.
differentthe
growth
factor
The level
expression
was
examined
by Testa and colleagues
and they demonstrated a higher level of IL-3R α chain in leukemic stem cells (LSC) and a low
2.expression
PreclinicalonData
and hematopoietic
Experiments stem cells, which makes it a marker for LSC and a target for
normal
treatment
[2]. Using
genetic engineering,
the first 388
amino acid
residues
of the diphtheria
are
Previously,
investigators
fused the truncated
diphtheria
toxin
with GM-CSF
using a toxin
His-Met
fused
with
interleukin
3
via
H-M
linker
(DT
IL3).
The
mechanism
of
action
starts
when
the
ligand
linker. However, due to the toxicity profile388
and the hepatic side effects, they decided to explore ais
attachedgrowth
to the IL-3R
subunit
Along
with binding
to the IL-3R
subunit,
will trigger
different
factorα[21].
The (CD123).
level of IL-3R
expression
was examined
byβTesta
and this
colleagues
and
receptor-mediated
endocytosis
into
a
vesicle
which
will
deliver
the
diphtheria
toxin
to
the
endosomes
they demonstrated a higher level of IL-3R α chain in leukemic stem cells (LSC) and a low expression
on
where ithematopoietic
is cleaved by stem
furincells,
to generate
the A and
B fragments
of the
The
environment
of
normal
which makes
it a marker
for LSC
andtoxin.
a target
foracidic
treatment
[2]. Using
the
endosomes
will
partially
unfold
all
three
toxin
domains
to
facilitate
the
escape
of
the
A
fragment
genetic engineering, the first 388 amino acid residues of the diphtheria toxin are fused with interleukin
the(DT
catalytic
domain into the cytosol. Finally, the catalytic domain will ADP-ribosylate
3that
via contains
H-M linker
388IL3). The mechanism of action starts when the ligand is attached to the IL-3R α
the elongation
factor
2, which
lead to
synthesis
inhibition
deathreceptor-mediated
[22] (see Figure 1).
subunit
(CD123).
Along
with will
binding
toprotein
the IL-3R
β subunit,
this and
willcell
trigger
Additionally,
several
animal
studies
confirmed
the
in
vivo
efficacy
of
the
engineered
diphtheria
toxin
endocytosis into a vesicle which will deliver the diphtheria toxin to the endosomes where
it is cleaved
and
provided
more
information
about
the
appropriate
dose
and
its
readiness
for
the
first
human
clinical
by furin to generate the A and B fragments of the toxin. The acidic environment of the endosomes will
trials [14,15].
To all
examine
the level
of expression
on different
myeloid
researchers
from Mayo
partially
unfold
three toxin
domains
to facilitate
the escape
of thelineage,
A fragment
that contains
the
Clinic
performed
a
study
of
CD123
expression
on
myeloproliferative
neoplasm
subtypes
with
flow
catalytic domain into the cytosol. Finally, the catalytic domain will ADP-ribosylate the elongation
factor
expression
on systemic
mastocytosis,
and aseveral
minor
2,cytometry.
which willThey
leadreported
to protein
synthesismostly
inhibition
and cell death
[22] (see clonal
Figureeosinophilia,
1). Additionally,
population
of
myelofibrosis
[16].
This
broadened
the
potential
activity
on
several
myeloid
disorders,
animal studies confirmed the in vivo efficacy of the engineered diphtheria toxin and provided more
along with the
previously
described
leukemic
stem cellsfor
in acute
myeloid
To understand
information
about
the appropriate
dose
and its readiness
the first
humanleukemia.
clinical trials
[14,15]. To
more
about
the
bone
marrow
microenvironment
and
its
effect
on
disease
pathogenesis,
along
with
examine the level of expression on different myeloid lineage, researchers from Mayo Clinic performed
the
possible
use
of
this
agent
as
an
investigational
drug
in
clinical
trials,
researchers
at
Dana-Farber
a study of CD123 expression on myeloproliferative neoplasm subtypes with flow cytometry. They
Cancer Institute
demonstrated
plasmacytoid
dendritic
cells
(pDCs) play
a role
in protecting
clonal
reported
expression
mostly on that
systemic
mastocytosis,
clonal
eosinophilia,
and
a minor
population
of
plasma
cells
in
multiple
myeloma
from
treatment.
Additionally,
they
reported
that
SL-401
induces
myelofibrosis [16]. This broadened the potential activity on several myeloid disorders, along with the
pDC apoptosis,
which
resultedstem
in overcoming
treatment
resistance
inTo
multiple
myeloma
previously
described
leukemic
cells in acute
myeloid
leukemia.
understand
more[17].
about the
bone marrow microenvironment and its effect on disease pathogenesis, along with the possible use of
3. Clinical Trials
this agent as an investigational drug in clinical trials, researchers at Dana-Farber Cancer Institute
The first that
in human
trial was
presented
the American
Society
of Clinical
Oncology
demonstrated
plasmacytoid
dendritic
cellsat(pDCs)
play a role
in protecting
clonal
plasma (ASCO)
cells in
annual
meeting
in
2006.
The
preliminary
findings
showed
manageable
toxicity
in
elderly
patients
with
multiple myeloma from treatment. Additionally, they reported that SL-401 induces pDC apoptosis,
acute myeloid
(AML)
or in those
with relapsed
or refractory
disease
which
resulted leukemia
in overcoming
treatment
resistance
in multiple
myeloma
[17]. [18]. The study included
Biomedicines 2019, 7, 6
4 of 9
31 patients who received one cycle of SL-401. The findings showed mild toxicity and promising
biologic activity. At the 2007 ASCO annual meeting, investigators reported a patient with AML
who achieved complete remission (CR) for eight months as well as two cases of partial response
(PR) [19]. These patients were heavily pretreated, including bone marrow transplantation, and the
cohort included patients with secondary AML either from a previous myelodysplastic syndrome or
therapy-related AML. The major side effects reported in this abstract were mild to moderate and
included transient fever, chills, hypotension, and hypoalbuminemia [19]. Table 1 highlights the clinical
trials for SL-401, adverse events, and response rates.
Phase I data were first published in 2008 [20] and highlighted the following points. First, the
maximum tolerated dose is 12.5 µg/kg/day, which can be infused safely over 15 min, twice daily
every 48 h with a total of six doses as one cycle. Second, toxicity and side effects were manageable,
and no grade IV/V adverse events were reported. Grade II/III adverse events included transaminitis,
fever, hypoalbuminemia, hypotension, and hypocalcemia. This cohort included elderly patients, most
of whom had a disease that was refractory to standard therapy. The median age was 67 years, and
only 11% of patients had previously untreated AML. In addition, 96% of patients had an intermediate
cytogenetic profile. The trial reported one case of CR and two cases of PR (Both AML and MDS). All of
the patients who showed some form of response had relapsed or refractory disease and the patients
with previously untreated AML showed no response to the experimental treatment [20].
The high level of IL-3R expression on BPDCN [23] provided the basis for investigation of SL-401
activity in this type of myeloid disease. Because BPDCN is an aggressive tumor, unfortunately, patients
have a short survival, irrespective of the intensity of chemotherapy [24]. This accelerated the pathway
to move SL-401 from in vitro studies to more early phase clinical trials [13,25]. The first prospective
trial in BPDCN using SL-401 published its results and outcomes in 2014. Frankel et al. reported the
detailed toxicity profile and response rates for the phase I/II trial [26]. The maximum tolerated dose
was 12.5 µg/kg/day for five days in 11 patients with BPDCN. The reported adverse events were grade
II/III, including edema, hypoalbuminemia, fever, chills, and transaminitis as well as one case of grade
IV thrombocytopenia that was not related to an underlying thrombotic microangiopathy. Another
adverse event that the investigators described in their outcomes data is vascular leak syndrome (VLS).
This was reported as mild to moderate, and it manifested clinically with edema, with laboratory
findings showing hypoalbuminemia. The VLS phenomenon had been reported in other fusion proteins
with toxins [27]. Of the patients who underwent evaluation for response, 78% had an objective
response, including 55% CR after the first cycle. Because of the nature of the drug, there is a question
about whether the antibody developed because of previous diphtheria vaccination during childhood
or whether the infused drug itself may trigger antibody development that might inactivate SL-401.
The investigators examined this theory and reported that pretreatment titers were positive as a result
of previous vaccination. A repeat evaluation after therapy showed an increased titer; however, this did
not affect toxicity events, pharmacokinetics, or the clinical response rate [26].
Biomedicines 2019, 7, 6
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Table 1. Summary of SL-401 clinical trials in various hematologic neoplasms, baseline characteristics, adverse events and clinical outcomes (See page 14). Abbreviations:
NCT, National Clinical Trial; R/R, Relapsed or refractory; AML, Acute myeloid leukemia; MDS, Myelodysplastic syndrome; CMML, Chronic myelomonocytic
leukemia; BPDCN, Blastic plasmacytoid dendritic cell neoplasm; MM, Multiple myeloma; ORR, Overall response rate; CR, Complete response; PR, Partial response;
LFT, Liver function tests; ASCO, American Society of Clinical Oncology; ASH, American Society of Hematology; CLS, Capillary leak syndrome; NA, Not available.
SL-401 Clinical
Trial Registry
Numbers
Primary
Malignancy
Status
Study
Start Date
Phase
Published
Data
Number
of
Patients
Number
of Cycles
Age
Male/Female
Adverse
Cytogenetics
Intermediate
Cytogenetics
Relapsed
(any) and
Refractory
NA
R/R or elderly
AML and high
risk MDS
Completed
NA
I
Leukemia
and
Lymphoma
2007
45
1
67
(32–81)
23/22
17
25
35
NCT00397579
BPDCN
Completed
May 2013
I/II
Blood 2014
for the
BPDCN
11
1
70
(40–77)
11/0
NA
NA
7
NA
RR/AML and
BPDCN
NA
NA
NA
ASH 2015
17
multiple
63
NA
NA
NA
NA
Recruiting
February
2015
I/II
NA
NA
NA
NA
NA
NA
NA
NA
Recruiting
December
2014
I/II
ASH 2016
19
multiple
69
(42–81)
NA
NA
NA
Recruiting
June 2017
I
NA
NA
NA
NA
NA
NA
NCT02270463
NCT02268253
NCT03113643
Consolidation
Rx in adverse
risk AML CR1
Advanced high
risk MNP (SM,
PED, MF,
CMML)
With AZA for
Rx naïve
AML/high risk
MDS not
eligible for
standard Rx
Adverse Events
ORR
CR
PR
NA
1
3
NA
5
0
NA
4
NA
NA
NA
NA
NA
19
Grade III
thrombocytopenia
and anemia
NA
1
NA
NA
NA
NA
NA
NA
NA
Grade III LFTs,
Grade II fever,
chills, low albumin
and hypotension
Grade IV
thrombocytopenia,
Grade III LFTs and
neutropenia
Grade V CLS,
Grade IV CLS,
Grade III LFTs
NCT02661022
R/R MM
Recruiting
January
2016
I/II
ASH 2016
2
multiple
65
(63–67)
NA
NA
NA
2
Grade II
thrombocytopenia
and
hypoalbuminemia
NA
0
2
NCT02113982
BPDCN and
R/R AML
Recruiting
September
2014
I/II
ASCO
2016 (only
BPDCN)
18
multiple
70
(45–82)
NA
NA
NA
10
CLS and
thrombocytopenia
87%
8
NA
Biomedicines 2019, 7, 6
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The current advances in measuring minimal residual disease (MRD) have shown that conventional
chemotherapy may not provide the deepest response or subsequently a better outcome. For example,
the use of traditional consolidation chemotherapy after achieving CR in AML might not be enough
to reduce the risk of relapse. For that, new treatment strategies are needed. Currently, a phase
I/II study (NCT02270463) is underway in patients with AML who are at risk for relapse based
on intermediate-high risk cytogenetics and are not candidates for allogeneic stem cell transplant.
The following initial results were reported at the 2016 ASH annual meeting. First, the adverse
event profile was similar to that of other clinical trials of SL-401, with a maximum tolerated dose of
12 µg/kg/day [28]. Second, the concept of targeting MRD-positive AML with SL-401 is promising,
and the trial will continue to recruit patients until December 2018 (www.clinicaltrials.gov).
For multiple myeloma, studies are still in the early phase, with evidence showing that SL-401
targets myeloma stem cells [29]. As with pDC in the marrow microenvironment, those cells express
CD123 and have a role in the proliferation of myeloma, which makes SL-401 a promising agent for
myeloma backbone therapy. Currently, a phase I/II trial (NCT02661022) is recruiting patients with
relapsed or refractory myeloma to be treated with pomalidomide, dexamethasone, and SL-401 [30].
The current evidence from early clinical trials indicates that side effects are tolerable and
manageable. Additionally, clinical activity is seen in tumors that express CD123, especially BPDCN.
Recently, investigators showed robust data at the American Society of Hematology annual meeting in
December 2017 for a phase II trial of single-agent SL-401 in BPDCN, with an 84% overall response rate.
Additionally, two abstracts were presented at the European Society of Hematology annual meeting
in June 2018. The first one described chronic myelomonocytic leukemia, and all patients showed a
spleen reduction response, while 12.5% showed marrow CR. The second abstract described primary
myelofibrosis, and 50% of patients showed a spleen response. In summary, the results of trials of
SL-401 indicate no serious adverse events, unlike other forms of immunotherapy, and a convening
clinical activity in various hematologic malignancies, most effectively in BPDCN. In the future, we will
likely see more positive results from phase III trials.
4. Side Effects and Tolerability
The currently reported side effects and clinical activity from early-phase trials are shown in
Table 1. To review those in detail, this is a recombinant protein from diphtheria toxin conjugated with
IL-3 to internalize the toxin inside the tumor cells. Based on the nature of this agent, some form of
immune or infusion reaction would be expected. The most serious adverse event that was reported in
human clinical trials was capillary leak syndrome (CLS), previously called VLS, which is manifested
by edema, hypoalbuminemia, hypotension, and fatigue. This was reported as grade ≤3; however,
three cases of fatal CLS were reported in the BPDCN trial (www.ashclinicalnews.org).
Other significant side effects were reported up to grade 4, including thrombocytopenia. However,
it was reversible and was not associated with a major bleeding event. In addition, less than grade 3
neutropenia was reported. Patients with marrow-involved malignancies are susceptible to cytopenia
because of disease expansion and the side effects of treatment. As shown in Table 1, the rest of the side
effects were tolerable and reversible, so one could argue that these patients have refractory disease
and are heavily pretreated, and this by itself can explain the cytopenia. Additionally, transaminitis has
been reported across trials, but it was reversible and less than or equal to grade 3.
5. Pediatric Experience
Although BPDCN is a disease of the elderly, a few patients who were younger than 18 years
old were included for compassionate use because their disease was refractory to standard therapy.
Sun et al. from the City of Hope reported the first three cases of pediatric patients with this type of
malignancy. Overall, the treatment was very well tolerated, with side effects similar to those of CLS
and infusion reaction [31]. The number of patients in this report was very small, so a conclusion about
the efficacy of this treatment cannot be made, although all patients showed some response.
Biomedicines 2019, 7, 6
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6. Conclusions
The concept of using diphtheria toxin in cancer treatment is not new but evolving.
Alternative therapies to traditional chemotherapy are needed to improve outcomes, especially in
patients with primary refractory or relapsing hematologic malignancies. Using IL-3 as a conjugate offers
an advantage because multiple hematologic malignancies express CD123 and CDw131. Therefore,
having a target that is highly expressed on tumor cells will increase the likelihood that the drug will
exhibit anti-tumor activity by internalizing the diphtheria toxin and inducing apoptosis. The most
striking activity was noted in BPDCN, with excellent responses in the affected organs, including bone
marrow, skin, and lymph nodes. Outcomes in other hematologic malignancies, such as multiple
myeloma and chronic myelomonocytic leukemia, are still in early clinical trials, and these studies are
providing a better understanding of the bone marrow microenvironment and the role of pDC in tumor
progression and treatment resistance. It has been reported that pDCs have anti-tumor activity through
the production of interferon alfa [32].
Funding: This research received no external funding.
Acknowledgments: All sources of funding of the study should be disclosed. Please clearly indicate grants that
you have received in support of your research work. Clearly state if you received funds for covering the costs to
publish in open access.
Conflicts of Interest: Omar Alkharabsheh has no conflict of interest. Arthur E. Frankel has an intellectual property
interest in Stemline Therapeutics, Inc.
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© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Inhibition of HIV Virus by Neutralizing Vhh Attached to Dual Functional Liposomes Encapsulating Dapivirine
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Nanoscale research letters
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© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. Inhibition of HIV Virus by Neutralizing Vhh
Attached to Dual Functional Liposomes
Encapsulating Dapivirine Scarlet Xiaoyan Wang1*, Johan Michiels2, Kevin K. Ariën2, Roger New3, Guido Vanham2 and Ivan Roitt1 Scarlet Xiaoyan Wang1*, Johan Michiels2, Kevin K. Ariën2, Roger New3, Guido Vanham2 and Iva Wang et al. Nanoscale Research Letters (2016) 11:350
DOI 10.1186/s11671-016-1558-7 Open Access Open Access Abstract Although highly active antiretroviral therapy (HAART) has greatly improved the life expectancy of HIV/AIDS patients,
the treatment is not curative. It is a global challenge which fosters an urgent need to develop an effective drug or
neutralizing antibody delivery approach for the prevention and treatment of this disease. Due to the low density of
envelope spikes with restricted mobility present on the surface of HIV virus, which limit the antibody potency and
allow virus mutation and escape from the immune system, it is important for a neutralizing antibody to form bivalent
or multivalent bonds with the virus. Liposome constructs could fulfil this need due to the flexible mobility of the
membrane with its attached antibodies and the capacity for drug encapsulation. In this study, we evaluated the
neutralization activity of a range of liposome formulations in different sizes coated with anti-gp120 llama antibody
fragments (Vhhs) conjugated via either non-covalent metal chelation or a covalent linkage. The non-covalent construct
demonstrated identical binding affinity to HIV-1 envelope glycoprotein gp120 and neutralizing ability for HIV virus as
free Vhh. Although covalently linked Vhh showed significant binding affinity to gp120, it unexpectedly had a lower
neutralization potency. This may be due to the comparability in size of the viral and liposome particles restricting
the number which can be bound to the liposome surface so involving only a fraction of the antibodies, whereas
non-covalently attached antibodies dissociate from the surface after acting with gp120 and free the remainder to
bind further viruses. Covalently conjugated Vhh might also trigger the cellular uptake of a liposome-virion complex. To
explore the possible ability of the antibody-coated liposomes to have a further function, we encapsulated the
hydrophobic antiviral drug dapivirine into both of the non-covalently and covalently conjugated liposome
formulations, both of which revealed high efficacy in reducing viral replication in vitro. Thus, dual function
liposomes may lead to a novel strategy for the prophylaxis of HIV/AIDS by combining the neutralizing activity
of Vhh with antiviral effects of high drug concentrations. Keywords: HIV/AIDS, Liposome, Neutralizing antibody, Drug delivery, Prophylaxis * Correspondence: x.wang@mdx.ac.uk
1Department of Natural Sciences, Middlesex University, London, UK
Full list of author information is available at the end of the article Background HIV-1 is more pathogenic and transmissible and is mainly
responsible for the global AIDS pandemic [4]. HIV infec-
tion has continued spreading by the transfer of body fluids
due to exposure to blood or blood products, by homo- or
heterosexual contact, or prenatally from mother-to-child. Since the start of the epidemic, around 78 million people
have become infected with HIV and 35 million people have
died of AIDS-related illnesses according to the global statis-
tical report from UNAIDS in 2016. In addition, 2.1 million
people became newly infected with HIV and 36.7 million
people were living with HIV globally in 2015. Human immunodeficiency virus (HIV), responsible for
the acquired immunodeficiency syndrome (AIDS), is a
lentivirus belonging to the retrovirus family. Infection
causes extensive destruction of T-helper cells, macro-
phages, dendritic cells and other cellular components asso-
ciated with cell-mediated immunity, eventually leading to
AIDS [1, 2]. As a consequence, HIV-infected patients are
more susceptible to opportunistic infections [3]. At present,
HIV-1 and HIV-2 are the two known types of HIV [4]. Currently, the major means of efficient management
of HIV and prevention of its progression towards AIDS * Correspondence: x.wang@mdx.ac.uk
1Department of Natural Sciences, Middlesex University, London, UK
Full list of author information is available at the end of the article * Correspondence: x.wang@mdx.ac.uk
1Department of Natural Sciences, Middlesex University, London, UK
Full list of author information is available at the end of the article Wang et al. Nanoscale Research Letters (2016) 11:350 Page 2 of 10 Page 2 of 10 Page 2 of 10 is highly active antiretroviral therapy (HAART), which
combines a minimum of three antiretroviral drugs from
at least two classes. These include nucleoside reverse
transcriptase inhibitors (NRTIs), non-nucleoside reverse
transcriptase inhibitors (NNRTIs), protease inhibitors
(PIs), fusion inhibitor, integrase inhibitors and/or entry
inhibitors [5]. Although the therapy has improved long-
term survival rate, there are associated toxic side effects
and emergence of multi-drug resistance. Furthermore,
there is a limited access to anti-HIV drugs in the devel-
oping countries due to their high costs [6]. Clearly, there
remains an urgent demand for an effective and low-cost
system for prophylaxis and treatment of HIV infection. liposome platform due to their specificity, high stability
and low production cost [31, 32]. Moreover, these anti-
body fragments showed very efficient ability to penetrate
human tissue by diffusing into the mucosal layer when
placed in a vaginal ring [31]. Background In this study, multivalent liposomal antibody constructs
were developed by grafting Vhh (J3), which achieves broad
and potent neutralization of HIV-1 via interaction with the
CD4-binding site of HIV-1 env [33], onto the liposome
membrane via either non-covalent metal chelation or a co-
valent linkage. The neutralization potency of multivalent
presentation of these fragments by liposomes was analyzed
by antiviral assays with TZM-bl cells. The results indicated
that the avidity of the Vhh was significantly increased while
the neutralization potency was compromised when the
Vhh was covalently conjugated to the lipid. The additional
ability of the liposome construct to effectively deliver
dapivirine, a potent non-nucleoside reverse transcriptase
inhibitor presently being developed as a vaginal HIV
microbicide, was further investigated. y
y
The challenge of developing an effective vaccine or
entry-inhibitor drug for HIV infection lies in the rapid
mutation of the glycoproteins comprising gp120 and
gp41 and the structural features that facilitate antibody
evasion [7–13]. Another recent explanation is the spatial
arrangement of the envelope spikes on HIV virus, which
contributes to its ability to escape the neutralization by
antibodies [14]. As is known, most neutralization activities
can be attributed to antibody-antigen interactions and the
avidity resulting from bivalent binding between two Fabs
and two physically linked antigens greatly increase the
neutralization potency of the antibody [15–17]. The pub-
lished studies indicated that there are 4 to 35 spikes per
HIV virus particle [18–22] with very restricted mobility
[23, 24]. This makes the antibody cross-linking very un-
likely because the hinge (10–15 nm) between the two Fabs
is not long enough to reach the two spikes randomly dis-
tributed on the surface of the virus. When the anti-HIV
antibody b12 was tested as an IgA, IgM or IgG, equivalent
neutralization potency was observed [14]. Actually, a
number of studies have demonstrated that neutralizing
antibodies against HIV cannot take the advantage of avid-
ity effects resulting from the IgG cross-linking epitopes on
the surface of virus [15–17], while antibodies have been
shown to bind bivalently to other viruses comprising
closely spaced epitopes [25, 26]. Materials Soy phosphatidylcholine (PC), 1,2-dioleoyl-sn-glycero-3-
[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl]
(18:1 DGS-NTA(Ni)) and 1,2-distearoyl-sn-glycero-3-phos-
phoethanolamine-N-[maleimide(polyethylene glycol)-2000]
(malDSPE-PEG2000) were purchased from Avanti Polar
Lipids. Methoxypolyethyleneglycol-di-stearoyl-phosphatidyl-
ethanolamine (DSPE-PEG2000, with mPEG MW2000Da)
was obtained from Genzyme. Cholesterol (Chol), PBS and
Sephadex G25 and G50 were purchased from Sigma. Acetone, methanol and dichloromethane were from Fisher
Scientific. RPMI1640, L-glutamine, penicillin-streptomycin
and foetal bovine serum (FBS) were from Invitrogen Life
Technologies. Dulbecco’s minimum essential medium
(DMEM) was purchased from Lonza. Vhhs, the variable
region fragment of single heavy-chain llama antibodies,
including J3 and lab5, were purchased from Quality in
Antibodies (Netherlands). Dapivirine, a non-nucleoside re-
verse transcriptase inhibitor (NNRTI), was kindly provided
by the authors Prof. Guido Vanham and Dr. Kevin Arien. CN54 gp140 was kindly provided by Prof. Robin Weiss
from University College London. Liposomes are artificially prepared vesicles composed
of a lipid bilayer and have been used extensively for drug
delivery due to their unique properties [27]. Liposomes
are also platforms for the development of multivalent
antibody constructs by coupling antibody molecules onto
liposome membranes. Chiu et al. have demonstrated that
the antibody potency was increased 25-fold by employing
the multivalent liposome formulation [28], which even
induced cascade effects by cross-linking the antibody
complex [29]. Cells, Virus and Reagents Cells, Virus and Reagents
TZM-bl cells (obtained through the NIH AIDS Research
and Reference Reagent Program, Germantown, MD)
were used for the evaluation of the anti-HIV activity of
free and liposome conjugated Vhh with and without
dapivirine. TZM-bl cells express high densities of CD4,
CCR5 and CXCR4. They contain a luciferase reporter
gene under control of HIV LTR, which will be tran-
scribed and translated into luciferase protein, if the cells Recently, llama heavy-chain antibody fragments (Vhh),
which compete with surface CD4 for binding to the
HIV-1envelope glycoprotein, have been described as po-
tent cross-clade HIV-1 candidate microbicides [30, 31]. Such Vhh would be excellent candidates for multivalent Wang et al. Nanoscale Research Letters (2016) 11:350 Page 3 of 10 Page 3 of 10 Page 3 of 10 get infected with HIV and start producing Tat. TZM-bl
cells were cultured in DMEM supplemented with 10 %
heat-inactivated FBS and 50 μg gentamicin/ml medium,
at 37 °C in a humidified 7 % CO2 environment. Cells
were split twice a week and plated at 106 cells in tissue
culture flasks and at 104 cells/well in 96-well plates. 10 mM DTT for 30 min at room temperature. Covalent
immunoliposomes were prepared via interaction be-
tween J3-cys and maleimide-derivatized lipid inserted
in lipo3. The mixture of J3-cys and liposomes with the
maleimide group was allowed to conjugate at room
temperature overnight. Unreacted maleimide groups
were subsequently saturated by adding cysteine. The
immunoliposomes were purified by running through a
Sephadex G50 column to remove excess cysteine or/
and unbound protein. The subtype B CCR-5 using reference HIV-1 strain
Ba-L was obtained from the NIH AIDS Research and
Reference Reagent Program, Germantown, MD. It was
grown and titered in human activated peripheral blood
mononuclear cells (PBMC). PBMC were isolated from
buffy coats from HIV-seronegative blood donors (provided
by the Antwerp Blood Transfusion Centre) using Ficoll
density gradient centrifugation. PBMC were stimulated for
48 h with 2 μg/ml of phytohemagglutinin (PHA) (Remel,
Kent, UK) in RPMI 1640 medium containing 10 % FBS,
50 μg/ml gentamicin and 2 μg/ml polybrene (Sigma-
Aldrich, Bornem, Belgium). Subsequently, PBMC were
activated for 24 h with 1 ng/ml of interleukin-2 (IL-2)
(Gentaur, Brussels, Belgium) in RPMI 1640 medium
supplemented with 10 % FBS, 50 μg/ml gentamicin, 2 μg/
ml polybrene and 5 μg/ml hydrocortisone (Calbiochem,
Leuven, Belgium). PHA, IL-2 activated PBMC. Characterization of Liposomes The phospholipid, mainly PC, in the liposome was quan-
tified by the Stewart assay [34]. The number of lipo-
somes per ml was estimated by assuming there were
80,000 phospholipid molecules per liposome of 100 nm
or 320,000 phospholipid molecules per liposome of 200-
nm diameter [35]. The mean particle size distribution
and polydispersity index of liposome suspensions were
determined by dynamic light scattering in a Malvern
Zetasizer Nano-2S (Malvern Instruments, Malvern, UK). The buffer for zeta potential is MilliQ water pH 7. The
stability of liposomes was evaluated by storing the lipo-
somes at 4 °C for 1 month, and samples were taken for
size measurement. Liposome Preparation Liposome compositions used in this study with diame-
ters in brackets were as follows: (a) lipo1 (200 nm), PC/
Chol/DSPE-PEG2000/18:1 DGS-NTA in proportions
65/33/1/1 mol%; (b) lipo2 (100 nm), PC/Chol/DSPE-
PEG2000/18:1 DGS-NTA in proportions 65/33/1/1 mol%
and (c) lipo3 (100 nm), PC/Chol/DSPE-PEG2000/mal-
DSPE-PEG2000 in proportions 65/33.5/0/1.5 mol%. Briefly,
the lipids with or without dapivirine were dissolved in
methanol: dichloromethane 1:2 (v/v) at room temperature. The lipid mixtures were deposited on the side wall of a
rotary glass vial by removing the solvent with nitrogen. The resulting dried lipid films were hydrated in 10-mM
sodium phosphate buffer pH 7.4. This process led to the
spontaneous formation of pegylated liposomes. The lipo-
somes were then extruded through 0.2 or 0.1 μm Anotop
10 filters (Whatman). Excess dapivirine was removed on a
Sephadex G25 column [34]. SDS-PAGE was employed to confirm the conjugation
of Vhh to the liposomes. Fluorescamine was used to
quantify the protein conjugated to liposomes [36]. Fluor-
escamine is a non-fluorescent compound, which reacts
rapidly with primary amines in proteins, such as the ter-
minal amino group of peptides and the ε-amino group
of lysine, to form highly fluorescent moieties. Basically,
the liposome suspension was diluted 1:20 in PBS pH 7. An aliquot of 25 μl of 2 mg/ml fluorescamine was added
to a total assay volume of 100-μl diluted liposome suspen-
sion. The fluorescence was measured at ex 390 nm/em
475 nm with a cut-off at 455 nm, and the background was
subtracted. The concentration of conjugated protein was
calculated by comparison with standard samples of the
same protein. The concentration of dapivirine in lipo-
somes was determined by measuring the absorbance at
310 nm after incubating the liposomes in ethanol [37, 38]. J3 is a neutralizing Vhh, while lab5 is an irrelevant
Vhh (irr-Vhh). J3 and lab5 were polyhistidinylated (J3-
His and lab5-His) for the non-covalent attachment to
liposomes via nitrilotriacetic acid (NTA) linkage. Mean-
while another form of J3 (J3-cys) with extra cysteine in
the C-terminus was generated for covalent conjugation
to liposomes via interaction with a maleimide group. Vhhs were produced as described before [31]. Non-
covalent immunoliposomes were produced by mixing
Vhh-His with the liposome suspension in a 1:10 M ra-
tio at room temperature for 1 h on a bench roller. Free
thiol groups were generated by incubating J3-cys with Enzyme-Linked Immunosorbent Assay An enzyme-linked immunosorbent assay (ELISA) was
carried out to determine the interaction between liposome-
linked Vhh and gp140 which is a derivative of gp160 that
can be cleaved into gp120 and gp41. Briefly, 1 μg/ml CN54
gp140 was coated at 4 °C overnight on 96-well Maxisorp
plates (NUNC, Denmark). After blocking with 200 μl/well
Microwell Blocking Buffer with Stabilizer (Rockland), serial
dilutions of immunoliposomes in PBS pH 7, free Vhh and
negative control liposomes coated with irr-Vhh (lab5) were
added to the plates for 1 h. After three washes with PBS, Wang et al. Nanoscale Research Letters (2016) 11:350 Page 4 of 10 Page 4 of 10 Page 4 of 10 Vhh density of the liposomes will contribute to the
neutralization efficiency of the liposomes for HIV virus. Lipo1 was of 200-nm diameter while lipo2 and lipo3
were of 100-nm diameter. His-tagged Vhh (J3-His and
lab5-His) were conjugated to lipo1 or lipo2 by inter-
action with the nickel-chelating lipid 18:1 DGS-NTA-Ni
which was incorporated into the liposome bilayer (Fig. 1a). The sulphydryl-reactive lipid (malDSPE-PEG2000) in lipo3
allowed covalent attachment of the reduced J3-cys to the
liposome (Fig. 1b). Immunoliposomes were then purified
by size exclusion chromatography to remove the unbound
Vhh and impurities. The conjugation and purification
were successful in all cases, and the resulting products
were analyzed by SDS-PAGE (Fig. 2). The conjugated
Vhhs gave bands of higher molecular weight than the free
Vhhs, and this was due to the contribution of the conju-
gated lipids including 18:1 DGS-NTA-Ni or malDSPE-
PEG2000. More than 95 % of the Vhh was shown to be
conjugated to the liposomes by the fluorescamine method
described above [34, 36]. The density of Vhh on liposome
surfaces was shown in Table 1. anti-myc-horse radish peroxidase (HRP) (Roche), which
targets the myc-tag in Vhh, was diluted 1:1000 in HRP
Conjugate Stabilizer (Rockland) and added to detect the
bound Vhh. 3,3′,5,5′-Tetramethylbenzidine liquid (Sigma)
substrate was added and left to develop for 30 min at room
temperature; after which, the reaction was stopped with
2 M H2SO4. Absorbance of the product was measured at
450 nm. Antiviral Assays for Antibody-Mediated Neutralization
and Effectiveness of Liposomal Dapivirine Drug Delivery Antiviral Assays for Antibody-Mediated Neutralization
and Effectiveness of Liposomal Dapivirine Drug Delivery
The antiviral activity of the compounds was determined
by pre-incubating 104 TZMbl cells/well in a 96-well
plate for 2 h at 37 °C and 7 % CO2 to maintain the opti-
mal pH with or without a serial dilution of compounds. Next, 200 TCID50 of HIV-1 BaL virus was added to each
well, and cultures were incubated for 48 h before luciferase
activity was quantified. To this end, 120 μL of supernatant
was removed, 75 μl of the luciferase substrate Steadylite
(Perkin Elmer, Life Sciences, Zaventem, Belgium) was
added to the wells, and the plates were incubated at room
temperature on an orbital shaker for 10 min. Next, the
luciferase activity was measured using a TriStar LB941
luminometer (Berthold Technologies GmbH & Co. KG., Bad Wildbad, Germany) and expressed in relative
light units (RLU). The final immunoliposomes were characterized, and
the data was shown in Table 1. Phospholipid (PC) was
detected in the liposomes by Stewart assay. It was esti-
mated to result in approximately 160 Vhh molecules per
200-nm liposome or 95–125 Vhh molecules per 100-nm
liposome on the basis that one 100-nm liposome contains
80,000 phospholipid molecules and one 200-nm liposome
contains 320,000 phospholipid molecules [29, 35]. This resulted in similar Vhh density on the surface of
the liposomes. PEGylated liposome suspensions were prepared by the
lipid film rehydration method [34] and extrusion tech-
nique, with a final mean diameter of 200–230 nm or
120–140 nm (polydispersity index of 0.1–0.2). The nega-
tive charge of the liposome formulations were attributed
to the DSPE-PEG2000 or malDSPE-PEG2000which ac-
counts for 1–1.5 % mol of the lipid. The liposomes were
stable for at least 1 month, with no significant change in
size or charge (Fig. 3). Each compound was tested in triplicate and each ex-
periment was repeated in three independent runs. Anti-
viral activity was expressed as the percentage of viral
growth compared to the control and plotted against the
compound concentration. Next, non-linear regression
analysis was used to calculate the EC50, using GraphPad
Prism 5.03 using non-linear regression (GraphPad Software,
San Diego, CA, USA). In Vitro Cytotoxicity Human cancer cells, Hela cells (Sigma-Aldrich, UK), were
cultured in 96-well plates overnight and treated with differ-
ent concentrations of liposomes for 48 h. Medium was then
removed from the plates, and the cells washed twice with
PBS. Fifty microlitres of 3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyltetrazolium bromide (MTT) was added per well. Cells were incubated for 30 min at 37 °C with 95 % O2 and
5 % CO2. MTT was removed carefully before 100-μl pro-
panol was added per well to dissolve crystals and incubated
for at least 30 min. The absorbance of this coloured solu-
tion was quantified by measuring at a wavelength of
570 nm by FLUOstar Omega (BMG Labtech, Aylesbury,
UK). Each treatment was conducted in triplicate. Immunoliposomes with or without NNRTI dapivirine
were produced to investigate whether conjugated Vhh
could show higher avidity to antigen and improve the ef-
ficacy of Vhh in HIV viral inhibition, as compared to
free Vhh. Dapivirine was dissolved in the solvent with
lipid mixture when formulating the liposomes. Unincor-
porated dapivirine was removed by size exclusion chro-
matography, and the concentration of dapivirine was
determined by UV spectrometry. Liposome Preparation and Characterization Potential cytotoxicity of the liposomes was estimated
from cell viability relative to control Hela cells treated
with medium only. MTT assay demonstrated that cell Three groups of liposome suspensions: lipo1, lipo2 and
lipo3, were prepared to investigate whether the size or Wang et al. Nanoscale Research Letters (2016) 11:350 Page 5 of 10 Fig. 1 Structural basis of conjugation. a Non-covalent ligation between Vhh-His and nickel-chelating lipid 18:1 DGS-NTA-Ni. b Covalent ligation
between Vhh-cys and malDSPE-PEG2000 lipid Fig. 1 Structural basis of conjugation. a Non-covalent ligation between Vhh-His and nickel-chelating lipid 18:1 DGS-NTA-Ni. b Covalent ligation
between Vhh-cys and malDSPE-PEG2000 lipid viability was between 80 and 120 % in relation to the
control samples (Fig. 4). One-way ANOVA statistical
analysis showed there is no evidence of liposome toxicity
at concentrations up to 1 mg/ml (p > 0.05). the free J3 at the same concentrations, together with the
irrelevant Vhh (lab5-His) and liposome-coated the same
irrelevant Vhh as negative controls. J3-His and J3-cys
revealed similar binding affinities for gp140 as expected
(Fig. 5). No improved or compromised affinity was ob-
served for lipo1-J3 and lipo2-J3 compared to free J3-His,
implying size differences are not influencing the avidity
despite similar Vhh densities on the liposome surfaces. Meanwhile, one-way ANOVA statistical analysis showed
that J3 conjugated to lipo3 demonstrated much higher
avidity to gp140 than free J3-cys (p < 0.001), indicating
covalent attachment greatly facilitated J3 interacting with Characterization of Neutralizing Antibody by Antiviral y
The neutralization activity of liposomal Vhhs and free
Vhhs for HIV virus was evaluated by subsequent incuba-
tion with TZM-bl cells. Lipo1-J3 and lipo2-J3 revealed
similar potencies for neutralization compared to free J3. Surprisingly, lipo3-J3 only provided 60 % of viral inhib-
ition at J3 concentration of 286 ng/ml, whereas lipo1-J3
and lipo2-J3 inhibited approximately 90 % of viral growth
at the same concentration (Fig. 6a). No viral growth reduc-
tion was observed for control liposomes, liposomes coated
with irrelevant Vhhs or free Vhhs alone. There was no
significant difference in neutralizing activities between Analysis of Binding of Liposomal Vhhs to Recombinant
HIV gp140 by ELISA As Vhh competed with soluble CD4 (sCD4) for binding
to gp120 [32], an ELISA assay was carried out to deter-
mine whether the Vhh conjugated to liposomes showed
compromised binding affinity or increased avidity to
gp140 (CN54). Liposome-linked J3 was compared with Fig. 2 Vhh and conjugated Vhh in liposomes analyzed by SDS-PAGE. Lanes: 1, molecular weight marker; 2, J3-His; 3, lipo1-J3; 4, lipo2-J3; 5, lab5;
6, lipo1-lab5; 7, lipo2-lab5; 8, J3-cys and 9, lipo3-J3 Fig. 2 Vhh and conjugated Vhh in liposomes analyzed by SDS-PAGE. Lanes: 1, molecular weight marker; 2, J3-His; 3, lipo1-J3; 4, lipo2-J3; 5, lab5;
6, lipo1-lab5; 7, lipo2-lab5; 8, J3-cys and 9, lipo3-J3 Wang et al. Nanoscale Research Letters (2016) 11:350 Page 6 of 10 Table 1 Characterisation of the immunoliposomes
Liposome
PC conc. (mg/ml)a
Dapivirine (μM)
No. of Vhh molecules per liposome
Vhh (μg/ml)
Diameter (nm)a
Zeta potential (mV)a
Lipo1-lab5
11.937 ± 0.001
–
160
126.3
228.0 ± 1.0
−45.0 ± 0.9
Lipo1-J3
11.708 ± 0.008
–
160
118.4
223.4 ± 4.5
−37.4 ± 0.5
Lipo2-lab5
5.974 ± 0.005
–
95
150.0
130.9 ± 1.4
−38.7 ± 0.7
Lipo2-J3
6.149 ± 0.003
–
108
159.9
131.2 ± 1.9
−38.5 ± 0.2
Lipo2-dapi
6.478 ± 0.001
6.5
–
–
122.9 ± 1.8
−46.0 ± 0.9
Lipo2-dapi-J3
5.548 ± 0.002
5.5
125
169.6
123.4 ± 1.8
−38.5 ± 0.4
Lipo3 control
7.573 ± 0.003
–
–
–
130.9 ± 1.7
−42.5 ± 0.4
Lipo3-J3
6.456 ± 0.004
–
121
168.2
142.2 ± 3.2
−36.4 ± 0.4
Lipo3-dapi
6.675 ± 0.003
6.7
–
–
128.2 ± 2.6
−41.7 ± 0.5
Lipo3-dapi-J3
6.806 ± 0.001
6.8
108
157.0
139.1 ± 1.8
−37.8 ± 0.6
Dapi: dapivirine
aData are means ± standard deviations of three replicate measurements in one representative experiment of three independent experiments gp140. In contrast, irrelevant negative controls demon-
strated no binding to gp140. lipo1-J3 and lipo2-J3 compared to free J3-His (Fig. 6b),
revealing no contribution from the size differences. However, covalently conjugated J3 (lipo3-J3) had a sig-
nificantly higher EC 50 (Fig. 6b), indicating less potency
for neutralization than non-covalently attached J3 or
free J3. The result is unexpected because the ELISA
data revealed significantly increased binding affinity of
lipo3-J3 to gp140 which should contribute to the
neutralization activity of conjugated J3. Discussion The treatment of HIV remains a serious challenge due
to the high genetic variation of the virus and associated
toxic side effects of the antiviral drugs. HIV-1 neutraliz-
ing antibodies have been considered to be critical for
vaccine development and prevention of HIV infection. Liposomes provided another approach shown to have
potential for HIV prophylaxis [39]. In the present study,
a combined platform with both liposome and antibody
fragments was developed to investigate the antiviral effi-
ciency of the multivalent liposomal formulation. y
p
Previous studies have demonstrated that Vhhs produced
by llamas (lama glama) exhibited comparable affinity and
specificity for gp120 to conventional immunoglobulins
despite their much smaller size. This smaller size of Vhhs
is an advantage over conventional immunoglobulins as it
has been shown to reduce their immunogenicity [33]. In
particular, J3 Vhh, which achieved broad and potent
neutralization of HIV-1 via interaction with the CD4-
binding site, neutralized 96 of 100 tested HIV-1 strains
and chimeric simian-HIV strains with HIV subtypes B and
C env [33]. Unlike most of the enveloped viruses which
express large numbers of closely spaced spikes on the sur-
face, HIV virus has a low spike surface density (4 to 35
spikes) and the arrangement of these spikes is likely to be
static over the time periods relevant to neutralization [14]. Therefore, the location and arrangement of the neutraliz-
ing Vhhs on the liposome surfaces is critical for their
interaction with the virus particles. Fig. 4 Cell viability of Hela cells after 48 h incubation with control
liposomes or immunoliposomes. No significant difference in cell
viability was observed against the media only treatment from one-way
ANOVA (p > 0.05). Data are means ± standard deviations of three
replicate measurements in one representative experiment of at
least two independent experiments. Dapi: dapivirine Fig. 4 Cell viability of Hela cells after 48 h incubation with control
liposomes or immunoliposomes. No significant difference in cell
viability was observed against the media only treatment from one-way
ANOVA (p > 0.05). Data are means ± standard deviations of three
replicate measurements in one representative experiment of at
least two independent experiments. Dapi: dapivirine although no significant difference was observed between
lipo2 and lipo3 with or without neutralizing Vhhs (Fig. 7a). Comparison of Antiviral Effects of Liposomal Dapivirine
with Free Dapivirine We compared the antiviral effects of different formula-
tions of liposomal dapivirine with free dapivirine. The
dose-response curves indicated increased antiviral effect
of liposomal dapivirine compared with free dapivirine, Fig. 3 Diameter and zeta potential of the liposome formulations over 1-month period. Data are means ± standard deviations of three replicate
measurements in one representative experiment of at least two independent experiments. Using unpaired t test (p > 0.05), no significant change
in diameter or zeta potential of liposomes was observed after 1-month storage at 4 °C. Dapi: dapivirine Fig. 3 Diameter and zeta potential of the liposome formulations over 1-month period. Data are means ± standard deviations of three replicate
measurements in one representative experiment of at least two independent experiments. Using unpaired t test (p > 0.05), no significant change
in diameter or zeta potential of liposomes was observed after 1-month storage at 4 °C. Dapi: dapivirine Wang et al. Nanoscale Research Letters (2016) 11:350 Page 7 of 10 Page 7 of 10 Fig. 4 Cell viability of Hela cells after 48 h incubation with control
liposomes or immunoliposomes. No significant difference in cell
viability was observed against the media only treatment from one-way
ANOVA (p > 0.05). Data are means ± standard deviations of three
replicate measurements in one representative experiment of at
least two independent experiments. Dapi: dapivirine Discussion Furthermore, there was approximately 76 % of viral growth
at a concentration of 1-nM free dapivirine, whereas
only 50–56 % of viral growth was detected with liposomal
dapivirine at this concentration. Calculated EC50 (Fig. 7b)
confirmed that liposomal dapivirine is more effective than
free dapivirine in inhibiting HIV virus. However, the anti-
viral effects were dominated by dapivirine since no differ-
ence was observed between the liposome preparations
with or without neutralizing J3. To optimize the size of the liposomal platform, J3 was
non-covalently conjugated to the surface of two lipo-
some formulations with diameters of 100 or 200 nm,
each with approximately the same Vhh densities. Metal
chelation via NTA has been employed to attach peptide
and protein antigens to liposomes to compare with cova-
lent linkage for their ability to elicit antibody responses
[40]. Pegylated liposomes were employed to ensure the
stability and minimal immunogenicity of the platform. To check that J3 was available and not masked by the
PEG coating on the liposome surface, appropriate access
of J3 to gp140 was verified by ELISA (Fig. 5). The identi-
cal binding curves demonstrated that lipo1-J3 and lipo2-
J3 bound to gp140 with the same avidity as free J3,
showing that the different geometrical location of J3 on
the surface of spherical liposomes did not compromise
its interaction with gp140. Surprisingly, no enhanced
avidity was observed for these liposomal J3 constructs
compared to free J3. This may be due to the charge on
the nickel-histidine complex which would inhibit close
association of the J3 antibody fragments to generate
multivalent attachment to the gp140 on ELISA surface. This presumption is confirmed by the neutralization
results indicating lipo1-J3 and lipo2-J3 inhibited HIV
infection in a dose-response manner similar to free J3 Fig. 5 The binding of free J3/J3 linked to liposomes to recombinant
gp140 (CN54) was compared in ELISA. Gp140 was immobilized on
the plate, and Vhh was detected with anti-myc-HRP. Irrelevant Vhh
(lab5) and liposome-irrelevant Vhh were included as negative
controls. Values are means ± standard deviations of three replicate
measurements in one representative experiment of three independent
experiments. *From one-way ANOVA, significant differences were
observed (p < 0.001) between lipo3-J3 and the other liposome
formulations or free Vhhs Fig. 5 The binding of free J3/J3 linked to liposomes to recombinant
gp140 (CN54) was compared in ELISA. Gp140 was immobilized on
the plate, and Vhh was detected with anti-myc-HRP. Discussion Irrelevant Vhh
(lab5) and liposome-irrelevant Vhh were included as negative
controls. Values are means ± standard deviations of three replicate
measurements in one representative experiment of three independent
experiments. *From one-way ANOVA, significant differences were
observed (p < 0.001) between lipo3-J3 and the other liposome
formulations or free Vhhs Wang et al. Nanoscale Research Letters (2016) 11:350 Page 8 of 10 a
b
Fig. 6 Antiviral effect of different liposomal formulations and free Vhhs. a Dose-response curves for the antiviral effect of different liposomal formulations
and free Vhhs. b Comparison of calculated EC50 of each liposomal formulation and free J3. Liposome preparations were pre-incubated with TZM-bl cells
for 2 h before virus was added and incubated for 48 h. Values are means ± standard deviations of three replicate measurements in one representative
experiment of three independent experiments. *From one-way ANOVA, significant differences (p < 0.001) in EC50 were observed between lipo3-J3 and the
other liposome formulations or free Vhhs b a
Fig. and f
for 2
expe
othe a b a Fig. 6 Antiviral effect of different liposomal formulations and free Vhhs. a Dose-response curves for the antiviral effect of different liposomal formulations
and free Vhhs. b Comparison of calculated EC50 of each liposomal formulation and free J3. Liposome preparations were pre-incubated with TZM-bl cells
for 2 h before virus was added and incubated for 48 h. Values are means ± standard deviations of three replicate measurements in one representative
experiment of three independent experiments. *From one-way ANOVA, significant differences (p < 0.001) in EC50 were observed between lipo3-J3 and the
other liposome formulations or free Vhhs J3 were distinct from each other, indicating that their inter-
actions with gp140 were qualitatively different with superior
avidity for lipo3-J3. This implied that a multivalent platform
had been formed with cross-linking or multimerization of
Vhhs. This is not surprising since the absence of the nickel
complex allows the free movement and geometrical ar-
rangement of J3-lipid complex within the lipid liposome
membrane so permitting the formation of multivalent lipo-
somal Vhh constructs through cross-linking the envelope in antiviral assay (Fig. 6). There was no significant differ-
ence in the EC50 between lipo1-J3, lipo2-J3 and free J3,
suggesting no improved antiviral potency for the non-
covalently anchored Vhh independent of liposome sizes. J3-cys was then covalently attached to liposomes to
produce lipo3-J3 (Fig. 1). Discussion One hundred-nanometre diam-
eter liposomes were used for economic reasons and since
liposomes of this size are broadly applied as drug delivery
system [41, 42]. The binding curves for lipo2-J3 and lipo3- a
b
Fig. 7 Antiviral effect of different liposomal formulations and free dapivirine. a Dose-response curves for the antiviral effect of different liposomal
formulations and free dapivirine. b Comparison of calculated EC50 of each liposomal formulation and free dapivirine. Liposome preparations were
pre-incubated with TZM-bl cells for 2 h before virus was added and incubated for 48 h. Values are means ± standard deviations of three replicate
measurements in one representative experiment of three independent experiments. *From one-way ANOVA, significant differences (p < 0.001) in
EC50 were observed between free dapivirine and the liposomal dapivirine. Dapi: dapivirine a
Fig. form
pre-i
meas
EC50 a
b a b Fig. 7 Antiviral effect of different liposomal formulations and free dapivirine. a Dose-response curves for the antiviral effect of different liposomal
formulations and free dapivirine. b Comparison of calculated EC50 of each liposomal formulation and free dapivirine. Liposome preparations were
pre-incubated with TZM-bl cells for 2 h before virus was added and incubated for 48 h. Values are means ± standard deviations of three replicate
measurements in one representative experiment of three independent experiments. *From one-way ANOVA, significant differences (p < 0.001) in
EC50 were observed between free dapivirine and the liposomal dapivirine. Dapi: dapivirine Fig. 7 Antiviral effect of different liposomal formulations and free dapivirine. a Dose-response curves for the antiviral effect of different liposomal
formulations and free dapivirine. b Comparison of calculated EC50 of each liposomal formulation and free dapivirine. Liposome preparations were
pre-incubated with TZM-bl cells for 2 h before virus was added and incubated for 48 h. Values are means ± standard deviations of three replicate
measurements in one representative experiment of three independent experiments. *From one-way ANOVA, significant differences (p < 0.001) in
EC50 were observed between free dapivirine and the liposomal dapivirine. Dapi: dapivirine Wang et al. Nanoscale Research Letters (2016) 11:350 Page 9 of 10 Page 9 of 10 spikes which are sparsely distributed on the virion surface
and remained static over the time periods. Unexpectedly,
the antiviral potency of lipo3-J3 was remarkably reduced as
evaluated in the antiviral assay compared to lipo1-J3, lipo2-
J3 or free J3, the EC50 for lipo3-J3 being approximately
four times higher than that for lipo2-J3 or free J3. Competing Interests
Th
h
d
l
h The authors declare that they have no competing interests. Authors’ Contributions SXW and IR are working in Middlesex University. SXW has designed the study,
prepared the liposome formulations for all the experiments in this study and
wrote the manuscript. IR provided consultation on the whole study including
the design and use of the liposome and Vhh antibody fragments. JM, KKA
and GV are working in Institute of Tropical Medicine, Belgium. JM has carried
out the antiviral assay in this study. KKA has designed the antiviral assay. GV
provided consultation on HIV infection and treatment. RN is the chief scientist
in Proxima Concepts Ltd. He has provided consultation on the liposome
preparation. All authors read and approved the final manuscript. References 1. Wyatt R, Sodroski J (1998) The HIV-1 envelope glycoproteins: fusogens,
antigens, and immunogens. Science 280:1884–8 2. Chan DC, Kim PS (1998) HIV entry and its inhibition. Cell 93:681–4 3. Wong HL, Chattopadhyay N, Wu XY, Bendayan R (2010) Nanotechnology
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Thi
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t This work was supported by the CHAARM EU consortium to develop vaginal
HIV microbicides. We would like to thank Dr. Lei Wang for his assistance in
statistical analysis. Author details
1
f 1Department of Natural Sciences, Middlesex University, London, UK. 2Institute
of Tropical Medicine, Antwerp, Belgium. 3Proxima Concepts. Ltd, London, UK. In addition to the neutralization capacity of antibody-
coated liposomes, we investigated whether the liposomes
could have the extra-function of delivering antiviral
drugs intracellularly, in particular using high doses of
otherwise poorly soluble hydrophobic drugs whose encap-
sulation in the liposome would limit its general toxicity. All of these dapivirine containing liposomes revealed
higher efficacy in reducing viral replication than free dapi-
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activities of J3 were masked by the high dose of dapivirine
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HIV virus as efficiently as free J3. It was also shown that
incorporation of the lipophilic drug dapivirine in lipo-
somes resulted in a higher level of virus inactivation in
vitro than observed after exposure of cells to free drug
dissolved in DMSO—an effective method of solubilizing
the drug, but not one which can be used in vivo. Since
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glycoprotein in complex with the CD4 receptor and a neutralizing human
antibody. Nature 393:648–59 10. Labrijn AF, Poignard P, Raja A et al (2003) Access of antibody molecules
to the conserved coreceptor binding site on glycoprotein gp120 is
sterically restricted on primary human immunodeficiency virus type 1. J Virol 77:10557–65 10. Labrijn AF, Poignard P, Raja A et al (2003) Access of antibody molecules
to the conserved coreceptor binding site on glycoprotein gp120 is
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38.
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39.
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42.
Gaddy DF, Lee H, Zheng J et al (2015) Whole-body organ-level and kidney
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fragments with cross-subtype human immunodeficiency virus type 1
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82:12069–81. Submit your manuscript to a
journal and benefi t from:
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Submit your next manuscript at 7 springeropen.com Submit your manuscript to a
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Submit your next manuscript at 7 springeropen.com Submit your manuscript to a
journal and benefi t from: Submit your manuscript to a
journal and benefi t from: 33. McCoy LE, Quigley AF, Strokappe NM et al (2012) Potent and broad
neutralization of HIV-1 by a llama antibody elicited by immunization. J Exp Med 209:1091–103 34. New RR (1990) Liposomes a practical approach. 1st ed. Oxford: Oxford
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https://openalex.org/W2178981046
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English
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Walks, Partitions, and Normal Ordering
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The Electronic journal of combinatorics/The journal of combinatorics
| 2,015
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cc-by
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Abstract We describe the relation between graph decompositions into walks and the nor-
mal ordering of differential operators in the n-th Weyl algebra. Under several spec-
ifications, we study new types of restricted set partitions, and a generalization of
Stirling numbers, which we call the λ-Stirling numbers. Keywords: Decompositions of graphs; differential operators; Weyl algebra; set
partitions; Stirling numbers dzhuma@hotmail.com
yeldamir@gmail.com Submitted: Apr 11, 2015; Accepted: Sep 30, 2015; Published: Oct 16, 2015
Mathematics Subject Classifications: 05A18, 05A30, 11B73 the electronic journal of combinatorics 22(4) (2015), #P4.10 1
Introduction Let G = (V, E) be a digraph with an ordered set of edges E = (e1, . . . , em). A walk of G
is any sequence of edges ei1 . . . eiℓsuch that the terminal vertex of eik coincides with the
initial vertex of eik+1 for all k < ℓ. The walk ei1 . . . eiℓis called increasing if i1 < · · · < iℓ. +
Consider decompositions of G into edge-disjoint increasing walks. This setting gen-
eralizes set partitions, because when G has only one vertex and m labeled loops (1, 1),
decompositions into increasing walks correspond to partitions of the set [m] := {1, . . . , m}
into subsets. If G is decomposed into one walk (with distinct edges), it is an Euler tour. If G is a path and edges along the path are labeled by an arbitrary permutation σ ∈Sm,
decompositions into minimal number of increasing walks index the descent set of σ. This interpretation arises from the normal ordering problem in the Weyl algebra. The
n-th Weyl algebra An is an associative algebra with 2n generators x1, . . . , xn, ∂1, . . . , ∂n
subject to relations [∂i, xj] = δi,j, [xi, xj] = 0, [∂i, ∂j] = 0, where [a, b] = ab −ba is the
commutator and δi,j is the Kronecker delta. The element w ∈An is normally ordered if
it is expressed in the form w = P
k ck
Q
i xi
Q
j ∂j. For the digraph G, the special case of
our normal ordering formula is −→
m
Y
ℓ=1
xiℓ∂jℓ=
X
decompositions into increasing
walks with sources I
Y
i∈I
xj
Y
j∈J
∂j, 1 1
2
3
e1
e2
e3
Figure 1: A digraph with an ordered set edges. 1
2
3
e1
e2
e3 Figure 1: A digraph with an ordered set edges. where (iℓ, jℓ) is an ℓ-th edge of G and J is the corresponding multiset of sinks (which is
determined uniquely from the given sources I). For example, the graph in Figure 1 has
four decompositions into increasing walks: e1e2e3,
e1e2 ∪e3,
e1 ∪e2e3,
e1 ∪e2 ∪e3. This corresponds to the normal ordering This corresponds to the normal ordering x1∂2x2∂3x3∂1 = x1∂1 + x1x3∂3∂1 + x1x2∂2∂1 + x1x2x3∂2∂3∂1, where the sources and sinks of walks are exactly the indices of terms. 1
Introduction In fact, this
formula is a graph-theoretic version of Olshanski’s analog of Wick’s formula [26] (cf. [27]). Collecting the terms we can rewrite where the sources and sinks of walks are exactly the indices of terms. In fact, this
formula is a graph-theoretic version of Olshanski’s analog of Wick’s formula [26] (cf. [27]). Collecting the terms we can rewrite −→
m
Y
ℓ=1
xiℓ∂jℓ=
X
I
SG(I)
Y
i∈I
xj
Y
j∈J
∂j, where SG(I) is the number of decompositions of G into increasing walks with multiset
of sources I. In the case of the first Weyl algebra A1 this formula gives the well-known
expansion where SG(I) is the number of decompositions of G into increasing walks with multiset
of sources I. In the case of the first Weyl algebra A1 this formula gives the well-known
expansion (x∂)m =
X
i
S(m, i)xi∂i for Stirling numbers of the second kind S(m, i). We introduced this combinatorial model in a more general setting in [15], where we
studied algebraic applications, polynomial identities and commutators on Weyl algebra. In this paper we focus on combinatorial aspects of walk decompositions and various spec-
ifications of the normal ordering interpretations, such as restricted set partitions and
generalized Stirling numbers. We study walk decompositions and the G-Stirling func-
tions which enumerate decompositions by sources of walks. As we see, the values of the
G-Stirling function serve as connection constants in the normal ordering problem. The setting of decomposing graphs into walks is a source for certain types of restricted
set partitions. For example, assume that digraph has 2 vertices and edges e1, e3, . . . going
from vertex 1 to 2 and the remaining edges e2, e4, . . . going back from 2 to 1. Decomposi-
tions on this model generate set partitions with the property that each block (when sorted)
is parity alternating (i.e. odd, even, odd, etc.). We show that the total number of parity 2 the electronic journal of combinatorics 22(4) (2015), #P4.10 alternating partitions of the set [m], satisfies the formula a(m) = B⌊(m+1)/2⌋B⌈(m+1)/2⌉,
where Bk is the k-th Bell number, the number of partitions of [k]. Note that the latter
formula is not an obvious fact from the definition of a(m). We apply the composition of
operators in Weyl algebra to compute the number of such decompositions. 1
Introduction This approach
is illustrated for a new special type of set partitions, which we call the residue alternating
partitions (a general version of the parity alternating partitions). The elements in every
block of these partitions form a consecutive (cyclic) interval modulo n and their total
number is the product of Bell numbers. We show both algebraic and bijective proofs to
this fact. We then specify our interpretations to the case n = 1, for which there are many related
studies, e.g. [3, 4, 12, 18, 20, 21, 24, 30] and refer to a recent book [22] on the subject
of normal ordering. We introduce and study the λ-Stirling numbers. These numbers
of second kind naturally appear in decompositions of graphs with one vertex and many
loops. For the given sequence λ = (λ1 ⩾λ2 ⩾· · · ), the λ-Stirling numbers of the
second kind Sλ(n, k) is the number of partitions of [n] into k blocks such that the first
λ1 elements of [n] are in distinct blocks, the next λ2 elements are in distinct blocks, and
so on. This definition is a natural generalization of the r-Stirling numbers [5] and it was
studied in [25]. We show how these numbers Sλ(n, k) arise from our general graph setting
as well as from differential operators in the first Weyl algebra. On the other hand, we also
define the corresponding dual λ-Stirling numbers of the first kind Cλ(n, k), and show a
combinatorial interpretation to them. Namely, Cλ(n, k) is the number of permutations of
[n] having k cycles such that non-minimal elements of the first λ1 cycles are greater than
all minimal elements of these λ1 cycles; non-minimal elements of the next λ2 cycles are
greater than all minimal elements of these λ2 cycles, and so on (some of the remaining
cycles are singletons). The classical Stirling numbers are defined on two parameters: the
number of elements and the number of blocks (cycles). For these generalizations, one can
see that the λ-sequence affects on the first parameter in λ-Stirling numbers of the second
kind, and affects on the second parameter (cycles) in λ-Stirling numbers of the first kind. We obtain many properties of Sλ(n, k), Cλ(n, k) analogous to the properties of the usual
Stirling numbers. 2
Decompositions into increasing walks Suppose that edges of the digraph G = (V, E) are ordered (or labeled), E = (e1, . . . , em). A k-decomposition is a decompositions of E into k edge-disjoint walks. We say that the
k-decomposition E = P1 ∪. . . ∪Pk is principal if every walk Pi = eℓ1 . . . eℓs (1 ⩽i ⩽k) is
increasing, i.e. we have ℓ1 < . . . < ℓs. When V = {1} and graph has m labeled loop edges (1, 1), principal decompositions
correspond to partitions of the set [m] into disjoint subsets. Further, we suppose that the
digraph G is presented by the vertex set V = [n]. A block (or p-block if p is specified) is a distinguished set of edges {e1, . . . , ep}. If graph
is built up from several (disjoint) blocks, then we will require that the edges in each block
must lie in distinct walks. 3 the electronic journal of combinatorics 22(4) (2015), #P4.10 1
2
3
4
e2
e3
e1
e4
e5
e6
Figure 2: A digraph built of three edge blocks (red, blue, black). 1
2
3
4
e2
e3
e1
e4
e5
e6 Figure 2: A digraph built of three edge blocks (red, blue, black). For example, digraph in Figure 2 which is built from three blocks B1 = {e1}, B2 =
{e2, e3}, B3 = {e4, e5, e6}, has a principal 4-decomposition e1e5 ∪e2 ∪e3e4 ∪e6. Note that
decomposition e1e5 ∪e2e3e4 ∪e6 cannot be used here since e2, e3 are from the same block
B1 and thus cannot be in the same path. For each vertex v ∈V consider the sets In(v) = {ei1, . . . , eis},
Out(v) = {ej1, . . . , ejℓ} of its incoming and outcoming edges, respectively (loops (v, v) are included in both sets). Suppose that the edges of G are built up from some partition of E into blocks. Consider
the matchings between In(v) and Out(v) defined as follows. We allow elements ei ∈In(v)
and ej ∈Out(v) to be matched if i < j and ei, ej are not in the same block. A matching
now is defined as some set of such matched pairs, where every edge is used at most once
from In(v) and at most once from Out(v) (so, only loops can be used once for both In(v),
Out(v)). position 2.1. There is a bijection between the sets M(1) × · · · × M(n) and PD(G Proposition 2.1. There is a bijection between the sets M(1) × · · · × M(n) and PD(G). Proposition 2.1. There is a bijection between the sets M(1) × · · · × M(n) and PD(G). Proof. Let us take an arbitrary matching for every vertex v (1 ⩽v ⩽n) and construct
a principal graph decomposition. If ei ∈In(v) and ej ∈Out(v) are matched, then we
define them to be a fragment of a path eiej. Otherwise, if ei ∈In(v) or ej ∈Out(v) are
unmatched edges, then define them as final and initial edges of their corresponding paths,
respectively. One can easily verify that this map defines a principal decomposition and
its inverse defines a matching for every vertex. Corollary 2.2. |PD(G)| = Qn
i=1 |M(i)|. Corollary 2.2. |PD(G)| = Qn
i=1 |M(i)|. 2
Decompositions into increasing walks Let M(v) be the set of all possible matchings (not necessarily maximal, and
including an empty matching) and M(v) be the set of maximal matchings. For example,
at vertex 2 of the graph in Figure 2 we have In(2) = {e1, e2},
Out(2) = {e3, e5},
M(2) = {∅, {(e1, e3)}, {(e1, e5)}, {(e2, e5)}, {(e1, e3), (e2, e5)}},
M(2) = {(e1, e3), (e2, e5)}. Let PD(G) be the set of principal decompositions of G. the electronic journal of combinatorics 22(4) (2015), #P4.10 2.1
On general walk and path decompositions h G = (V, E), define flux(v) := in(v) −out(v), v ∈V,
V + := {v ∈V | flux(v) > 0},
V −:= {v ∈V | flux(v) < 0} and the total flux as f(G) :=
X
v∈V +
flux(v) =
X
v∈V −
−flux(v). The basic properties of walk decompositions such as existence criteria, simply rely on
the usual Euler tours. We just add a new vertex so that for all v ∈V we get flux(v) = 0. This also allows to compute (or bound) the total number of walk decompositions using
the BEST theorem and the Matrix-tree theorem. The basic properties of walk decompositions such as existence criteria, simply rely on
the usual Euler tours. We just add a new vertex so that for all v ∈V we get flux(v) = 0. This also allows to compute (or bound) the total number of walk decompositions using
the BEST theorem and the Matrix-tree theorem. Decompositions into walks have the following matroid structure studied in [23]. Sets of
edges that are in distinct walks in a certain (minimal) decomposition are independent sets
of a matroid of rank f(G) (adding with the number of components of G). This matroid
is isomorphic to a cotransversal matroid. In fact, the sets of source (initial) edges of all
f(G)-decompositions also form a collection of matroid bases. For (simple) path decompositions there are many studies (e.g. [1, 2, 8, 13, 16, 17, 19]),
most of which are around Gallai’s conjecture. Note that f(G) is attainable minimal
number of paths in a decomposition for acyclic digraphs; it was also shown in [1] that
the minimal number of paths needed to decompose a transitive tournament digraph on n
vertices is ⌊n2/4⌋. Gallai’s conjecture states that every connected simple undirected graph
with n vertices can be decomposed into at most ⌈n/2⌉paths; it is known that every such
graph can be decomposed into at most ⌊n/2⌋paths or cycles [19]. Corollary 2.2. |PD(G)| = Qn
i=1 |M(i)|. Denote in(v) = |In(v)|, out(v) = |Out(v)|. Denote in(v) = |In(v)|, out(v) = |Out(v)|. 4 the electronic journal of combinatorics 22(4) (2015), #P4.10 Corollary 2.3. If G is acyclic digraph, then there exists a labeling of its edges such that
every path is increasing. Therefore, every decomposition of G is a principal decomposition
for such labeling. For every vertex v we can match some i edges from In(v) with some i
edges of In(v) in
in(v)
i
out(v)
i
i! ways. Therefore, M(v) =
X
i⩾0
in(v)
i
out(v)
i
i! and the total number of decompositions of the acyclic digraph G is and the total number of decompositions of the acyclic digraph G is n
Y
v=1
X
i⩾0
in(v)
i
out(v)
i
i! ! . 2.1
On general walk and path decompositions
For a digraph G = (V, E), define flux(v) := in(v) −out(v), v ∈V,
V + := {v ∈V | flux(v) > 0},
V −:= {v ∈V | flux(v) < 0} 2.1
On general walk and path decompositions 2.1
On general walk and path decompositions 3
G-Stirling functions We use the following notation for multisets: A −X is a difference, e.g. {13, 22, 3, 43} −
{12, 2, 4} = {1, 2, 3, 42}; A ⊎X is a merge, e.g. {12, 2, 42} ⊎{1, 22, 3} = {13, 23, 3, 42}. We
also write G −e if edge e is eliminated from G or G −B if block B is removed. We use the following notation for multisets: A −X is a difference, e.g. {13, 22, 3, 43} −
{12, 2, 4} = {1, 2, 3, 42}; A ⊎X is a merge, e.g. {12, 2, 42} ⊎{1, 22, 3} = {13, 23, 3, 42}. We
also write G −e if edge e is eliminated from G or G −B if block B is removed. 5 the electronic journal of combinatorics 22(4) (2015), #P4.10 For a given digraph G, let Vout := {1out(1), . . . , nout(n)},
Vin := {1in(1), . . . , nin(n)},
Mout(G) := {I | I ⊆Vout}, Vout := {1out(1), . . . , nout(n)},
Vin := {1in(1), . . . , nin(n)}, Mout(G) := {I | I ⊆Vout}, i.e., Mout is the set of all sub(multi)sets of Vout. Note that if for a k-decomposition, we have the sources I, then the corresponding
sinks J = Vin ⊎I −Vout are determined uniquely. (Further, for any sources I we will just
write sinks as J meaning that J = Vin ⊎I −Vout.) )
fine the G-Stirling function SG : Mout(G) →Z⩾0 as follows )
Define the G-Stirling function SG : Mout(G) →Z⩾0 as follows SG(I) := the number of principal decompositions of G with sources I. If n = 1, then SG(I) corresponds to Stirling number of the second kind S(m, k) where
|I| = k and digraph G has m labeled loops (1, 1). Proposition 3.1. If I = {1ℓ1, . . . , nℓn}, then Proposition 3.1. If I = {1ℓ1, . . . , nℓn}, then Proposition 3.1. If I = {1ℓ1, . . . , nℓn}, then SG(I) =
n
Y
i=1
Si(out(i) −ℓi),
(1) (1) (1) where Si(k) the number of matchings in M(i) of size k. Proof. Since Si(k) the number of matchings in M(i) of size k, the vertex i is unmatched
out(i) −k times. Thus, from the bijection of Proposition 2.1, the vertex i is used exactly
out(i) −ℓi times as a source. 3
G-Stirling functions Therefore, we get the formula by considering this argument
for every vertex i = 1, . . . , n and using Proposition 2.1. Theorem 3.2 ([15]). The G-Stirling function SG satisfies the following properties: (i) SG(Vout) = 1; (ii) if SG(I) > 0 for some I ⊂Vout, then for any I′, such that Vout ⊇I′ ⊃I, we have
SG(I′) > 0; (ii) if SG(I) > 0 for some I ⊂Vout, then for any I′, such that Vout ⊇I′ ⊃I, we have
SG(I′) > 0; (iii) Suppose that digraph G is built up from blocks B1, . . . , Bm so that the indices of
edges increase with respect to the order of blocks. Let e = (i, j) ∈Bm, G′ = G −e,
I′ = I −{i}. Let ki be the number of repetitions of i in (J −{j}) ⊎{i} and re be
the number of edges in Bm −e that end by i. Then the following recurrence relation
holds for SG(I). (iii) Suppose that digraph G is built up from blocks B1, . . . , Bm so that the indices of
edges increase with respect to the order of blocks. Let e = (i, j) ∈Bm, G′ = G −e,
I′ = I −{i}. Let ki be the number of repetitions of i in (J −{j}) ⊎{i} and re be
the number of edges in Bm −e that end by i. Then the following recurrence relation
holds for SG(I). SG(I) = SG′(I′) + (ki −re)SG′(I). (2) (2) Corollary 3.3. If G has edges e1, . . . , em (without blocks), then for em = (i, j), G′ =
G −em, I′ = I −{i} and ki the number of repetitions of i in (J −{j}) ⊎{i}, we have Corollary 3.3. If G has edges e1, . . . , em (without blocks), then for em = (i, j), G′ =
G −em, I′ = I −{i} and ki the number of repetitions of i in (J −{j}) ⊎{i}, we have SG(I) = SG′(I′) + kiSG′(I). (3) (3) 6 the electronic journal of combinatorics 22(4) (2015), #P4.10 6 Corollary 3.4. If G has n = 1 vertex and m loops (1, 1), then SG(I) = S(m, k) if |I| = k,
where S(m, k) is Stirling number of the second kind. Relation (3) becomes the well-known
recurrence Corollary 3.4. 3
G-Stirling functions If G has n = 1 vertex and m loops (1, 1), then SG(I) = S(m, k) if |I| = k,
where S(m, k) is Stirling number of the second kind. Relation (3) becomes the well-known
recurrence S(m, k) = S(m −1, k −1) + kS(m −1, k). Remark 3.5. The G-Stirling function SG(I) is a graph generalization of Stirling number
of the second kind. Note that SG is different from Stirling (and Bell) numbers for graphs
studied in [14], which count partitions of graph vertex set into independent sets. Although,
for n = 1 (and several blocks) there is a correspondence between these definitions as noted
in section 6. In fact, Mout(G) can be considered as a poset ordered by inclusion. Let us consider a
subdomain of Mout(G) at which SG takes positive values; define the poset PG := {I | SG(I) > 0},
(4) (4) whose elements (multisets) are ordered by inclusion. Proposition 3.6. Let m(v) = |M(v)| be the size of a maximal matching. The Proposition 3.6. Let m(v) = |M(v)| be the size of a maximal matching. Then (i) PG has a unique maximal element Vout, unique minimal element V0, where V0 = Vout −{1m(1), . . . , nm(n)}. V0 = Vout −{1m(1), . . . , nm(n)}. (ii) PG is isomorphic to m(1) × · · · × m(n). (ii) PG is isomorphic to m(1) × · · · × m(n). Note that PG is isomorphic to a divisibility poset, and µ(I, J) = (−1)|J−I| if J −I is a
set and 0 if J −I is a multiset. Hence we get Note that PG is isomorphic to a divisibility poset, and µ(I, J) = (−1)|J−I| if J −I is a
set and 0 if J −I is a multiset. Hence we get SG(J) =
X
I⊆[n]
(−1)|J−I|BG(J −I) and the kind of recurrence and the kind of recurrence BG = 1 −
X
∅⊂I⊆[n]
(−1)|Vout−I|BG(Vout −I). (ii) PG is isomorphic to m(1) × · · · × m(n). Here by m we denote the chain poset of m elements and m × s is the poset of ms
elements defined as (cartesian) product of posets m, s (if s is empty, then put m×s = m). (See e.g. [29]) Proof. First, from (i), (ii) of Theorem 3.2, Vout is a unique maximal element and if V0 is
some minimal element, then for all V0 ⊆I ⊆Vout. Let us prove that V0 = {1out(1)−m(1), . . . , nout(n)−m(n)} V0 = {1out(1)−m(1), . . . , nout(n)−m(n)} is a unique minimal element. From Theorem 2.1, there is a principal decomposition with
sources V0, so V0 ∈PG. If there is another minimal element V ′, then iout(i)−m(i) ̸∈V ′ for
some i. This means that the vertex i has a matching of size greater than m(i), which is
impossible. So, both items (i), (ii) clearly imply from these arguments. is a unique minimal element. From Theorem 2.1, there is a principal decomposition with
sources V0, so V0 ∈PG. If there is another minimal element V ′, then iout(i)−m(i) ̸∈V ′ for
some i. This means that the vertex i has a matching of size greater than m(i), which is
impossible. So, both items (i), (ii) clearly imply from these arguments. Corollary 3.7. If G is a cycle graph with edge labels given by 1 →e1 2 →e2 · · · →en−1 n →en 1, then m(1) = 0, and m(i) = 2 for i = 2, . . . , n. Hence, PG is isomorphic to 2 × · · · × 2 =
2[n−1] then m(1) = 0, and m(i) = 2 for i = 2, . . . , n. Hence, PG is isomorphic to 2 × · · · × 2 =
2[n−1]. 7 the electronic journal of combinatorics 22(4) (2015), #P4.10 7 The total number of principal decompositions |PD(G)| serves as a generalized Bell
number. Let BG = |PD(G)|, then we have BG =
X
V0⊆I⊆Vout
SG(I). (5) (5) If we now define an extension BG(J) as BG(J) :=
X
V0⊆I⊆J
SG(I),
(6) (6) then applying the M¨obius inversion formula on the poset PG we get SG(J) =
X
I⊆J
µ(I, J)BG(I) and in particular SG(Vout) = 1 =
X
I⊆Vout
µ(I, J)BG(I). 4
Normal ordering in the Weyl algebra Let K be a field of characteristic 0. The n-th Weyl algebra An is an associative algebra
over K defined by 2n generators x1, . . . , xn, ∂1, . . . , ∂n and relations [xi, xj] = [∂i, ∂j] = 0, [∂i, xj] = δi,j for 1 ⩽i, j ⩽n, where [a, b] = ab −ba is the commutator and δi,j is the Kronecker symbol. The typical
example of An is the polynomial algebra with ∂i considered as partial derivations d/dxi. The elements of types xα∂β := xα1
1 · · · xαn
n ∂β1
1 · · · ∂βn
n with α = (α1, . . . , αn), β = (β1, . . . , βn) ∈Zn
⩾0 are called monomials. We will also write
monomials in the equivalent form xi1 . . . xis∂j1 . . . ∂jp. All monomials xα∂β form a linear
vector space basis of An. When the element w of An is expressed as a linear combination w =
X
α,β
c(α, β)xα∂β,
c(α, β) ∈K, the electronic journal of combinatorics 22(4) (2015), #P4.10 the electronic journal of combinatorics 22(4) (2015), #P4.10 8 we say that w is normally ordered. Define the following subspaces of An: A(p,q)
n
:= ⟨xα∂β : |α| = p, |β| = q⟩,
A(0)
n :=
M
i⩾1
A(i,i)
n
, where |α| = Pn
i=1 αi. Note that A(0)
n
is the subalgebra of An. We show that combinatorial meaning of coefficients in the normal ordering can be in-
terpreted in terms of graph decompositions. Furthermore, we will consider the monomials
of subspace A(0)
n
(otherwise, we may add fictive elements as shown in subsection 6.1). We
associate every monomial w = xi1 . . . xip∂j1 . . . ∂jp ∈A(0)
n
with the p-block of a graph in
the following way: block(w) := {(i1, j1), . . . , (ip, jp)}. block(w) := {(i1, j1), . . . , (ip, jp)}. Theorem 4.1 ([15]). Let w1, . . . , wm ∈A(0)
n
be monomials. Then we have Theorem 4.1 ([15]). Let w1, . . . , wm ∈A(0)
n
be monomials. Then we have w1 · · · wm =
X
I⊆Vout
SG(I)
Y
i∈I
xi
Y
j∈J
∂j,
(7) (7) where G = ([n], E) with E = (block(w1), . . . , block(wm)) (i.e. where
m
m1,...,mk
=
m!
m1!···mk! is a multinomial coefficient. Proof. From monomials x1∂2, x2∂3, . . . , xm−1∂m, xm∂m+1, the digraph G is a single path
1 →2 →· · · →m →m + 1. Taking all permutations σ ∈Sm means that we permute
the edges of G up to σ. Consider decompositions into increasing walks in that case. Suppose that we break the paths at vertices 2 ⩽i1 < · · · < ik ⩽m. Then the number
of permutations σ ∈Sm for which the fragments 1 →· · · →i1/i1 →· · · →i2/ · · · /ik →
· · · →m+1 are all increasing, is clearly equal to
m
i1−1,i2−i1,...,m+1−ik
and the corresponding
monomial is x1xi1 · · · xik∂i1 · · · ∂ik∂m+1 and so we obtain the formula. Remark 4.6. Note that the last formula is computationally effective; we compute 2m−1
terms instead of calculating s+
m with m! summands. 4
Normal ordering in the Weyl algebra the indices of edges increase
with respect to the order of blocks) and J = Vin ⊎I −Vout. Corollary 4.2. For a digraph G = ([n], E) with E = (e1, . . . , em), we have Corollary 4.2. For a digraph G = ([n], E) with E = (e1, . . . , em), we have −→
m
Y
ℓ=1
xiℓ∂jℓ=
X
I
SG(I)
Y
i∈I
xi
Y
j∈J
∂j,
(8) (8) where eℓ= (iℓ, jℓ) and the sum runs over all (multi)sets of sources I. where eℓ= (iℓ, jℓ) and the sum runs over all (multi)sets of sources I. Corollary 4.3. If n = 1, then (8) gives the well-known formula (x∂)m =
m
X
i=0
S(m, i)xi∂i. where S(m, i) is Stirling number of the second kind. where S(m, i) is Stirling number of the second kind. Analogous to formula (8), the operators xi∂j + xj∂i give a decomposition formula for
undirected graphs. Namely, we obtain the following result. Theorem 4.4. For an undirected graph G = ([n], E) with E = (e1, . . . , em), eℓ= (iℓ, jℓ),
we have
−→ m
Y
ℓ=1
(xiℓ∂jℓ+ xjℓ∂iℓ) =
X
decompositions into increasing
walks with sources I
Y
i∈I
xi
Y
j∈J
∂j. (9) (9) the electronic journal of combinatorics 22(4) (2015), #P4.10 9 The normal ordering decomposition formulas like (8), (9) are in particular useful when
we sum through all decompositions of G or symmetrize over all permutations of the edge
set. For w1, . . . , wm ∈An, let s+
m(w1, . . . , wm) :=
X
σ∈Sm
wσ(1) · · · wσ(m). s+
m(w1, . . . , wm) :=
X
σ∈Sm
wσ(1) · · · wσ(m). Then for undirected graph G we have Then for undirected graph G we have s+
m(xi1∂j1+xj1∂i1, . . . , xim∂jm+xjm∂im) = (# Euler tours i →j) xi∂j+higher order s+
m(xi1∂j1+xj1∂i1, . . . , xim∂jm+xjm∂im) = (# Euler tours i →j) xi∂j+higher order terms. Consider a case of computing the sum s+
m. Proposition 4.5. s+
m(x1∂2, x2∂3, . . . , xm−1∂m, xm∂m+1) s+
m(x1∂2, x2∂3, . . . , xm−1∂m, xm∂m+1)
= x1
X
2⩽i1<···<ik⩽m
m
i1 −1, i2 −i1, . . . , m + 1 −ik
k
Y
ℓ=1
xiℓ
k
Y
ℓ=1
∂iℓ
! ∂m+1, = x1
X
2⩽i1<···<ik⩽m
m
i1 −1, i2 −i1, . . . , m + 1 −ik
k
Y
ℓ=1
xiℓ
k
Y
ℓ=1
∂iℓ
! ∂m+1, where
m
m1,...,mk
=
m! m1!···mk! is a multinomial coefficient. 5
Cyclic multigraphs and residue alternating partitions Consider the digraph Cm,n with n vertices and m edges (m > n) that consecutively form
a cycle (12 · · · n), i.e. the edges {e1, . . . , em} in order are e1 = (1, 2), e2 = (2, 3), . . . , en = (n, 1), en+1 = (1, 2), . . . The principal decompositions on such graph correspond to the following type of set parti-
tions. Say that the set partition ∪Xi = [m] is residue alternating if for every i (1 ⩽i ⩽k)
and Xi = {a1, . . . , ar} with a1 < · · · < ar, we have ai+1 −ai ≡1 (mod n) for i = 1, . . . , r
(ar+1 = a1). Let A(m, n) be the number of residue alternating partitions (or the number of decom-
positions of digraph Cm,n). 10 10 the electronic journal of combinatorics 22(4) (2015), #P4.10 Theorem 5.1. The following formulas hold for A(m, n) Theorem 5.1. The following formulas hold for A(m, n) A(nk, n) = Bk(Bk+1)n−1 and for 0 < r < n A(nk + r, n) = (Bk+1)n−r+1(Bk+2)r−1, where Bk is the Bell number, the number of partitions of set [k]. where Bk is the Bell number, the number of partitions of set [k]. Proof. First we examine the approach using composition of differential operators. Note
that A(m, n) corresponds to the number BG = P
I SG(I) (see def. (5)) of all principal
decompositions of G = Cm,n. Therefore, we can use the normal ordering expansion of
differential operators to calculate A(m, n) = BG. (
)
If m = nk, then the composition of operators is (x1∂2 · · · xn∂1) · · · (x1∂2 · · · xn∂1) = (x1∂2 · · · xn∂1)k = (x1∂1)k(∂2x2)k · · · (∂nxn)k. In fact, A(m, n) is the sum of coefficients in the normal ordering expansion of the last
expression. The sum of coefficients in expansion (x1∂1)k is In fact, A(m, n) is the sum of coefficients in the normal ordering expansion of the last
expression. 5
Cyclic multigraphs and residue alternating partitions Construct set partition of [k + 1] as follows: (1) if (ean+i−1, ebn+i) ∈M, where a ⩽b, then put a + 1, b + 2 in the same block (1) if (ean+i−1, ebn+i) ∈M, where a ⩽b, then put a + 1, b + 2 in the same block; (2) the remaining elements of [k + 1] (that were not considered yet), put in separate
blocks. For example, if k = 5, In(i) = {e1, e3, e5, e7, e9}, Out(i) = {e2, e4, e6, e8, e10},
M = {(e1, e6), (e3, e4), (e5, e8)}, then (e1, e6) means that we should put 1, 4 in the same block; for (e3, e4) the elements 2, 3
are in the same block; and for (e5, e8) the elements 3, 5 are in the same block. Therefore,
we have the partition {1, 4}{2, 3, 5}{6}. then (e1, e6) means that we should put 1, 4 in the same block; for (e3, e4) the elements 2, 3
are in the same block; and for (e5, e8) the elements 3, 5 are in the same block. Therefore,
we have the partition {1, 4}{2, 3, 5}{6}. The inverse procedure can be described as follows: take any block of the partition,
{a1 < · · · < ar} and for every j = 1, . . . , r −1 match the edges (e(aj−1)n+i−1, e(aj+1−2)n+i). One can see that this properly defines the bijection. By applying a similar argument one can show that |M(1)| = Bk and therefore, by
Theorem 2.1, A(nk, n) = Bk(Bk+1)n−1. The formula for A(nk + r, n) implies analogously. 5
Cyclic multigraphs and residue alternating partitions The sum of coefficients in expansion (x1∂1)k is k
X
i=0
S(k, i) = Bk k
X
i=0
S(k, i) = Bk k
X
i=0
S(k, i) = Bk and the sum of coefficients in the normal ordering of (∂ℓxℓ)k is k
X
i=0
k
i
Bi, k
X
i=0
k
i
Bi, since since
(∂ℓxℓ)k = (1 + xℓ∂ℓ)k =
k
X
i=0
k
i
(xℓ∂ℓ)i. (∂ℓxℓ)k = (1 + xℓ∂ℓ)k =
k
X
i=0
k
i
(xℓ∂ℓ)i. It remains to use the well-known recurrence for Bell numbers Bk+1 =
k
X
i=0
k
i
Bi. Bk+1 =
k
X
i=0
k
i
Bi. So, the sum of coefficients A(nk, n) in the normal ordering of (x1∂1)k(∂2x2)k · · · (∂nxn)k is
Bk(Bk+1)n−1. So, the sum of coefficients A(nk, n) in the normal ordering of (x1∂1)k(∂2x2)k · · · (∂nxn)k is
Bk(Bk+1)n−1. Similarly, if m = nk + r we have the composition So, the sum of coefficients A(nk, n) in the normal ordering of (x1∂1)k(∂2x2)k · · · (∂nxn)k is
Bk(Bk+1)n−1. k(
k+1)
Similarly, if m = nk + r we have the composition +1)
ilarly, if m = nk + r we have the composition (
+ )
Similarly, if m = nk + r we have the composition (x1∂2 · · · xn∂1) · · · (x1∂2 · · · xn∂1)x1∂2 · · · xr∂r+1
= x1(∂1x1)k
(∂2x2)k+1 · · · (∂rxr)k+1
(∂r+1xr+1)k∂r+1
(∂r+2xr+2)k · · · (∂nxn)k
and by the same argument it follows that A(nk + r, n) = (Bk+1)n−r+1(Bk+2)r−1. the electronic journal of combinatorics 22(4) (2015), #P4.10
11 the electronic journal of combinatorics 22(4) (2015), #P4.10 11 Bijective proof. Using Corollary 2.2 of Theorem 2.1 we look at matchings of edges at every
vertex. Suppose m = nk and consider the vertex i, where 2 ⩽i ⩽n. We have In(i) = {ei−1, en+i−1, e2n+i−1, . . . , e(k−1)n+i−1},
Out(i) = {ei, en+i, e2n+i, . . . , e(k−1)n+i}. In(i) = {ei−1, en+i−1, e2n+i−1, . . . , e(k−1)n+i−1},
Out(i) = {ei, en+i, e2n+i, . . . , e(k−1)n+i}. We will prove that |M(i)| = Bk+1 by establishing a bijection between matchings in M(i)
and partitions of set [k + 1]. Let M ∈M(i) be any matching between In(i), Out(i). the electronic journal of combinatorics 22(4) (2015), #P4.10 Proposition 5.3. Recurrence relations for a(m, k, i) are given by Proposition 5.3. Recurrence relations for a(m, k, i) ar ition 5.3. Recurrence relations for a(m, k, i) are given by
a(1, 1, 0) = 1, a(m, k, i) = 0 for m < k or k < i,
a(2m + 1, k, i) = a(2m, k −1, i) + (i + 1)a(2m, k, i + 1),
a(2m, k, i) = a(2m −1, k −1, i −1) + (k −i + 1)a(2m −1, k, i −1). Proof. Consider parity alternating partitions of set [2m + 1] into k blocks, i of whose
maximal elements are even. The element 2m + 1 can form a separate block contributing
a(2m, k −1, i) ways. Otherwise, 2m + 1 can be placed into blocks of parity alternating
partitions of [2m] having k blocks and (i + 1) maximal even elements (since 2m + 1 will
change the parity of one maximal element); this gives (i + 1)a(2m, k, i + 1) ways. Similarly, if we look at partitions of [2m], then 2m can form a separate block in
a(2m−1, k−1, i−1) ways and can be placed into other blocks in (k−i+1)a(2m−1, k, i−1)
ways. 5.1
Parity alternating partitions For n = 2 we have the graph model with 2 vertices and n edges {e1, . . . , en} such that all
odd-indexed e2i−1 are of type (1, 2) and all even-indexed e2i are of type (2, 1). All principal decompositions on this graph can be considered as partitions X1∪· · ·∪Xk
of [m] that have the following property: For every i(1 ⩽i ⩽k) and Xi = {a1, . . . , ar}
with a1 < · · · < ar, the sequence a1, . . . , ar is parity alternating (i.e. even, odd, even, odd,
etc. or similarly beginning with odd). Let us call such partitions of sets parity alternating
partitions. For example, the parity alternating partitions of {1, 2, 3, 4} into two blocks are {1}{2, 3, 4};
{4}{1, 2, 3};
{1, 2}{3, 4};
{1, 4}{2, 3}. Denote by a(m) = A(m, 2) the total number of parity alternating partitions of [m]. Then the following formulas hold Denote by a(m) = A(m, 2) the total number of parity alternating partitions of [m]. Then the following formulas hold a(2k) = BkBk+1,
a(2k + 1) = B2
k+1. the electronic journal of combinatorics 22(4) (2015), #P4.10 12 the electronic journal of combinatorics 22(4) (2015), #P4.10 Remark 5.2. The latter formulas mean that the number of parity alternating partitions of
[m] is equal to the number of partitions of [m + 1] where elements in each block have the
same parity. (The second sequence appears in OEIS, A124419 [28]). Bijectively, this fact
can be described as follows: x, y are successive elements in the block of parity alternating
partitions iffx, y + 1 are successive elements of the second type of partitions. Note that
such reduction algorithm was applied to regular (noncrossing) set partitions in [6]. The values of a(m) can also be computed by the number of blocks. Let a(m, k) be
the number of parity alternating partitions into k blocks and a(m, k, i) the number of
parity alternating partitions into k blocks i of which have even maximal elements. We
have a(m, k) = Pk
i=0 a(m, k, i), a(m) = Pn
k=1 a(m, k). 5.1
Parity alternating partitions m\k
1
2
3
4
5
6
7
8
a(m)
1
1
1
2
1
1
2
3
1
2
1
4
4
1
4
4
1
10
5
1
6
11
6
1
25
6
1
10
28
26
9
1
75
7
1
14
61
86
50
12
1
225
8
1
22
136
276
236
92
16
1
780
Table 1: Small values of a(m) and a(m, k). Table 1: Small values of a(m) and a(m, k). 6.1
Normal ordering for n = 1. Combinatorial interpretations of coefficients in the normal ordering expansion xr1∂s1 · · · xrt∂st =
X
i
SG(i)xi∂j
(10) (10) 13 the electronic journal of combinatorics 22(4) (2015), #P4.10 the electronic journal of combinatorics 22(4) (2015), #P4.10 can be extracted from Theorem 4.1 as follows. The elements xri∂si are not from A(0)
1
=
⟨xi∂i : i = 0, 1, . . .⟩in general (i.e. if ri ̸= si). To deal with this situation, we add fictive
|ri −si| new variables xi+1 or ∂i+1 so that the monomial will belong to A(0)
1 , and the graph
scheme can be applied. For example, x2∂5 is transformed to x3
2x2∂5. Note that the new
variables commute with all other and so we can freely move them in the normal ordering
expansion. Using these new monomials, we construct the graph G according to the rules
above. Thus, combinatorial meaning of SG(i) can be described as the number of principal
decompositions of G having i sources at vertex 1. This interpretation is similar to the
graph models studied in [3] (the model there is acyclic which is different to ours since for
n = 1 we have loops). Remark 6.1. In fact, the normal ordering in the n-th Weyl algebra can be computed using
the n = 1 case. For instance, we can restructure compositions as follows x1x2∂1∂3x2x3∂1∂3x1∂2 = (x1∂2
1x1)(x2
2∂2)(∂3x3∂3). This view also helps to refine all possible multisets of sources and sinks I, J, since what
coefficients are nonzero in every composition like (10) can be found. This view also helps to refine all possible multisets of sources and sinks I, J, since what
coefficients are nonzero in every composition like (10) can be found. 6.2
The λ-Stirling numbers • Cλ(n, k) is the number of permutations of [n] with k cycles such that non-minimal
elements of the first λ1 cycles are greater than all minimal elements of these λ1
cycles; non-minimal elements of the next λ2 cycles are greater than all minimal
elements of these λ2 cycles, and so on; and the remaining rk cycles are singletons
(i.e. consist of one element). • Cλ(n, k) is the number of permutations of [n] with k cycles such that non-minimal
elements of the first λ1 cycles are greater than all minimal elements of these λ1
cycles; non-minimal elements of the next λ2 cycles are greater than all minimal
elements of these λ2 cycles, and so on; and the remaining rk cycles are singletons
(i.e. consist of one element). By definition, the sequence λ affects on the first argument n in Sλ(n, k) and on the
second argument k in Cλ(n, k). Further we will see that Sλ(n, k), Cλ(n, k) are dual to
each other. Remark 6.2. For permutation σ of [n] having k cycles we associate representation in
the form σ = (σ(1)) · · · (σ(k)), where σ(1), . . . , σ(k) are cycles written in increasing order
of their minimal elements. For example, the permutation (5, 6, 1, 7, 3, 2, 4) is written as
(1, 5, 3)(2, 6)(4, 7). So we divide the set [n] according to the integer partition n = λ1 + . . . + λq + r: So we divide the set [n] according to the integer partition n = λ1 + . . . + λq + r:
[n] = {1, . . . , λ1
|
{z
}
1st part
, . . . , λ1 + · · · + λq−1 + 1, . . . , λ1 + · · · + λq
|
{z
}
q-th part
, n −λq + 1, . . . , n
|
{z
}
last r elements
} [n] = {1, . . . , λ1
|
{z
}
1st part
, . . . , λ1 + · · · + λq−1 + 1, . . . , λ1 + · · · + λq
|
{z
}
q-th part
, n −λq + 1, . . . 6.2
The λ-Stirling numbers Consider the graph with n = 1 vertex and suppose it is built up from blocks of loops
(1, 1) of λ1, λ2, . . . edge sizes. Principal decompositions on this model require that the
edges within one block cannot lie on the same walk. This setting clearly corresponds
to partitions of the set [m] (m is a total number of edges), where the first λ1 elements
are in distinct subsets, the next λ2 elements are also in distinct subsets, and so on. The
coefficients SG(I) present a generalization of Stirling numbers of the second kind on such
restricted partitions. In this section we study these generalized Stirling numbers. We also
introduce a generalization of Stirling numbers of the first kind, which can be considered
as dual to the second. We call these numbers the λ-Stirling numbers. Fix the sequence λ0 = 0,
λ = (λ1, λ2, . . .),
λ1 ⩾λ2 ⩾· · · of nonnegative integers, and let of nonnegative integers, and let q = qn := max{i | λ0 + · · · + λi ⩽n},
r = rn := n −λ0 −· · · −λq. q = qn := max{i | λ0 + · · · + λi ⩽n}, r = rn := n −λ0 −· · · −λq. So λ is a kind of ‘infinite’ integer partition, q is the analog of quotient and r is the analog
of remainder. So λ is a kind of ‘infinite’ integer partition, q is the analog of quotient and r is the analog
of remainder. Definition. The λ-Stirling numbers of second and first kinds Sλ(n, k), Cλ(n, k) are defined
as follows: the electronic journal of combinatorics 22(4) (2015), #P4.10 14 • Sλ(n, k) is the number of partitions of [n] into k blocks such that the first λ1 elements
of [n] are in distinct blocks, the next λ2 elements are in distinct blocks, and so on;
the remaining rn elements are also in distinct blocks. • Sλ(n, k) is the number of partitions of [n] into k blocks such that the first λ1 elements
of [n] are in distinct blocks, the next λ2 elements are in distinct blocks, and so on;
the remaining rn elements are also in distinct blocks. 6.2
The λ-Stirling numbers , n
|
{z
}
last r elements
} and consider only those set partitions which restrict the elements of the same part to be
in the same subset. and consider only those set partitions which restrict the elements of the same part to be
in the same subset. For the case of permutations, we take the partition k = λ1 +. . .+λqk +rk and consider
permutations of the following type: σ(1), . . . , σ(λ1)
|
{z
}
∀non-min > ∀min
| · · · | σ(λ1+···+λqk−1+1), . . . , σ(λ1+···+λqk)
|
{z
}
∀non-min > ∀min
| σ(k−rk+1), . . . , σ(k)
|
{z
}
last rk singletons {z
∀non-min > ∀min {z
∀non-min > ∀min Consider examples. If λ = (3, 2, 1, 1, . . 6.2
The λ-Stirling numbers .), then Sλ(6, 4) = 30 and allowed configurations
of partitions of {1, 2, 3, 4, 5, 6} into 4 blocks can be described as follows: • If 1, 2, 3, 4 are blocks minima; then 5 can be put with 1, 2, 3 (except restriction with
4) and 6 with 1, 2, 3, 4 which totally gives 3 × 4 = 12 ways; If 1, 2, 3, 4 are blocks minima; then 5 can be put with 1, 2, 3 (except restriction wit
4) and 6 with 1 2 3 4 which totally gives 3 × 4 = 12 ways; • If 1, 2, 3, 4 are blocks minima; then 5 can be put with 1, 2, 3 (except restriction with
4) and 6 with 1, 2, 3, 4 which totally gives 3 × 4 = 12 ways; • If 1, 2, 3, 5 are blocks minima; then 4 can be put with 1, 2, 3 and 6 with 1, 2, 3, 4
which totally gives 3 × 4 = 12 ways; • If 1, 2, 3, 5 are blocks minima; then 4 can be put with 1, 2, 3 and 6 with 1, 2, 3, 4
which totally gives 3 × 4 = 12 ways; • If 1, 2, 3, 6 are blocks minima; then 4 can be put with 1, 2, 3 and 5 with 1, 2, 3 except
the block with 4, which totally gives 3 × 2 = 6 ways; • If 1, 2, 3, 6 are blocks minima; then 4 can be put with 1, 2, 3 and 5 with 1, 2, 3 except
the block with 4, which totally gives 3 × 2 = 6 ways; • If 1, 2, 3, 6 are blocks minima; then 4 can be put with 1, 2, 3 and 5 with 1, 2, 3 except
the block with 4, which totally gives 3 × 2 = 6 ways; So, there are totally 12 + 12 + 6 = 30 ways to arrange desired partitions. Cλ(6, 4) = 36 and configurations of permutations of (1, . . . , 6) with 4 cycles can be
described as follows: So, there are totally 12 + 12 + 6 = 30 ways to arrange desired partitions. Cλ(6, 4) = 36 and configurations of permutations of (1, . . . 6.2
The λ-Stirling numbers , 6) with 4 cycles can be
described as follows:
(
h
ld
l
b
l
d
l
b
l
) So, there are totally 12 + 12 + 6 = 30 ways to arrange desired partitions. Cλ(6, 4) = 36 and configurations of permutations of (1, . . . , 6) with 4 cycles can be
described as follows: 6, 4) = 36 and configurations of permutations of (1, . . . , 6) with 4 cycles can be
ed as follows: (1, 2, 3 should always be cycle minima; and cycle number 4 is singleton) 15 the electronic journal of combinatorics 22(4) (2015), #P4.10 • If 1, 2, 3, 4 are cycle minima; then 5 can be put with 1, 2, 3 and 6 can be put after
1, 2, 3, 5, which gives 3 × 4 = 12 ways; • If 1, 2, 3, 4 are cycle minima; then 5 can be put with 1, 2, 3 and 6 can be put after
1, 2, 3, 5, which gives 3 × 4 = 12 ways; • If 1, 2, 3, 5 are cycle minima; then 4 can be put with 1, 2, 3 and 6 can be put after
1, 2, 3, 4, which gives 3 × 4 = 12 ways; • If 1, 2, 3, 5 are cycle minima; then 4 can be put with 1, 2, 3 and 6 can be put after
1, 2, 3, 4, which gives 3 × 4 = 12 ways; • If 1, 2, 3, 6 are cycle minima; then 5 can be put with 1, 2, 3 and 4 can be put after
1, 2, 3, 5, which gives 3 × 4 = 12 ways. • If 1, 2, 3, 6 are cycle minima; then 5 can be put with 1, 2, 3 and 4 can be put after
1, 2, 3, 5, which gives 3 × 4 = 12 ways. So, there are totally 12 + 12 + 12 = 36 possible permutations with 4 cycles. So, there are totally 12 + 12 + 12 = 36 possible permutations with 4 cycles. Clearly if λ = (1, 1, . . .), then Cλ(n, k), Sλ(n, k) are just the usual Stirling numbers. The case λ = (r, 1, 1, . . .) corresponds to the r-Stirling numbers of first and second kinds
introduced in [5]. 6.2
The λ-Stirling numbers Generalized Stirling numbers of the second kind Sr,s(n, k) that arise
from the expansion xr1∂s1 · · · xrn∂sn = x
P ri−di X
k
Sr,s(n, k)xk∂k. k have been studied in the bosons normal ordering problem [4, 24] (see also [12]). Our
definition gives a natural (and simple) combinatorial interpretation to the case Sr,r(n, k)
and also solves an inverse problem, where Stirling numbers of the first kind arise. Note
that the general formulas for Sr,s(n, k) given in [24] can be used to compute our numbers
SG, BG (defined in Section 2). Interpretation to Sr,r(n, k) with colorings of complete
graphs introduced in [7] is very close to the meaning of Sλ(n, k) (λ = (r, r, . . .)), since
any r elements that cannot be in the same subset can be viewed as proper colorings of a
component complete graph Kr; on the other hand, this definition corresponds to Stirling
numbers for graphs in the sense of counting partitions of a vertex set into independent sets
(see [14]). The numbers Sλ(n, k) were introduced in [25] as (r1, . . . , rp)-Stirling numbers
of the second kind. In order to be consistent with the corresponding Stirling numbers
of the first kind Cλ(n, k), we define these numbers over a general sequence λ relating
it with integer partitions, since it somehow acts (as integer partition) on first (or resp. second) argument of these Stirling numbers. In fact, by polynomial relations (18), (19)
shown below, the λ-Stirling numbers correspond to a case of the multiparameter non-
central Stirling numbers introduced in [11]. This also leads to a case of a general study
of connection constants between persistent sequences of polynomials [10]. [
]
The numbers Sλ(n, k) also related to compositions (ordered partitions) of multisets in
the following way. For n = λ1 + · · · + λq + r consider a multiset n := {1λ1, . . . , qλq, (q + 1)r}. n := {1λ1, . . . , qλq, (q + 1)r}. Then the number of ways to distribute the elements of n into k ordered nonempty sets is
equal to Then the number of ways to distribute the elements of n into k ordered nonempty sets is
equal to Sλ(n, k)k! λ1! · · · λq!r!. Sλ(n, k)k! λ1! · · · λq!r!. 6.2
The λ-Stirling numbers .)
n\k
2
3
4
5
6
2
1
3
2
1
4
2
2
1
5
4
14
8
1
6
4
32
38
12
1
n\k
2
3
4
5
6
2
1
3
2
1
4
6
4
1
5
24
18
8
1
6
120
96
58
12
1
Sλ(n, k) for 2 ⩽n, k ⩽6
Cλ(n, k) for 2 ⩽n, k ⩽6. λ = (3, 2, 1, . . .)
n\k
3
4
5
6
3
1
4
3
1
5
6
6
1
6
18
30
11
1
n\k
3
4
5
6
3
1
4
3
1
5
12
6
1
6
60
36
11
1
Sλ(n, k) for 3 ⩽n, k ⩽6
Cλ(n, k) for 3 ⩽n, k ⩽6. Table 2: Some tables for Sλ(n, k), Cλ(n, k).1 Table 2: Some tables for Sλ(n, k), Cλ(n, k).1 6.3
Main properties 1Sλ(n, k) = Cλ(n, k) = 0 if k < λ1 or k > n. If λ = (1, 1, . . .) or λ = (r, 1, 1, . . .) then the λ-Stirling
numbers are referred to the ordinary Stirling numbers (A008275, A008277, A048994 in OEIS) or to
the r-Stirling numbers (A143494, A143495, A143496, A193685, A143491, A143492, A143493 in OEIS
[28]), respectively. Some values of Sλ(n, k) (of second kind) are referred to A078739, A008297, A035342,
A078740, A078741, A090214 in OEIS [28]. 6.2
The λ-Stirling numbers the electronic journal of combinatorics 22(4) (2015), #P4.10
16 16 the electronic journal of combinatorics 22(4) (2015), #P4.10 This easily implies from the combinatorial interpretation of Sλ(n, k). If blocks are ordered,
then we multiply the number of ways by k!. The elements λ1+· · ·+λj−1+1, . . . , λ1+· · ·+λj
(which all in distinct blocks) can be changed to the repetition jλj; that was calculated λj! times. λ = (2, 2, 1, 1, . . .)
n\k
2
3
4
5
6
2
1
3
2
1
4
2
4
1
5
4
14
8
1
6
8
46
46
13
1
n\k
2
3
4
5
6
2
1
3
2
1
4
6
4
1
5
24
18
8
1
6
120
96
58
13
1
Sλ(n, k) for 2 ⩽n, k ⩽6
Cλ(n, k) for 2 ⩽n, k ⩽6. λ = (2, 2, 2, . . .)
n\k
2
3
4
5
6
2
1
3
2
1
4
2
2
1
5
4
14
8
1
6
4
32
38
12
1
n\k
2
3
4
5
6
2
1
3
2
1
4
6
4
1
5
24
18
8
1
6
120
96
58
12
1
Sλ(n, k) for 2 ⩽n, k ⩽6
Cλ(n, k) for 2 ⩽n, k ⩽6. λ = (3, 2, 1, . . .)
n\k
3
4
5
6
3
1
4
3
1
5
6
6
1
6
18
30
11
1
n\k
3
4
5
6
3
1
4
3
1
5
12
6
1
6
60
36
11
1
Sλ(n, k) for 3 ⩽n, k ⩽6
Cλ(n, k) for 3 ⩽n, k ⩽6. Table 2: Some tables for Sλ(n, k), Cλ(n, k).1 λ = (2, 2, 1, 1, . . .) λ = (2, 2, 1, 1, . . .)
n\k
2
3
4
5
6
2
1
3
2
1
4
2
4
1
5
4
14
8
1
6
8
46
46
13
1
n\k
2
3
4
5
6
2
1
3
2
1
4
6
4
1
5
24
18
8
1
6
120
96
58
13
1
Sλ(n, k) for 2 ⩽n, k ⩽6
Cλ(n, k) for 2 ⩽n, k ⩽6. λ = (2, 2, 2, . . 6.3
Main properties Let us define (x)0 := 1;
(x)m := x(x −1) · · · (x −m + 1);
(x)λ
n := (x)λ0 · · · (x)λqn(x)rn. Denote Dℓ:= xℓ∂ℓ, then Denote Dℓ:= xℓ∂ℓ, then Dℓ1Dℓ2 = Dℓ2Dℓ1 Dℓ1Dℓ2 = Dℓ2Dℓ1 or more precisely or more precisely Dℓ1Dℓ2 =
X
i
i! ℓ1
i
ℓ2
i
Dℓ1+ℓ2−i. (11) (11) 1Sλ(n, k) = Cλ(n, k) = 0 if k < λ1 or k > n. If λ = (1, 1, . . .) or λ = (r, 1, 1, . . .) then the λ-Stirling
numbers are referred to the ordinary Stirling numbers (A008275, A008277, A048994 in OEIS) or to
the r-Stirling numbers (A143494, A143495, A143496, A193685, A143491, A143492, A143493 in OEIS
[28]), respectively. Some values of Sλ(n, k) (of second kind) are referred to A078739, A008297, A035342,
A078740, A078741, A090214 in OEIS [28]. the electronic journal of combinatorics 22(4) (2015), #P4.10 17 The last formula gives
Dℓ= Dℓ−1D1 −(ℓ−1)Dℓ−1. (12) Dℓ= Dℓ−1D1 −(ℓ−1)Dℓ−1. (12) (12) Dℓ= Dℓ−1D1 −(ℓ−1)Dℓ−1. Let Dλ
n be the operator defined as Let Dλ
n be the operator defined as Dλ
n := Dλ0 · · · DλqDr. Dλ
n := Dλ0 · · · DλqDr. Similar to equation (12) we can obtain that Similar to equation (12) we can obtain that Similar to equation (12) we can obtain that Dλ
n = Dλ
n−1D1 −rn−1Dλ
n−1. (13) (13) Theorem 6.3. The numbers Sλ(n, k), Cλ(n, k) have the following properties. (i) Recurrence relations Theorem 6.3. The numbers Sλ(n, k), Cλ(n, k) have the following properties. (i) Recurrence relations Sλ(n, k) = Sλ(n −1, k −1) + (k −rn−1)Sλ(n −1, k)
(14)
with Sλ(n, k) = 0 if k < λ1 or k > n; Sλ(n, n) = 1. Cλ(n, k) = Cλ(n −1, k −1) + (n −1 −rk)Cλ(n −1, k)
(15) (14) (15) with Cλ(n, k) = 0 if k < λ1 or k > n; Cλ(n, n) = 1. (ii) Expansions with differential operators with Cλ(n, k) = 0 if k < λ1 or k > n; Cλ(n, n) = 1. (ii) Expansions with differential operators with Cλ(n, k) = 0 if k < λ1 or k > n; Cλ(n, n) = 1. (ii) Expansions with differential operators Dλ
n =
X
k
Sλ(n, k)Dk,
(16)
Dn =
X
k
(−1)n−kCλ(n, k)Dλ
k. 6.3
Main properties (17) Dλ
n =
X
k
Sλ(n, k)Dk,
(16) (16) k
Dn =
X
k
(−1)n−kCλ(n, k)Dλ
k. (17) Dn =
X
k
(−1)n−kCλ(n, k)Dλ
k. (17) (iii) Polynomial expansions (iii) Polynomial expansions (iii) Polynomial expansions (x)λ
n =
n
X
k=0
Sλ(n, k)(x)k,
(18) (x)λ
n =
n
X
k=0
Sλ(n, k)(x)k,
(18)
(x)n =
n
X
k=0
(−1)n−kCλ(n, k)(x)λ
k
(19) (x)λ
n =
n
X
k=0
Sλ(n, k)(x)k, (x)λ
n =
n
X
k=0
Sλ(n, k)(x)k,
(x)n =
n
X
k=0
(−1)n−kCλ(n, k)(x)λ
k (18) (x)n =
n
X
k=0
(−1)n−kCλ(n, k)(x)λ
k (19) (iv) Orthogonality relations (iv) Orthogonality relations X
k
(−1)k−mSλ(n, k)Cλ(k, m) = δn,m,
X
k
(−1)k−mCλ(n, k)Sλ(k, m) = δn,m. (20) (21) (v) Symmetric functions related formulas 2 (v) Symmetric functions related formulas 2 (v) Symmetric functions related formulas 2 Sλ(n, k) =
X
1⩽i1⩽···⩽in−k⩽k
n−k
Y
j=1
(ij −rij+j−1), (22) 2These are related to complementary symmetric functions studied in [9]. 2These are related to complementary symmetric functions studied in [9]. 2These are related to complementary symmetric functions studied in [9]. the electronic journal of combinatorics 22(4) (2015), #P4.10 the electronic journal of combinatorics 22(4) (2015), #P4.10 18 Cλ(n, k) =
X
1⩽i1<···<in−k⩽n
n−k
Y
j=1
(ij −rij+1−j). (23) (23) (vi) The general formula for Sλ(n, k) is given by (vi) The general formula for Sλ(n, k) is given by Sλ(n, k) = 1
k! k
X
ℓ=0
k
ℓ
(−1)k−ℓ(ℓ)λ
n. (24) (24) (vii) The following recurrence relations hold (viii) The formulas (viii) The formulas (viii) The formulas Sλ(n, k) =
X
j1,...,jqn+1
rn
jqn+1
(k −jqn+1)rn−jqn+1
qn
Y
ℓ=1
λℓ
jℓ
(k −jℓ−· · · −jqn+1)λℓ−jℓ. (27) (27) Cλ(n, k) =
X
j1,...,jqk+1
jqk+1
rk
(n −rk −1)jqk+1−rk
qk
Y
ℓ=1
jℓ
λℓ
(n −jℓ−· · · −jqk+1 −rℓ−1)jℓ−rℓ. (
) j1,...,jqk+1
ℓ=1
(28) (28) the electronic journal of combinatorics 22(4) (2015), #P4.10 (vii) The following recurrence relations hold Sλ(n, k) =
rn
X
j=0
rn
j
(k −j)rn−jSλ(n −rn, k −j),
(25) (25) Cλ(n, k) =
n
X
j=rk
j
rk
(n −rk −1)j−rkCλ(n −j, k −rk). (26) (26) Proof. (i) Recurrence relations. We can show that the number of described partitions has the same recurrence as (14). Note that the number of ways is 0 when k < λ1 or k > n. If we consider the element
n, then two cases are possible. If n forms a separate block, then we have the number of
ways to partition [n −1] into k −1 parts over partition λ. If n if placed in the block with
some other elements except the restricted; this can be done in (k −rn−1) ways of any of
partitions of [n −1] into k blocks over partitions λ. This argument implies the needed
recurrence for Sλ(n, k). We show that the described number of ways satisfies the same recurrence as (15). Note
that the number of ways is 0 when k < λ1 or k > n. Consider the element n and two
cases. If n form a singleton separate cycle, then the number of corresponding ways is the
number of permutations of [n −1] having k −1 cycles with the properties for λ partition
of k −1. If n is in cycle with the other elements, then we can put n in cycles after any
element except last rk singletons. This gives (n −1 −rk) ways for any permutation of
[n −1] with k cycles and the described partition property. This argument clearly implies
the needed recurrence for Cλ(n, k). 19 the electronic journal of combinatorics 22(4) (2015), #P4.10 (ii) Expansions with differential operators. The first relation
n Dλ
n =
n
X
k=0
Sλ(n, k)Dk implies from our general Theorem 4.1 on graph partitions. From equation (12) we may easily obtain that implies from our general Theorem 4.1 on graph partitions. From equation (12) we may easily obtain that implies from our general Theorem 4.1 on graph partitions. g
g
From equation (12) we may easily obtain that Dλ
n = Dλ
n−1D1 −rn−1Dλ
n−1. tronic journal of combinatorics 22(4) (2015), #P4.10
20 Proof. (i) Recurrence relations. For the companion expansion if for we know for n −1 For the companion expansion if for we know for n −1 Dn−1 =
n−1
X
k=0
(−1)n−1−kCλ(n −1, k)Dλ
k, then for n we get then for n we get Dn = Dn−1D1 −(n −1)Dn−1
=
n−1
X
k=0
(−1)n−1−kCλ(n −1, k)Dλ
kD1 −
n−1
X
k=0
(−1)n−1−k(n −1)Cλ(n −1, k)Dλ
k
=
n−1
X
k=0
(−1)n−1−kCλ(n −1, k)(Dλ
k+1 + rkDλ
k) −
n−1
X
k=0
(−1)n−1−k(n −1)Cλ(n −1, k)Dλ
k
=
n
X
k=0
(−1)n−k(Cλ(n −1, k −1) + (n −1 −rk)Cλ(n −1, k))Dλ
k
=
n
X
k=0
(−1)n−kCλ(n, k)Dλ
k. (iii) Polynomial expansions. (vii) Recurrence relations. (vii) Recurrence relations. (vii) Recurrence relations. Recurrence (25). Suppose that j elements of the last rn in [n] are singleton blocks. We can choose these elements in
rn
j
ways. The remaining (rn −j) elements should be Recurrence (25). Suppose that j elements of the last rn in [n] are singleton blocks. We can choose these elements in
rn
j
ways. The remaining (rn −j) elements should be
put in distinct (k −j) blocks of any of Sλ(n −rn, k −j) partitions, which can be done
(k −j)rn−j times. Recurrence (26). We may choose the needed rk singleton cycles from the last j elements
of [n]. This can be done in
j
rk
ways. The remaining (j −rk) elements should be put
in the first (n −j) cycles of any of Cλ(n −j, k −rk) permutations, which can be done
(n −rk −1)j−rk times. j
k
i) The last formulas (27), (28) imply from iterative use of relations (25), (26). j
k
(viii) The last formulas (27), (28) imply from iterative use of relations (25), (2 j
(viii) The last formulas (27), (28) imply from iterative use of relations (25), (26). [1] B. R. Alspach and N. J. Pullman. Path decompositions of digraphs. Bull. Austral.
Math. Soc., 10:421–427, 1974. (iii) Polynomial expansions. (iii) Polynomial expansions. Applying the derivation operation to the function xt with a real parameter t, expan-
(
) (
) (iii) Polynomial expansions. Applying the derivation operation to the function xt with a real parameter t, expan-
sions (16), (17) yield (t)λ0 · · · (t)λqn(t)rnxt =
n
X
k=0
Sλ(n, k)(t)kxt, (t)nxt =
n
X
k=0
(−1)n−kCλ(n, k)(t)λ0 · · · (t)λqk(t)rkxt. The last two identities are polynomial relations in t and hold for all t which imply (18),
(19). The last two identities are polynomial relations in t and hold for all t which imply (18),
(19) )
(iv) Orthogonality relations directly imply from the inverse expansions (16), (17) (
)
(iv) Orthogonality relations directly imply from the inverse expansions (16), (17). 20 the electronic journal of combinatorics 22(4) (2015), #P4.10 (v) Symmetric function related formulas can be easily obtained by induction and use
of the recurrence relations (14), (15). (v) Symmetric function related formulas can be easily obtained by induction and use
of the recurrence relations (14), (15). (vi) The general formula for Sλ(n, k). We will show that this formula holds using
combinatorial interpretation of Sλ(n, k) and the inclusion-exclusion principle. Suppose
that blocks are ordered. Let us enumerate them as 1, . . . , k. Denote by Ai (1 ⩽i ⩽k) the set of corresponding restricted (up to λ) arrangements
of [n] into k ordered blocks such that the i-th block is empty. Let A be the number of all
restricted arrangements of [n] into k blocks (some of them might be empty). Then it is
clear that k!Sλ(n, k) = A −|A1 ∪· · · ∪Ak|. Note that Note that A = (k)λ1 . . . (k)λqn(k)rn A = (k)λ1 . . . (k)λqn(k)rn and |Ai1 ∩· · · ∩Aik−ℓ| = (ℓ)λ1 . . . (ℓ)λqn(ℓ)rn for any set of indices 1 ⩽i1 < · · · < ik−ℓ⩽k. Therefore, using the inclusion-exclusion
principle we get for any set of indices 1 ⩽i1 < · · · < ik−ℓ⩽k. Therefore, using the inclusion-exclusion
principle we get k!Sλ(n, k) = A −|A1 ∪· · · ∪Ak|
= (k)λ1 . . . (k)λqn(k)rn −
k−1
X
ℓ=0
X
1⩽i1<···<ik−ℓ⩽k
(−1)k−1−ℓ|Ai1 ∩· · · ∩Aik−ℓ|
= (k)λ1 . . . (k)λqn(k)rn −
k−1
X
ℓ=0
(−1)k−1−ℓ
k
ℓ
(ℓ)λ1 . . . (ℓ)λqn(ℓ)rn
=
k
X
ℓ=0
(−1)k−ℓ
k
ℓ
(ℓ)λ1 . . . (ℓ)λqn(ℓ)rn. (vii) Recurrence relations. (vii) Recurrence relations. References [1] B. R. Alspach and N. J. Pullman. Path decompositions of digraphs. Bull. Austral. Math. Soc., 10:421–427, 1974. 21 the electronic journal of combinatorics 22(4) (2015), #P4.10 [2] P. Balister. Packing digraphs with directed closed trails. Combin. Probab. Comput.,
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and Bell numbers. Electron. J. Combin., 18:#P77, 2011. [21] T. Mansour, M. Schork and M. Shattuck. The generalized Stirling and Bell numbers
revisited. J. Integer Seq., 15:Article 12.8.3, 2012. [22] T. Mansour and M. Schork. Commutation Relations, Normal Ordering, and Stirling
Numbers. CRC Press, 2015. [23] C. J. H. McDiarmid. Path-partition structures of graphs and digraphs. Proc. Lond. Math. Soc., 3.4:750–767, 1974. [24] M. A. M´endez, P. Blasiak, and K. A. Penson. Combinatorial approach to generalized
Bell and Stirling numbers and boson normal ordering problem. J. Math. Phys.,
46:083511-1-8, 2005. [25] M. Mihoubi and M.S. Maamra. The (r1, . . . , rp)-Stirling numbers of the second kind. Integers, 12:#A35, 2012. [26] G. Olshanski. Representations of infinite-dimensional classical groups, limits of
enveloping algebras, and Yangians. Topics in representation theory, Advances in
Soviet Mathematics (A. Kirillov, ed.), vol. 2, AMS, Providence, RI, 1–66, 1991. [27] A. Okounkov. Young basis, Wick formula, and higher Capelli identities. Int. Math. Res. Not., 1996.17: 817-839, 1996. [28] The Online Encyclopedia of Integer Sequences (OEIS), http://oeis.org [29] R. Stanley. Enumerative Combinatorics, Vol. 1. Cambridge University Press, Cam-
bridge, 2011. [30] A. Varvak. Rook numbers and the normal ordering problem. J. Combin. Theory
Ser. A, 112:292–307, 2005. the electronic journal of combinatorics 22(4) (2015), #P4.10 23 the electronic journal of combinatorics 22(4) (2015), #P4.10
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Shape of Testosterone
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The journal of physical chemistry letters
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Shape of Testosterone Article Recommendations * Supporting Information *
sı * ABSTRACT: We have successfully characterized the structure of testosterone, one of the
essential steroids, through high-resolution rotational spectroscopy. A single conformer has
been detected, and a total of 404 transitions have been fitted, allowing a precise
determination of the rotational constants. It allowed us to unravel that the isolated structure
of testosterone adopts an extended disposition. The results obtained in this work highlight
how using laser ablation techniques in combination with Fourier transform microwave
techniques allow the study of large biomolecules or common pharmaceuticals. It is an
important step toward studying relevant biomolecules and developing new analytical
techniques with unprecedented sensitivity and resolution. T
estosterone (17β-hydroxyandrost-4-en-3-one, mp = 155
°C), shown in Scheme 1, is one of the most relevant T magnetic resonance (NMR) spectroscopic studies have been
conducted.5,6 These high-resolution studies conducted under
the solid-state 13C NMR technique show the existence of two
species. However, the authors highlight how care should be
taken when transferring conformational information from
crystalline to the solution states,6 since the conformational
landscape of a molecule can be perturbed due to the
surrounding solvent effects. An effective way of removing
such effects is the use of gas-phase spectroscopic techniques,
most notably supersonic expansions. The absence of any
solvent provides the conformational panorama unbiased by
perturbing agents.7−10 Scheme 1. Chemical Structure of Testosteronea
aThe labels a−d are used to reference the different rings in refs 5 and
6. Scheme 1. Chemical Structure of Testosteronea Scheme 1. Chemical Structure of Testosteronea aThe labels a−d are used to reference the different rings in refs 5 and
6. A significant limitation occurring in gas-phase studies is the
size of a molecule: as growing molecular size reduces the vapor
pressure, i.e., the larger the molecular size and the lower the
vapor pressure, more complex organic molecules cannot be
driven into the gas phase. It is essential for biomolecules that
cannot be studied using conventional heating methods due to
their thermolability and low vapor pressure. One exception is
estradiol, a vital steroid hormone. Its rotational spectrum has
been recently reported, and three conformers have been
characterized.11 In a first attempt, we tried to measure the
rotational spectrum of testosterone through heating, but no
spectral signature was obtained. Shape of Testosterone
Iker León,* Elena R. Alonso, Santiago Mata, and José L. Alonso
Cite This: J. Phys. Chem. Lett. 2021, 12, 6983−6987
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Article Recommendation The Journal of Physical Chemistry Letters pubs.acs.org/JPCL Table 1. Experimental Spectroscopic Parameters for
Testosterone’s Detected Conformer along with Plausible
Configurations I and IIa Calculated at the B3LYP-GD3/6-
311++G(d,p) Level of Theory
configuration I
configuration II
experimental
1
2
3
4
5
6
Ab
785.3463(11)h
789
787
788
667
665
666
B
168.66869(21)
168
168
169
182
182
182
C
153.72059(18)
153
153
153
171
171
172
|μa|
observed
3.4
3.8
4.9
3.0
3.6
4.4
|μb|
observed
0.8
0.7
0.6
0.5
1.2
0.9
|μc|
observed
1.1
2.8
0.8
2.1
3.6
1.4
σc
19.0
Nd
404
ΔEe
0
24
73
492
523
556
ΔEZPEf
0
45
64
546
595
598
ΔGg
0
67
62
596
664
642
aSee text for details. bA, B, and C represent the rotational constants
(in MHz); μa, μb, and μc are the electric dipole moment components
(in D). cRMS deviation of the fit (in kHz). dNumber of measured
transitions. eEnergies (in cm−1) relative to the global minimum
calculated at the B3LYP-GD3BJ/6-311++G(d,p) level of theory. fEnergies (in cm−1) relative to the global minimum, taking into
account the zero-point energy (ZPE), calculated at the B3LYP-
GD3BJ/6-311++G(d,p) level of theory. gGibbs energies (in cm−1)
relative to the global minimum calculated at 298 K at the B3LYP-
GD3BJ/6-311++G(d,p) level of theory. hStandard error in paren-
theses in units of the last digit. pubs.acs.org/JPCL
Letter Table 1. Experimental Spectroscopic Parameters for
Testosterone’s Detected Conformer along with Plausible
Configurations I and IIa Calculated at the B3LYP-GD3/6-
311++G(d,p) Level of Theory Table 1. Experimental Spectroscopic Parameters for
Testosterone’s Detected Conformer along with Plausible
Configurations I and IIa Calculated at the B3LYP-GD3/6-
311++G(d,p) Level of Theory methodology, we were successful in obtaining the first
rotational spectrum of testosterone. We used our laser ablation chirped-pulse Fourier transform
microwave (LA-CP-FTMW) spectrometer14,15 to obtain the
microwave spectrum of testosterone in the 1.5−6.5 GHz
frequency range as shown in Figure 1a. Obtaining the laser- configuration I
configuration II
experimental
1
2
3
4
5
6
Ab
785.3463(11)h
789
787
788
667
665
666
B
168.66869(21)
168
168
169
182
182
182
C
153.72059(18)
153
153
153
171
171
172
|μa|
observed
3.4
3.8
4.9
3.0
3.6
4.4
|μb|
observed
0.8
0.7
0.6
0.5
1.2
0.9
|μc|
observed
1.1
2.8
0.8
2.1
3.6
1.4
σc
19.0
Nd
404
ΔEe
0
24
73
492
523
556
ΔEZPEf
0
45
64
546
595
598
ΔGg
0
67
62
596
664
642
b Figure 1. The Journal of Physical Chemistry Letters (a) Broadband spectrum of testosterone in the 1.5−6.5
GHz frequency region using the LA-CP-FTMW spectrometer,
highlighting the a-type R-branch progressions (J' ←J) originated
by a single conformer of testosterone. (b) A comparison between a
selected range of the experimental spectrum with the simulated one,
with selected rotational transitions (J' ́K'a,K'c ←JKa,Kc). As can be seen,
there is an excellent agreement between theory and experiment. aSee text for details. bA, B, and C represent the rotational constants
(in MHz); μa, μb, and μc are the electric dipole moment components
(in D). cRMS deviation of the fit (in kHz). dNumber of measured
transitions. eEnergies (in cm−1) relative to the global minimum
calculated at the B3LYP-GD3BJ/6-311++G(d,p) level of theory. fEnergies (in cm−1) relative to the global minimum, taking into
account the zero-point energy (ZPE), calculated at the B3LYP-
GD3BJ/6-311++G(d,p) level of theory. gGibbs energies (in cm−1)
relative to the global minimum calculated at 298 K at the B3LYP-
GD3BJ/6-311++G(d,p) level of theory. hStandard error in paren-
theses in units of the last digit. To help identify the observed rotamer of testosterone, we
performed a conformational search using molecular mechanics
methods. A total of 8 molecular structures were screened
within an energy window of 2500 cm−1. These structures were
reoptimized using DFT methods (B3LYP-GD3/6-311++G-
(d,p)), and the six low-energy structures, those below 1000
cm−1, belong to the two configurations shown in Figure 2. In
configuration I, rings b and c (see Scheme 1) adopt a chair
conformation, while ring a takes a half-chair disposition. Ring a
is almost in the same plane as rings b, c, and d, and the
molecule adopts an extended form. Configuration II is in a
semifolded arrangement shown in Figure 2b, with the a ring
almost perpendicular to the b−c−d rings’ plane. The three
lowest-energy conformers of either configuration differ in the
hydroxyl group’s orientation for each configuration, as
indicated in Figure S1. As shown in Table 1, the three
conformers belonging to configuration I are considerably more
stable than those belonging to configuration II, resulting in
extra stability of 600 cm−1. Figure 1. (a) Broadband spectrum of testosterone in the 1.5−6.5
GHz frequency region using the LA-CP-FTMW spectrometer,
highlighting the a-type R-branch progressions (J' ←J) originated
by a single conformer of testosterone. (b) A comparison between a
selected range of the experimental spectrum with the simulated one,
with selected rotational transitions (J' ́K'a,K'c ←JKa,Kc). Shape of Testosterone This problem can be
overcome by using laser ablation techniques, which have
proven to be successful for many biomolecules.12,13 Using this steroids.1,2 It is a sex hormone that plays an essential role in the
body. It regulates sex drive (libido), fat distribution, body hair,
bone mass, muscle mass, and red blood cells in humans. It also
plays a crucial role in sperm production and developing male
reproductive tissues such as the testes and prostate. It is
biosynthesized from cholesterol through a series of steps, and a
small amount of circulating testosterone is transformed into
estradiol, a form of estrogen. Though it is also present in
females to a lesser extent, they are more sensitive to it. As men
age, the testosterone levels decrease, producing less estradiol. Thus, changes attributed to testosterone deficiency might be
partly or entirely due to the accompanying decline in estradiol. Additionally, testosterone is also used as a medication for
breast cancer treatment in women and low testosterone levels
in men. Received:
June 2, 2021
Accepted:
July 16, 2021
Published: July 20, 2021
https://doi.org/10.1021/acs.jpclett.1c01743
J. Phys. Chem. Lett. 2021, 12, 6983−6987 Received:
June 2, 2021
Accepted:
July 16, 2021
Published: July 20, 2021 Received:
June 2, 2021
Accepted:
July 16, 2021
Published: July 20, 2021 It is well-known that there is a direct relationship between a
simple or macromolecule’s structure and its particular function
or properties.3,4 Obtaining the precise structure of testosterone
is therefore needed to understand its activity. So far, nuclear © 2021 The Authors. Published by
American Chemical Society https://doi.org/10.1021/acs.jpclett.1c01743
J. Phys. Chem. Lett. 2021, 12, 6983−6987 https://doi.org/10.1021/acs.jpclett.1c01743
J. Phys. Chem. Lett. 2021, 12, 6983−6987 6983 Letter Letter https://doi.org/10.1021/acs.jpclett.1c01743
J. Phys. Chem. Lett. 2021, 12, 6983−6987 The Journal of Physical Chemistry Letters The barriers to hydroxyl conformer interconversion are below
400 cm−1, suggesting that neon should be capable of relaxing
structure 2 into structure 3 and the latter into structure 1. Nevertheless, at room temperature, testosterone should
participate in a dynamic equilibrium of the three conformers. We unambiguously determined that the extended form of
testosterone is the most stable configuration. Subsequently,
this knowledge allows for some conclusions about its biological
activity due to the structure−property correspondence. Explanation of epimers’ activity differences is likely to be
related to the substituents’ gross spatial orientation rather than
to subtle conformational changes in the steroid skeleton.6
From our results in Figure 2, it is clear that the semifolded
form of testosterone, in which ring a is out of plane from the
rest of the structure, is not suitable to establish a hydrogen
bond between subsequent testosterone molecules. Further-
more, it would not allow direct interactions between stacked
molecules, thus precluding van der Waals interactions between
different ribbons. The extended form, on the other hand,
allows both types of interactions. It is in excellent agreement y
Having deduced a plausible assignment of testosterone’s
observed rotamer, we conclude the spectral analysis by
explaining why we observe only a single conformer, turning
our attention to the properties of a supersonic expansion. By
examining the behavior of a variety of molecules, Ruoffet al. noted that, when a low potential energy barrier separates the
conformational species, the noble-gas collisions in the initial
stages of the expansion provide the energy required for their
interconversion.19,20 We used theoretical calculations to
perform the relaxed potential energy surface (PES) scan
shown in Figure 3 by rotating the C−C−O−H dihedral angle. The barriers to hydroxyl conformer interconversion are below
400 cm−1, suggesting that neon should be capable of relaxing
structure 2 into structure 3 and the latter into structure 1. Nevertheless, at room temperature, testosterone should
participate in a dynamic equilibrium of the three conformers. b
l
d
d h
h
d d f
f Figure 3. Relaxed PES rotating the C−C−O−H dihedral angle of the
three lowest-energy conformers of testosterone within configuration I. The low barrier separating them explains why a single conformer is
seen. A close-up view of the hydroxyl group position is shown, while a
larger view of the structures can be found in the SI. The Journal of Physical Chemistry Letters The Journal of Physical Chemistry Letters The Journal of Physical Chemistry Letters Letter pubs.acs.org/JPCL Figure 2. A comparison between the two plausible configurations of testosterone. A top and side view of (a) structure 1 within the extended
configuration and (b) structure 4 within the semifolded configuration. For each configuration, three conformers are possible differing in the
hydroxyl group’s orientation, as indicated by the green arrow. Figure 2. A comparison between the two plausible configurations of testosterone. A top and side view of (a) structure 1 within the extended
configuration and (b) structure 4 within the semifolded configuration. For each configuration, three conformers are possible differing in the
hydroxyl group’s orientation, as indicated by the green arrow. Figure 3. Relaxed PES rotating the C−C−O−H dihedral angle of the
three lowest-energy conformers of testosterone within configuration I. The low barrier separating them explains why a single conformer is
seen. A close-up view of the hydroxyl group position is shown, while a
larger view of the structures can be found in the SI. are directly connected with the selection rules and intensity of
the observed transitions. We estimate relative intensities of
6.5:1.0:1.5 for the a-, b-, anc c-type transitions, respectively. The intensity of the a-type transitions is significantly larger
than those of b- and c-type in good agreement with the dipole
moment selection rules predicted for structures 1 and 3 but in
sharp contrast to intense c-type transitions expected for
structure 2, which therefore can be ruled out. The distinction
between structures 1 and 3 is not definitive. However,
calculations using both DFT and MP2 methods predict
structure 1 to be the global minimum and, therefore, is most
likely the detected structure of testosterone. y
Having deduced a plausible assignment of testosterone’s
observed rotamer, we conclude the spectral analysis by
explaining why we observe only a single conformer, turning
our attention to the properties of a supersonic expansion. By
examining the behavior of a variety of molecules, Ruoffet al. noted that, when a low potential energy barrier separates the
conformational species, the noble-gas collisions in the initial
stages of the expansion provide the energy required for their
interconversion.19,20 We used theoretical calculations to
perform the relaxed potential energy surface (PES) scan
shown in Figure 3 by rotating the C−C−O−H dihedral angle. https://doi.org/10.1021/acs.jpclett.1c01743
J. Phys. Chem. Lett. 2021, 12, 6983−6987 The Journal of Physical Chemistry Letters As can be seen,
there is an excellent agreement between theory and experiment. ablated rotational spectrum of such a large biomolecule is not
easy and requires careful control of the experimental
parameters as well as its fragmentation to be minimized.12
As can be seen, the spectrum shows very intense rotational
lines. Initially, lines corresponding to known photofragment
species and water clusters were identified and removed.14,16,17
The remaining rotational spectrum still showed many
rotational transitions, subsequently attributed to testosterone. At first glance, the characteristic pattern of an a-type R-branch
progression arising from a dominant rotameric species (see
Figure 1a) was quickly identified. The first set of rotational
constants determined from a rigid rotor analysis18 helped us
quickly locate b- and c-type transitions with new predictions. A
total of 404 rotational transitions were assigned and measured
(Table S1 of the Supporting Information, SI), allowing us to
determine very accurate values of the rotational constants. Figure 1b shows excellent matching between experimental and
simulated spectra using the fitted values, which are listed in the
first column of Table 1. After the rotational lines of this
rotamer were removed, no significant signals remained
unassigned in the spectrum. Therefore, no spectral searches
for other conformational candidates were conducted. We used two different indicators for conformer identi-
fication: rotational constants and dipole moment components. The rotational constants provide information on the rotamer’s
mass distribution and are fundamental in obtaining conforma-
tional structures. The comparison of Table 1 between the
experimental rotational constants with the theoretically
predicted values unambiguously assigns the observed rotamer
to one of the three conformers of configuration I, i.e., the
extended form. Unfortunately, the values of the rotational
constants are not able to discern between the three structures
1, 2, and 3 that differ only in the orientation of the terminal
hydroxyl group. This small difference does not cause a
significant change in the mass distribution and, consequently,
in the rotational constants’ values. The missing information
can be obtained from the dipole moment components, which 6984 The Journal of Physical Chemistry Letters with the crystal testosterone’s structural arrangement:6 The
testosterone’s unit cell consists of two extended parallel
ribbons of the molecules extended into a sheet. These ribbons
are hydrogen-bonded sequentially between the hydroxyl group
of a molecule and the next molecule’s ketone group. Strong
van der Waals interactions bind both ribbons together, which
are in a head-to-tail disposition. It is important to note that
these results correlate very well with the arrangement observed
in steroids in the condensed phase.21,22 We unambiguously determined that the extended form of
testosterone is the most stable configuration. Subsequently,
this knowledge allows for some conclusions about its biological
activity due to the structure−property correspondence. Explanation of epimers’ activity differences is likely to be
related to the substituents’ gross spatial orientation rather than
to subtle conformational changes in the steroid skeleton.6
From our results in Figure 2, it is clear that the semifolded
form of testosterone, in which ring a is out of plane from the
rest of the structure, is not suitable to establish a hydrogen
bond between subsequent testosterone molecules. Further-
more, it would not allow direct interactions between stacked
molecules, thus precluding van der Waals interactions between
different ribbons. The extended form, on the other hand,
allows both types of interactions. It is in excellent agreement An interesting comparison is that of testosterone with
estradiol. Estradiol is similar to testosterone but with ring a
differing considerably: the methyl group in ring a is lost and it
has a hydroxyl group instead of the ketone group, leaving ring
a as a phenolic ring. This change makes estradiol lose the two
possible ring configurations in testosterone, which is now
planar. Effectively, the structure of estradiol has a single
configuration but six possible conformers that differ in the
hydroxyl groups’ orientations. The rotational spectrum of
estradiol11 shows two predominant structures that are similar 6985 Letter pubs.acs.org/JPCL The Journal of Physical Chemistry Letters Letter Santiago Mata −Grupo de Espectrocopía Molecular (GEM),
Edificio Quifima, Laboratorios de Espectroscopia y
Bioespectroscopia, Unidad Asociada CSIC, Parque Científico
UVa, Universidad de Valladolid, 47011 Valladolid, Spain; to our characterized structure but differing in the two possible
orientations of the hydroxyl group in ring a. This is another
point supporting our assignment. Additionally, a third
conformer is found, which is much weaker than expected
due to conformational interconversion. It further confirms our
assignment and discussion about conformational interconver-
sion. *
sı Supporting Information *
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jpclett.1c01743. (5) Hayamizu, K.; Kamo, O. Complete Assignments of the 1H and
13C NMR Spectra of Testosterone and 17α-methyltestosterone and
the 1H Parameters Obtained from 600 MHz Spectra. Magn. Reson. Chem. 1990, 28, 250−256. Detailed experimental and theoretical section; detailed
view of the configurations of testosterone (Figure S1);
measured frequencies for the 404 transitions of the
testosterone’s detected rotamer (Table S1) (PDF) (6) Roberts, P. J.; Pettersen, R. C.; Sheldrick, G. M.; Isaacs, N. W.;
Kennard, O. Crystal and Molecular Structure of 17β-Hydroxyandrost-
4-En-3-One (Testosterone). J. Chem. Soc., Perkin Trans. 2 1973, 2,
1978−1984. (7) Robertson, E. G.; Simons, J. P. Getting into Shape: Conforma-
tional and Supramolecular Landscapes in Small Biomolecules and
Their Hydrated Clusters. Phys. Chem. Chem. Phys. 2001, 3, 1−18. Corresponding Author Iker León −Grupo de Espectrocopía Molecular (GEM),
Edificio Quifima, Laboratorios de Espectroscopia y
Bioespectroscopia, Unidad Asociada CSIC, Parque Científico
UVa, Universidad de Valladolid, 47011 Valladolid, Spain;
orcid.org/0000-0002-1992-935X; Email: Iker.leon@
uva.es Iker León −Grupo de Espectrocopía Molecular (GEM),
Edificio Quifima, Laboratorios de Espectroscopia y
Bioespectroscopia, Unidad Asociada CSIC, Parque Científico
UVa, Universidad de Valladolid, 47011 Valladolid, Spain;
id
/0000 0002 1992 935X E
il Ik
l
@ (9) Oomens, J.; Steill, J. D.; Redlich, B. Gas-Phase IR of
Deprotonated Amino Acids. J. Am. Chem. Soc. 2009, 131, 4310−4319. (
) (10) Caminati, W. Nucleic Acid Bases in the Gas Phase. Angew. Chem., Int. Ed. 2009, 48, 9030−9033. orcid.org/0000-0002-1992-935X; Email: Iker.leon@
uva.es (11) Zinn, S.; Schnell, M. Flexibility at the Fringes: Conformations
of the Steroid Hormone β-Estradiol. ChemPhysChem 2018, 19, 2915−
2920. ■AUTHOR INFORMATION (8) De Vries, M. S.; Hobza, P. Gas-Phase Spectroscopy of
Biomolecular Building Blocks DFT: Density-Functional Theory. Annu. Rev. Phys. Chem. 2007, 58, 585−612. Notes The authors declare no competing financial interest. The authors declare no competing financial interest. The Journal of Physical Chemistry Letters Comparing both steroids is interesting, because the
characterized predominant species are very similar but have
entirely different biological functionalities. Because the only
structural difference is ring a, it must be responsible for their
ligand−protein binding in different receptors. The variation
from a carbonyl to a hydroxyl group probably forces a binding
with a receptor capable of a proton donor or proton acceptor,
respectively. Additionally, the receptor must be ready to adapt
to the dispersive forces or sterical effects caused by the methyl
group in testosterone. This comparison is another illustrative
example of the importance of the structure−property relation-
ship. Santiago Mata −Grupo de Espectrocopía Molecular (GEM),
Edificio Quifima, Laboratorios de Espectroscopia y
Bioespectroscopia, Unidad Asociada CSIC, Parque Científico
UVa, Universidad de Valladolid, 47011 Valladolid, Spain; orcid.org/0000-0002-1892-5015 José L. Alonso −Grupo de Espectrocopía Molecular (GEM),
Edificio Quifima, Laboratorios de Espectroscopia y
Bioespectroscopia, Unidad Asociada CSIC, Parque Científico
UVa, Universidad de Valladolid, 47011 Valladolid, Spain;
orcid org/0000 0002 3146 8250 José L. Alonso −Grupo de Espectrocopía Molecular (GEM),
Edificio Quifima, Laboratorios de Espectroscopia y
Bioespectroscopia, Unidad Asociada CSIC, Parque Científico
UVa, Universidad de Valladolid, 47011 Valladolid, Spain;
id
/0000 0002 3146 8250 orcid.org/0000-0002-3146-8250 Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jpclett.1c01743 ■REFERENCES (1) Mooradian, A. D.; Morley, J. E.; Korenman, S. G. Biological
Actions of Androgens. Endocr. Rev. 1987, 8, 1−28. (1) Mooradian, A. D.; Morley, J. E.; Korenman, S. G. Biological
Actions of Androgens. Endocr. Rev. 1987, 8, 1−28. (2) Rommerts, F. F. G. Testosterone: An Overview of Biosynthesis,
Transport, Metabolism and Nongenomic Actions. Testosterone 1998,
1. (3) Lehninger, A.; Nelson, D.; Cox, M. Lehninger Principles of
Biochemistry, 5th ed.; W. H. Freeman: New York, 2008. (4) Park, Y.; Helms, V. On the Derivation of Propensity Scales for
Predicting Exposed Transmembrane Residues of Helical Membrane
Proteins. Bioinformatics 2007, 23, 701−708. ■ACKNOWLEDGMENTS The financial fundings from the Ministerio de Ciencia e
Innovación (CTQ2016-76393-P and PID2019-111396GB-
I00), Junta de Castilla y León (Grants VA077U16 and
VA244P2), and European Research Council under the
European Union’s Seventh Framework Programme (FP/
2007-2013)/ERC-2013-SyG, Grant Agreement n. 610256
NANOCOSMOS, are gratefully acknowledged. E.R.A. ac-
knowledges the Ministerio de Ciencia e Innovación for a Juan
de la Cierva postdoctoral grant (FJC2018-037320-I). In summary, we have been able to transfer testosterone, a
large biomolecule, from its solid into the gas phase using laser
ablation techniques. It has allowed us to characterize the
structure of a relevant biomolecule such as testosterone by
rotational spectroscopy for the first time. This technique is one
of the most powerful spectroscopic techniques for structural
determination due to the direct relation of shape and spectral
position of 404 rotational lines. Interestingly, out of the two
possible configurations, the extended form is the predominant
one. Supersonic-jet analytical techniques combined with laser
ablation can be used to determine a sample’s composition
without dissolving the sample in a suitable medium that would
usually require chemical manipulations. Additionally, there is
no challenging or tedious preparation of the sample requiring
lengthy analysis times. As we show, the sensitivity reached
using our experimental procedure is sufficient to detect large
biomolecules or common pharmaceuticals with unrivaled
structural determination. https://doi.org/10.1021/acs.jpclett.1c01743
J. Phys. Chem. Lett. 2021, 12, 6983−6987 https://doi.org/10.1021/acs.jpclett.1c01743
J. Phys. Chem. Lett. 2021, 12, 6983−6987 Authors (12) Alonso, E. R.; León, I.; Alonso, J. L. The Role of the
Intramolecular Interactions in the Structural Behavior of Biomole-
cules: Insights from Rotational Spectroscopy. Intra- and Intermolecular
Interactions Between Non-covalently Bonded Species 2021, 93. Elena R. Alonso −Instituto Biofisika (UPV/EHU, CSIC),
University of the Basque Country, 48940 Leioa, Spain;
Departamento de Química Física, Facultad de Ciencia y
Tecnología, Universidad del País Vasco, 48940 Leioa, Spain;
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ECDSA White-Box Implementations: Attacks and Designs from CHES 2021 Challenge
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10.46586/tches.v2022.i4.527-552 ECDSA White-Box Implementations Citation for published version (APA):
Barbu, G., Beullens, W., Dottax, E., Giraud, C., Houzelot, A., Li, C., Mahzoun, M., Ranea, A., & Xie, J. (2022). ECDSA White-Box Implementations: Attacks and Designs from CHES 2021 Challenge. IACR Transactions on
Cryptographic Hardware and Embedded Systems, 2022(4), 527-552. https://doi.org/10.46586/tches.v2022.i4.527-552 Citation for published version (APA):
Barbu, G., Beullens, W., Dottax, E., Giraud, C., Houzelot, A., Li, C., Mahzoun, M., Ranea, A., & Xie, J. (2022). ECDSA White-Box Implementations: Attacks and Designs from CHES 2021 Challenge. IACR Transactions on
Cryptographic Hardware and Embedded Systems, 2022(4), 527-552. https://doi.org/10.46586/tches.v2022.i4.527-552 Download date: 24. Oct. 2024 www.tue.nl/taverne Take down policy
If you believe that this document breaches copyright please contact us at:
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providing details and we will investigate your claim. Download date: 24. Oct. 2024 IACR Transactions on Cryptographic Hardware and Embedded Systems
ISSN 2569-2925, Vol. 2022, No. 4, pp. 527–552. DOI:10.46586/tches.v2022.i4.527-552 Guillaume Barbu1 , Ward Beullens2, Emmanuelle Dottax1 ,
Christophe Giraud1 , Agathe Houzelot1,3 , Chaoyun Li4 ,
Mohammad Mahzoun5, Adrián Ranea4
and Jianrui Xie4 1 IDEMIA, Cryptography & Security Labs, Pessac, France
firstname.lastname@idemia.com
2 IBM Research, Zurich, Switzerland
wbe@zurich.ibm.com
3 LaBRI, CNRS, Université de Bordeaux, Bordeaux, France
4 imec-COSIC, KU Leuven, Leuven, Belgium
firstname.lastname@esat.kuleuven.be
5 Eindhoven University of Technology, Eindhoven, Netherlands
m.mahzoun@tue.nl Abstract. Despite the growing demand for software implementations of ECDSA
secure against attackers with full control of the execution environment, scientific
literature on ECDSA white-box design is scarce. The CHES 2021 WhibOx contest
was thus held to assess the state-of-the-art and encourage relevant practical research,
inviting developers to submit ECDSA white-box implementations and attackers to
break the corresponding submissions. In this work, attackers (team TheRealIdefix) and designers (team zerokey) join to
describe several attack techniques and designs used during this contest. We explain
the methods used by the team TheRealIdefix, which broke the most challenges, and we
show the efficiency of each of these methods against all the submitted implementations. Moreover, we describe the designs of the two winning challenges submitted by the
team zerokey; these designs represent the ECDSA signature algorithm by a sequence
of systems of low-degree equations, which are obfuscated with affine encodings and
extra random variables and equations. The WhibOx contest has shown that securing ECDSA in the white-box model is an
open and challenging problem, as no implementation survived more than two days. In this context, our designs provide a starting methodology for further research, and
our attacks highlight the weak points future work should address. Keywords: ECDSA · White-Box Cryptography · WhibOx Contest Keywords: ECDSA · White-Box Cryptography · WhibOx Contest Licensed under Creative Commons License CC-BY 4.0.
Received: 2022-04-15
Accepted: 2022-06-15 1
Introduction Cryptographic techniques are primarily designed to be secure in a context where the
confidentiality of secret keys is ensured with black-box access to the algorithm – only inputs
and outputs are available to the attacker. Confidence in security is built from detailed
studies, carefully defined security notions, and security proofs. Such a strong level of
confidence is now a standard expectation. However, real-life scenarios for implementations
might jeopardize initial assumptions, where attackers have access to additional information
via side channels (e.g., timing or power consumption) or can modify the algorithm execution Licensed under Creative Commons License CC-BY 4.0. Received: 2022-04-15
Accepted: 2022-06-15 Licensed under Creative Commons License CC-BY 4.0. Received: 2022-04-15
Accepted: 2022-06-15 Published: 2022-08-31 Published: 2022-08-31 528 Attacks and Designs from CHES 2021 Challenge and exploit faulty results. This is called the grey-box model. Developers have to put
countermeasures in place to reach the originally expected security level. and exploit faulty results. This is called the grey-box model. Developers have to put
countermeasures in place to reach the originally expected security level. In the context of mobile applications – contactless payments, cryptocurrency wallets,
streaming services – or connected objects, devices often lack secure storage to protect
secret keys, and their generally open execution environment exposes a large attack surface. This hostile environment is captured by the white-box model, which assumes an attacker
having control of every aspect of the implementation: execution flow, memory content
and addresses. The first white-box implementations were proposed in the early 2000s by
Chow et al. [CEJvO02,CEJv03], and the field has continuously developed since then, with
design proposals [BG03,BCD06,XL09,Kar11,DFLM18,RW19,SEL21,BCC21], attacks
[BGEC04,GMQ07,WMGP07,MGH09,DWP10,DRP13,LRD+14,AMR19,GRW20] and
efforts to define security notions [SWP09,DLPR14,AABM20]. The industry shows a growing interest in white-box cryptography owing to the
widespread usage of security-related applications on connected devices. The WhibOx
contest, attached to the CHES conference, has been held biennially since 2017 to encourage
practical experiments both from the designer and attacker perspectives. It lasts several
months, inviting coders to post white-box implementations and attackers to break them. Participants can remain anonymous and silent about any detail on their work. The first
two editions in 2017 and 2019 focused on white-box implementations of AES and exhibited
the community’s strong interest in this subject. Some candidates survived all attacks
in the second edition in 2019, showing a certain maturity for this algorithm. 1Guillaume Barbu, Emmanuelle Dottax, Christophe Giraud and Agathe Houzelot are part of the team
TheRealIdefix; Ward Beullens, Chaoyun Li, Mohammad Mahzoun, Adrián Ranea and Jianrui Xie compose
the team zerokey. 2
Rules of the WhibOx 2021 Contest Designers were required to post challenges computing ECDSA signatures on the NIST
P-256 curve under a hard-coded, freely chosen key, and accepting as input any 256-bit
message digest e = H(m). Notice that the cryptographic hash function H is excluded
from the intended white-box implementation of ECDSA and the message m is also not
provided. At the same time, attackers were encouraged to extract the private keys. In
addition, acceptance of submitted implementations was conditioned on some requirements: • the public key corresponding to the embedded private key, as well as a proof of
knowledge of the private key, had to be provided, • submissions had to be source code in portable C, • linking to external libraries was forbidden, except for the GNU Multi Precision
library [Gt20], • the signature algorithm had to be deterministic, • the execution time was limited to 3 seconds, the program size to 20 MB, and the
RAM usage to 20 MB as well. • the execution time was limited to 3 seconds, the program size to 20 MB, and the
RAM usage to 20 MB as well. There was an elaborate system with scoreboards to reward designers and attackers. A challenge gains strawberries as time goes by till broken. Challenges with a higher
performance score (measured in terms of execution time, code size, and RAM usage) gain
strawberries faster. Eventually, the challenge with the highest number of strawberries wins
the competition. Accordingly, when submitting a matching private key to the system,
attackers receive bananas, the number of which is determined by the number of strawberries
of the challenge at the time of the break. More detailed information can be found on the
contest website [CHE]. 1
Introduction Section 5 discloses the designs of Challenges 226 and
227 proposed by the team zerokey, and Section 6 concludes this paper. Outline. The paper is organized as follows. Section 2 outlines the rules of the WhibOx
2021 contest. Section 3 recalls the ECDSA algorithm and the state-of-the-art regarding
white-box implementations. Section 4 presents the different methods that have been used
by the team TheRealIdefix to break various implementations and some statistics regarding
the success rate of these methods. Section 5 discloses the designs of Challenges 226 and
227 proposed by the team zerokey, and Section 6 concludes this paper. 1
Introduction In 2021,
organisers changed the target and decided to consider the ECDSA signature algorithm,
whose white-box implementation is of substantial interest to the industry but virtually
lacks scientific literature. From May 17th to August 22nd 2021, 97 candidate implementations were submitted for
scrutiny by 37 (teams of) attackers. All challenges were broken within 35 hours, suggesting
the difficulty of achieving a secure white-box implementation of ECDSA. Thus, studying
the attacks would help to discern weak points inside the implementations. Besides, the
analysis of the design of the most resistant challenges, which successfully defeated most
attackers, would also give directions for future designs. Contributions. In this paper, teams TheRealIdefix — who broke the most challenges —
and zerokey — who proposed the two winning challenges — join to present how they
proceeded during the contest1. On the attack side, we describe a strategy to achieve
efficient attacks. As reverse engineering is a time-consuming task, automated attacks
are desirable. We consider different attack paths against ECDSA white-boxes: the ones
inherited from traditional cryptanalysis, the extensions of attacks in the grey-box model,
and the logical attacks of the software. We discuss the feasibility of automating each attack
path and provide detailed information regarding which attacks succeeded (or failed) on each
candidate. Our results show that, with few exceptions, it was sufficient to fully recover the
secret value by these automated attacks. On the design side, we describe the methodology
we used to build the two winning challenges, Challenges 226 and 227. It includes modifying
the implicit framework [RVP22] originally proposed for block ciphers, applying techniques
from multivariate public-key cryptosystems, and obfuscating the resulting C code with
a C obfuscator. Our design thus turns the ECDSA signature algorithm into a sequence
of systems of low-degree equations which are obfuscated with large affine encodings and
additional variables and equations. Finally, we show how to break Challenge 226 with
automated attacks and how to break Challenge 227 once reverse-engineered. 529 Barbu et al. Outline. The paper is organized as follows. Section 2 outlines the rules of the WhibOx
2021 contest. Section 3 recalls the ECDSA algorithm and the state-of-the-art regarding
white-box implementations. Section 4 presents the different methods that have been used
by the team TheRealIdefix to break various implementations and some statistics regarding
the success rate of these methods. 3.1
ECDSA In 1992, Vanstone introduced a variant of DSA based on elliptic curves. The result-
ing public-key signature algorithm is called Elliptic Curve Digital Signature Algorithm
(ECDSA) [Van92]. Its parameters are an elliptic curve E over a field Fq, a point G of prime
order n, and a cryptographic hash function H. The private key d is randomly drawn from
J1, n −1K, and the public key consists of the point Q = [d]G where [d]G corresponds to the
scalar multiplication of the point G by the scalar d. The ECDSA signature is described in
Algorithm 1, where Rx and Ry denote the coordinates of the point R. Note that the key d is not the only sensitive value in that scheme. Indeed, the recovery
of the nonce k allows the computation of d from the signature (r, s) and the message m: d = (ks −H(m))r−1 mod n . (1) d = (ks −H(m))r−1 mod n . (1) Attacks and Designs from CHES 2021 Challenge 530 Algorithm 1: ECDSA signature
Input
: the message m
Output : the signature (r, s)
1 e ←H(m)
2 k
$←−J1, n −1K
3 R = (Rx, Ry) ←[k]G
4 r ←Rx mod n
5 s ←k−1(e + rd) mod n
6 if r = 0 or s = 0 then
7
Go to step 2
8 end
9 Return (r, s) 1 e ←H(m)
2 k
$←−J1, n −1K
3 R = (Rx, Ry) ←[k]G
4 r ←Rx mod n
5 s ←k−1(e + rd) mod n
6 if r = 0 or s = 0 then
7
Go to step 2
8 end
9 Return (r, s) The nonce must not only remain secret but also differ for each execution of the algorithm. Indeed, an efficient way to recover its value is to find another signature (r′, s′) of a different
message m′ ̸= m using the same nonce, that is with k′ = k. In that case, we also have
r′ = r, so the adversary may compute k = (H(m′) −H(m))(s′ −s)−1 mod n . (2) (2) In the black-box model, the security of ECDSA is based on the difficulty of the Elliptic
Curve Discrete Logarithm Problem (ECDLP), i.e., on the difficulty of computing the
scalar k (resp. d) from the points G and R = [k]G (resp. Q = [d]G). 3.1
ECDSA To ensure that
this problem is difficult to solve, there are several standards to define elliptic curves,
e.g. [Loc10,Sta10,JOR11,FIP13]. However, there is a gap between the security of ECDSA
in theory and that of ECDSA implementations. Many grey-box attacks have been described
in the literature (see for example [FV12]). Some of them directly target the key d while
others aim at recovering some information on the nonce k. As explained previously, the
knowledge of the nonce allows an adversary to compute the secret key. Recovering a few
bits of the nonces associated to different signatures may be enough for an attacker. Indeed,
this allows the construction of a system of equations that can be solved using lattice-based
algorithms [BH19,JSSS20] or Bleichenbacher’s FFT-based approach [ANT+20]. These
bits could, for example, be recovered via side-channel analysis if the implementation is not
protected or simply guessed if the nonce is not drawn uniformly at random. These attacks
show that it is already complicated to achieve a secure implementation of ECDSA in the
grey-box model, and of course, things get worse in the white-box context. 4
Breaking the Challenges White-box implementations usually rely on encodings and other theoretically sound
approaches to protect the secret values and their manipulations. It is also very often
the case that code obfuscation techniques are used to make understanding the design a
time-consuming and challenging task. Extensive use of such obfuscation techniques in
the submitted source files causes independently reverse-engineering each challenge to be
overwhelming in time. We thus focused on designing attack methods that could be efficient
and easily automated. This section looks at the different attacks that can be automated in a white-box context
and gives the rationale for using and discarding them. We then present the results of
applying the selected methods to the whole set of submissions. 3.2
White-box Implementation of ECDSA The white-box model assumes that the attacker has total access to the executable: he can
read and modify it at will. He also has access to all the memory used during execution, so
a white-box designer does not only have to protect his implementation against grey-box
attacks but also against an adversary who can dump the memory and search for sensitive
values such as k or d. The first technique to prevent secret data from appearing in plain was
introduced by Chow et al. in [CEJv03]. Their idea is to embed the key into the algorithm,
and each operation is performed with the help of look-up tables protected by carefully
crafted encodings. Informally, the algorithm is split into low-level operations, and each
operation op is replaced by f −1 ◦op ◦f ′, where f and f ′ are bijections called respectively
input and output encodings. The drawback of this technique is that the required memory
drastically increases with the algorithm’s complexity. Using it to secure operations as 531 Barbu et al. complex as scalar multiplications or inversions while remaining efficient is thus a real
challenge. complex as scalar multiplications or inversions while remaining efficient is thus a real
challenge. Another challenge in white-box cryptography is the impossibility of relying on any
external source of randomness. An attacker could simply disable such a source and fix
its output to a constant value. For example, in the context of AES, this renders some
countermeasures against side-channel or fault attacks based on randomization techniques
completely inefficient. When one considers ECDSA signatures, disabling the source of
randomness yields multiple uses of the same nonce and, thus, easy recovery of the private
key, as seen in Sect. 3.1. The solution is to compute k as a function of the only source of
randomness available, the input message: k = f(m). In order to maintain the security of
the signature scheme, this mapping must be computationally indistinguishable from what
a randomly and uniformly chosen function would return. We will see in the next section
that many challenges of the WhibOx competition did not fulfill this requirement. 2https://man7.org/linux/man-pages/man8/ld.so.8.html 4.1.1
Hooking Shared Libraries The contest rules were a clear incentive for developers to use the GMP library for big
number arithmetic operations. A first attempt to break the submitted challenges was then
to search if sensitive values were manipulated in clear by the GMP library. In order to
perform this automatically, our approach has been to hook the calls to GMP functions
thanks to the so-called LD_PRELOAD trick. Pre-loading is a feature of the dynamic linker on UNIX systems that allows loading a
specific shared library before all other libraries linked to a given executable binary2. In
our specific case, we built a shared library defining the same function as the GMP library
(e.g. mpz_mul, mpz_mod or mpz_invert). Each of these functions simply updates a log of
the given parameters before calling the real GMP function, explicitly using the dynamic
linker (thanks to the <dlfcn.h> module) to ensure the correct execution of the white-box
implementation. It is then only necessary to add our shared library to the LD_PRELOAD
environment variable of the dynamic linker on our system before calling the ECDSA binary
to have our custom functions called in place of the genuine GMP ones. The corresponding
log is analysed in a second step to eventually reveal the secret key if d, k or related values
such as r · d or e + r · d are found in the log. Such an approach allowed us to break 32% of
the challenges. As a side note, this technique also jeopardizes implementations relying on system-
dependent random generators such as srand or mpz_XrandomX functions, or on other
sources such as time. 2https://man7.org/linux/man-pages/man8/ld.so.8.html 532 Attacks and Designs from CHES 2021 Challenge Attacks and Designs from CHES 2021 Challenge 4.1.2
Biased Nonces To sum up, the relations we used for our lattice attacks are the following (with ei
ranging from 0 to 999): • assuming l = 6 known most- or least-significant bits of the ephemeral key: kmsb2L + klsb = s−1(e + rd) mod n ,
(3) (3) with L = 256 −l for the MSB case and L = l for the LSB case (we considered both
cases where the known value is 0 or 63 = 26 −1), with L = 256 −l for the MSB case and L = l for the LSB case (we considered both
cases where the known value is 0 or 63 = 26 −1), • assuming the ephemeral keys are ki = tκi: t
= κ−1
0 (e0 + r0d) mod n,
κi
= t−1(s−1
i ei −s−1
i rir−1
0 e0) + κ0(s−1
i rir−1
0 s0) mod n,
(4) (4) with κi < 2248 and t an unknown constant scalar. with κi < 2248 and t an unknown constant scalar. with κi < 2248 and t an unknown constant scalar. Such an approach allowed us to break 72% of the challenges. 4.1.2
Biased Nonces As explained in Sect. 3.2, white-box designers usually generate the nonce k from the input. In the case of the WhibOx contest, the nonce is thus computed as a function of the hash,
i.e. k = f(e). However, if the function f is not carefully selected, it could happen that the
ki’s generated from different ei’s are not uniformly random. In the worst case, we have collisions such that different hash values e0 and e1 produce
the same nonce (k0 = k1). If such a collision occurs, one can recover the private key d as
explained in Sect. 3.1. Furthermore, collisions can be efficiently detected by looking at
the r part of the signature. To efficiently browse a subset of hash values in search of such
collisions, we limited ourselves to hash values with a Hamming weight equal to 1 or 2. We
thus considered 32 896 hash values and were able to break 60% of the challenges with this
technique. In those cases where we did not find any collision, we looked for biases in the nonce
generation. We used well-known lattice attacks derived from [NS03] and [FGR13] to exploit
such a potential weakness. Such attacks can recover an ECDSA private key only with the
knowledge of a few bits of the ephemeral keys of several signatures. A concrete example showing why such techniques can succeed in our context consists in
considering f = Id. Then ki = ei and with providing ei ranging from 0 to 99 we obtained
100 signatures for which the 249 most-significant bits of the nonces are 0. This bias is
more than enough for a lattice attack to recover the private key d. Lattice-based attacks can also be applied when the ephemeral key is the product of a
small random κ by another (large) constant scalar t. Such a design allows to efficiently
perform the scalar multiplication as R = [κ]T = [k]G, with T = [t]G a precomputed value. The point is that the small size of κ reduces the cost of the scalar multiplication. 4.1.4
Fault Injections Another attack method is to disturb the algorithm execution and exploit the resulting
faulty output. In the white-box context, faults can be easily induced since the attacker
can modify the binary or use debugging tools to stop the execution and, for example,
skip an instruction or modify the value of a particular register. Again, this attack can be
automated and does not require an earlier reverse engineering step. All the fault attacks that can be performed in the grey-box context are obviously also
a potential threat in the white-box context. In the case of ECDSA, different faults can be
induced on different variables to give an exploitable result. The most obvious attack is to
force the use of a weak elliptic curve during the scalar multiplication by disturbing the
curve parameters [BMM00] in order to solve the discrete logarithm problem easily. The
attacker can also force the use of biased nonces, for instance, by sticking a 32-bit word of
k at zero during several executions. The corresponding signatures can then be used to
obtain information on the key using lattice-based algorithms. Finally, modifying one byte
of d during the computation of rd may allow one to recover information on the key, as
shown in [GK04]. In addition, the white-box model offers new possibilities [PSS+18,ABF+18,DGH21]. They arise from the fact that deterministic versions of the scheme have to be implemented
due to the impossibility of relying on a source of randomness in this context. When the
algorithm is used twice on the same message, the same nonce k is derived. The attacker
may thus obtain a correct signature for a given digest e, and an erroneous one by modifying
a second execution of the same signature. To break the challenges of the WhibOx contest,
we mainly disturbed the computation of the first part of the signature r, obtaining faulty
results ˜r and ˜s = k−1(e + ˜rd) mod n. Some secret information can be deduced from the
correct and faulty signatures: (r −˜r)(s −˜s)−1 ≡(r −˜r)(k−1d(r −˜r))−1 ≡kd−1 mod n . (5) (5) Let α = kd−1 mod n. The adversary can then compute the private key: Let α = kd−1 mod n. The adversary can then compute the private key: d = e(αs −r)−1 mod n . (6) (6) It is also possible to disturb other variables, but still, the faulty value must be known
to exploit the result. 4.1.3
DCA In 2016, Bos et al. showed that although firstly described for the grey-box context, the
well-known side-channel attacks could be very well adapted to the white-box model. The
resulting attack [BHMT16] is called Differential Computational Analysis (DCA). The
principle is very similar to classical side-channel attacks: secret values are extracted from
leakage traces obtained during several executions of a cryptographic algorithm with the
help of statistical tools. The only difference relies upon the nature of the traces. Whereas
in the grey-box context, one can record the power consumption of the device in which the
algorithm is implemented, a white-box attacker can simply use software execution traces. By instrumenting the binary, he can record completely noiseless traces of all accessed
addresses and data over time, leading to much more efficient attacks. 533 Barbu et al. In theory, this attack is particularly devastating since it can be fully automated and
does not require any earlier reverse engineering step. In practice, it is quite difficult to
apply because of the size of the traces, in particular for cryptosystems such as ECDSA
that have relatively long execution time. Indeed, if the whole white-box execution were to
be recorded, each trace would easily reach several gigabytes. For instance, tracing n 64-bit
registers on a 3GHz machine during 3 seconds would lead to a single trace of 9 ∗8 ∗n Gb. Therefore, iterating over dozens of traces for a CPA would be overwhelming in time and
memory. A time-consuming step of reverse engineering allowing to select a smaller window
of the implementation before the attack is thus required, which is why we did not use this
technique to break the challenges of the WhibOx contest. 4.1.4
Fault Injections Interestingly, when one modifies the first part of the signature, if no
countermeasure is implemented, the faulty value is just given to the attacker as part of the
output. Furthermore, the attack surface is huge: the fault may happen anywhere during
the scalar multiplication. This is why we considered only this perturbation in the context
of this competition. This approach is the most successful one, allowing us to break 75% of
the challenges. 534 Attacks and Designs from CHES 2021 Challenge 4.2
Attacks Results When applying the various attack methods described above, we obtain the results presented
in Table 1. We observe that lattice and fault attacks are very efficient. Collision attacks
also give good results. Table 1: Success rate of each attack on the 97 challenges. Attack type
Percentage of
broken challenges
Hooking
32%
Bad nonce
- Collision
60%
- Lattice
72%
Fault Injection
75% We give in Appendix A the specific vulnerabilities of each of the 97 submitted challenges
as well as the corresponding private key. However, we noticed that many challenges had a low level of security, some of which
were even plain implementations. We thus excluded 30 challenges3 where the nonce and/or
the private key were manipulated in plain. Table 2 illustrates the efficiency of the attacks
presented in Sect. 4.1 on the remaining 67 challenges. We observe that hooking gives no
significant result anymore, collision and lattice attacks become less efficient, and fault
injection seems the most powerful attack. Table 2: Success rate of each attack on the 67 strongest challenges. Table 2: Success rate of each attack on the 67 strongest challenges. Attack type
Percentage of
broken challenges
Hooking
1%
Bad nonce
- Collision
49%
- Lattice
61%
Fault Injection
69% Among the 67 strongest challenges, Challenges 226 and 227 are the winning ones. In
the next section, we present the design of these two white-box implementations. 3The challenges 3, 4, 8, 10, 11, 32, 45, 54, 55, 57, 85, 97, 114, 135, 136, 139, 153, 157, 174, 185, 187,
231, 235, 267, 274, 299, 307, 320, 321, and 323 are considered as weak. 5.1
Implicit White-box Implementations The implicit framework is a method to obtain a white-box implementation of a block
cipher. Its main idea is to represent the round functions of the cipher by implicit functions
of low degree and to protect these implicit functions with large affine encodings. Before
introducing implicit white-box implementations, we need to introduce the notions of
encoding, encoded implementation, and quasilinear implicit functions. While these notions
are originally defined in [RVP22] for vectorial functions over the binary field, we extend
these notions for an arbitrary finite field. Let Fq be the finite field with q elements. A vectorial function F from the vector space
(Fq)l to (Fq)l′ is called a (l, l′) function over Fq, and its l′ component functions are denoted
by (F1, F2, . . . , Fl′). The degree of an (l, l′) function F denotes the maximum polynomial
degree of the l′ multivariate polynomials uniquely representing the component functions of
F. Definition 1. Let F be an (l, l′) function over Fq, A be an (l, l) permutation over Fq and
B be an (l′, l′) permutation over Fq. The function F = B ◦F ◦A is called an encoded
function of F, and A and B are called the input and output encodings respectively. Definition 2. Let F = F (t) ◦F (t−1) ◦· · ·◦F (1) be a vectorial function over Fq. An encoded
implementation of F, denoted by F, is an encoded function of F composed of encoded
functions of F (i), that is, F = F (t) ◦· · · ◦F (1) = (B(t) ◦F (t) ◦A(t)) ◦· · · ◦(B(1) ◦F (1) ◦A(1)) , where the input and output encodings (A(i), B(i)) are permutations over Fq such that
A(r+1) =
B(r)−1. The first and last encodings (A(1), B(t)) are called the external
encodings. where the input and output encodings (A(i), B(i)) are permutations over Fq such that
A(r+1) =
B(r)−1. The first and last encodings (A(1), B(t)) are called the external
encodings. Definition 3. Let F be an (l, l′) function over Fq. A (l + l′, l′′) function T is called an
implicit function of F if it satisfies T(u1, u2, . . . , ul, v1, v2, . . . , vl′) = 0 ⇐⇒F(u1, u2, . . . , ul) = v1, v2, . . . , vl′ . 5
Design of the Winning Challenges In this section, we describe the designs of the two winning challenges of the WhibOx
contest: Challenges 226 and 227. The designs of both challenges were inspired from
the white-box implicit framework [RVP22], which allows encoding the whole state with
large affine permutations efficiently. We implemented both challenges with the same
methodology; they only differ in some additional countermeasures used. As mentioned in Sect. 2, in the WhibOx contest, a challenge gains strawberries quadrat-
ically with time before being broken. The rule is that challenges that are either smaller,
faster, or less memory-consuming gain strawberries faster. As a result, we strategically 535 Barbu et al. posted two challenges with different trade-offs between security level and implementa-
tion cost. Challenge 227, our lightweight variant, was the winning implementation of
the contest, obtaining the highest number of strawberries (20.39). On the other hand,
Challenge 226, our hardened but heavier variant, achieved second place in the contest with
the second-highest number of strawberries (11.19). However, it stood unbroken for the
longest time (35 hours). Note that these challenges were specifically built for the WhibOx contest, where
attackers did not know the design. Against an attacker who knows the design details,
these challenges are easy to break once reverse-engineered. This section first introduces the implicit framework, then describes the shared design
approach of both challenges, and finally explains the additional countermeasures used in
each challenge. For access to the underlying software used to build these challenges, please
contact the authors from the team zerokey. 5.1
Implicit White-box Implementations An implicit implementation
of F with underlying encoded implementation F is a set of quasilinear implicit functions
{T (1), T (2), . . . , T (t)} where T (i) is an implicit function of F (i). 5.1
Implicit White-box Implementations In this case, T is said to be quasilinear if for any (u1, u2, . . . , ul) ∈(Fq)l, the function
(v1, v2, . . . , vl′) 7→T(u1, u2, . . . , ul, v1, v2, . . . , vl′) is affine over Fq. The following lemma from [RVP22] describes how the composition of affine permutations
translates to implicit functions. 536 Attacks and Designs from CHES 2021 Challenge Lemma 1. Let F be an (l, l′) function over Fq and T be a quasilinear implicit (l + l′, l′′)
function of F. Let A be an affine (l, l) permutation over Fq, B be an affine (l′, l′)
permutation over Fq, and M be a linear (l′′, l′′) permutation over Fq. Then, T ′ = M ◦T ◦
(A, B−1) is a quasilinear implicit function of F ′ = B ◦F ◦A. Lemma 1. Let F be an (l, l′) function over Fq and T be a quasilinear implicit (l + l′, l′′)
function of F. Let A be an affine (l, l) permutation over Fq, B be an affine (l′, l′)
permutation over Fq, and M be a linear (l′′, l′′) permutation over Fq. Then, T ′ = M ◦T ◦
(A, B−1) is a quasilinear implicit function of F ′ = B ◦F ◦A. The quasilinear property allows the implicit evaluation of F in a point (u1, u2, . . . , ul) by
solving the affine system T(u1, u2, . . . , ul, v1, v2, . . . , vl′) = 0 for the variables v1, v2, . . . , vl′. We are ready to present the definition of an implicit implementation. Definition 4. Let F = F (t) ◦F (t−1) ◦· · · ◦F (1) be a vectorial function over Fq, and let
F = F (t)◦F (t−1)◦· · ·◦F (1) be an encoded implementation of F. An implicit implementation
of F with underlying encoded implementation F is a set of quasilinear implicit functions
{T (1), T (2), . . . , T (t)} where T (i) is an implicit function of F (i). Definition 4. Let F = F (t) ◦F (t−1) ◦· · · ◦F (1) be a vectorial function over Fq, and let
F = F (t)◦F (t−1)◦· · ·◦F (1) be an encoded implementation of F. 5.2
White-boxing ECDSA Signature Algorithm Using the Implicit Frame
work In the WhibOx contest, designers submitted white-box implementations of the ECDSA
signature algorithm on the NIST P256 curve. As opposed to the standard ECDSA algorithm
(cf. Algorithm 1), the algorithm for the WhibOx contest (hereafter denoted by E) takes
as input the 256-bit message digest. The private key is not an input of the algorithm;
it is freely chosen by the designer, but it is fixed (hard-coded) in the implementation. Algorithm 2 depicts a high-level overview of this deterministic variant of ECDSA, where
the deterministic nonce derivation mechanism is chosen freely by the designer. Algorithm 2: Deterministic ECDSA signature algorithm for WhibOx contest
Input
: 256-bit message digest e
Output : the signature (r, s)
1 state ←e
2 k, state ←NonceDerivation(state)
3 R = (Rx, Ry) ←[k]G
4 r ←Rx mod n
5 s ←k−1(e + rd) mod n
6 if r = 0 or s = 0 then
7
Go to step 2
8 end
9 Return (r, s) Algorithm 2: Deterministic ECDSA signature algorithm for WhibOx contest
Input
: 256-bit message digest e
Output : the signature (r, s) 1 state ←e
2 k, state ←NonceDerivation(state)
3 R = (Rx, Ry) ←[k]G
4 r ←Rx mod n
5 s ←k−1(e + rd) mod n
6 if r = 0 or s = 0 then
7
Go to step 2
8 end
9 Ret
(
) The main steps of E can be represented by the functions E(1) and E(2). The Fp-function
E(1) is given by E(1)(e) = (Rx, k, e) ,
(7) (7) which takes as input e ∈Fp and computes the scalar multiplication R = [k]G over Fp. On
the other hand, the Fn-function E(2) can be written as E(2)(R′
x, k′, e′) = (r, s) ,
(8) (8) which takes as input (R′
x, k′, e′) = (Rx mod n, k mod n, e mod n) and computes (r, s) =
(R′
x, k−1(e + rd)) over Fn. Inspired from the implicit framework, we built the white-box implementations of
Challenges 226 and 227 by encoding E(1) and E(2) with affine permutations and obtaining
low-degree implicit round functions of E(1) and E(2), the encoded functions of E(1) and
E(2). We will first describe the implicit implementation of E(1) and then that of E(2). 537 Barbu et al. 5.2.1
White-boxing the Scalar Multiplication To build an implicit implementation of E(1), we need first to decompose E(1) as the
composition of Fp-functions that we call round functions. Then we explain how to encode
these round functions and how to obtain low-degree quasilinear implicit functions of the
encoded round functions. Decomposing E(1) into round functions. The function E(1)(e) = (Rx, k, e), mainly
consists of the scalar multiplication r = [k]G of the nonce k and the point G. For the
scalar multiplication, we perform the following subroutine. First, we precompute and
store a list of t random point pairs on the curve, i.e., (Gi,0 = [ki,0]G, Gi,1 = [ki,1]G) for
1 ≤i ≤t . Then, for each pair we select one of the two points together with its logarithm,
denoted as (Gi,bi, ki,bi), where bi ∈{0, 1} and 1 ≤i ≤t . We add the selected points and
the selected logarithms, obtaining the scalar multiplication G1,b1 + · · · + Gt,bt = [k1,b1 + · · · + kt,bt]G = [k]G ,
(9) (9) where k = k1,b1 + · · · + kt,bt. This selection is done in a deterministic way depending
on the bits (e1, e2, . . . , e256) of the hash e, the only source of entropy in the algorithm. Moreover, the selection is done with Fp-arithmetic operations rather than with conditional
instructions, so that each iteration only performs Fp operations. The subroutine is given
in Algorithm 3. It is worth pointing out that the values ki,j are chosen such that the sum of max(ki,0, ki,1)
for all i is always smaller than n. That is, we have k < n. Hence, r and s are never 0. In
this way, we avoid the trivial case, i.e., avoid going to Step 7 in Algorithm 2. Algorithm 3: Round-based scalar multiplication used in E(1)
Input
: the bits (e1, e2, . . . 5.2.1
White-boxing the Scalar Multiplication , e256) of the hash e (little-endian order)
Output : x-coordinate of [k]G and the message-dependent scalar k
/* Round 1:
input e1, k1,0, k1,1, G1,0, G1,1 embedded values
*/
1 R ←[1 −e1]G1,0 + [e1]G1,1
2 k ←(1 −e1)k1,0 + e1k1,1
/* Round i:
input (R, k, ei), ki,0, ki,1, Gi,0, Gi,1 embedded values
*/
3 for 2 ≤i ≤t do
4
R ←R + [1 −ei]Gi,0 + [ei]Gi,1
5
k ←k + (1 −ei)ki,0 + eiki,1
6 end
7 return Rx, k
// (Rx, Ry) = R = [k]G /* Round 1:
input e1, k1,0, k1,1, G1,0, G1,1 embedded values
1 R ←[1 −e1]G1,0 + [e1]G1,1
k ←(1
)k
+
k [
]
,
[
]
,
2 k ←(1 −e1)k1,0 + e1k1,1 /* Round i:
input (R, k, ei), ki,0, ki,1, Gi,0, Gi,1 embedded values
3 for 2 ≤i ≤t do /* Round i:
input (R, k, ei), ki,0, ki,1, Gi,0, Gi,1 embedded values
3 for 2 ≤i ≤t do 4
R ←R + [1 −ei]Gi,0 + [ei]Gi,1
5
k ←k + (1 −ei)ki,0 + eiki,1 6 end // (Rx, Ry) = R = [k]G 7 return Rx, k By considering the precomputed points Gi,j and their logarithms ki,j as fixed values
and by representing the elliptic curve additions by operations over Fp, we can represent
E(1) given by Algorithm 3 as an iterated function over Fp, that is, E(1) = F (t) ◦· · · ◦F (2) ◦F (1) ,
(10) (10) where each (4, 4) round function F (i) is given by the following component functions F (i)
1,2(u1, u2, u3, u4) = (u1, u2) + [1 −ei]Gi,0 + [ei]Gi,1
F (i)
3 (u1, u2, u3, u4) = u3 + (1 −ei)ki,0 + eiki,1 ,
F (i)
4 (u1, u2, u3, u4) = u4 + ei2i . (11) (11) F (i)
4 (u1, u2, u3, u4) = u4 + ei2i . 538 Attacks and Designs from CHES 2021 Challenge Note that the input value of F (1) is (0, 0, 0, 0), and each round function F (i) takes the
hash bit ei as an additional input value. The pair of component functions F (i)
1,2 return a
point on the elliptic curve with F (i)
1
and F (i)
2
the x- and y- coordinates respectively. 5.2.1
White-boxing the Scalar Multiplication The
component function F (i)
3
updates and returns the current nonce, while F (i)
4
updates the
current hash. The hash e is recomputed so that E(1) can output the hash e and provide it
in an encoded form to E(2) . Encoding the round functions. To protect the round functions, we encode each round
with random Fp-affine permutations A(i), obtaining the encoded round functions F (i) = A(i) ◦F (i) ◦(A(i−1))−1, 1 ≤i ≤t . (12) (12) In other words, the input and output encodings of F (i) are
(A(i−1))−1, A(i)
, and the
composition of the round functions cancels all intermediate encodings except (A(0))−1 and
A(t), that is, E(1) = F (t) ◦· · · ◦F (2) ◦F (1) = A(t) ◦F (t) ◦· · · ◦F (2) ◦F (1) ◦(A(0))−1 ,
(13)
is the number of rounds. The input encoding (A(0))−1 of F (1) is set as the identity
g to preserve the input-output behaviour of E. E(1) = F (t) ◦· · · ◦F (2) ◦F (1) = A(t) ◦F (t) ◦· · · ◦F (2) ◦F (1) ◦(A(0))−1 ,
(13)
is the number of rounds The input encoding (A(0))−1 of F (1) is set as the identity E(1) = F (t) ◦· · · ◦F (2) ◦F (1) = A(t) ◦F (t) ◦· · · ◦F (2) ◦F (1) ◦(A(0))−1 ,
(13) (13) where t is the number of rounds. The input encoding (A(0))−1 of F (1) is set as the identity
mapping to preserve the input-output behaviour of E. Obtaining the implicit round functions. Now we proceed to obtain an implicit round
function T (i) of each encoded round function F (i). To this end, we first show how to derive
an implicit function of the elliptic curve addition. Let ADD(Px, Py, Qx, Qy) = (Rx, Ry) be the vectorial Fp-function denoting the elliptic
curve addition P + Q = R where P and Q are not the point at infinity and where P and
Q have different x-coordinates4. In this case, R can be written as [KL14] Rx = (Qy −Py)2((Qx −Px)2)−1 −Px −Qx
Ry = (Qy −Py)(Px −Rx)(Qx −Px)−1 −Py . (14) (14) From Eq. (14) it is easy to see that P + Q = R holds if and only if the relations From Eq. 4The only elliptic curve additions performed in E(1) are the additions between the random points Gi,j,
and the probability that these additions involve points at infinity or points with the same x-coordinate is
negligible. 5.2.2
White-boxing the Computation of s Now we turn our attention to E(2), the second step of the signing algorithm, where we
compute r = Rx mod n and s = k−1(e + dRx) mod n, and output the signature (r, s). As opposed to E(1), we do not decompose E(2) but build a single (vectorial) quasilinear
implicit function of E(2) = E(2) ◦(A(t))−1, the encoded version of E(2). (
)
The vectorial Fn-function T (t+1) defined as (
)
The vectorial Fn-function T (t+1) defined as (
T (t+1)
1
(Rx, Ry, k, e; s, r) = ks −e −dRx
T (t+1)
2
(Rx, Ry, k, e; s, r) = r −Rx
(19) (19) is a quasilinear implicit function of E(2). In other words, the polynomial system T (t+1) =
{T (t+1)
1
, T (t+1)
2
} implicitly defines E(2) because (s, r) = E(2)(Rx, Ry, k, e) if and only if
T (t+1)(Rx, Ry, k, e; s, r) = 0. Moreover, the system is affine in r and s, so after plugging
in values for Rx, Ry, k and e, the system can be solved for r, s efficiently. The encoded version E(2) gets as input u = A(t)(Rx, Ry, k, e), where A(t) is the affine
function that protects the last round of E(1). By Lemma 1, we build the implicit round
function of E(2) as T (t+1)(u; s, r) = M · T (t+1)((A(t))−1(u); s, r) ,
(20) (20) where (A(t))−1 is the inverse of A(t) mod n, and where M is a random invertible 2-by-2
matrix mod n. The function T (t+1) is quasilinear, and we can implicitly evaluate E(2)
on input u = A(t)(Rx, Ry, k, e) by plugging u in the first slot of T (t+1) and solving the
remaining system (which is affine) for r and s over Fn. However, the fact that E(1) works in Fp while E(2) works in Fn causes a problem. The
input to E(2) is u = A(t)(Rx, Ry, k, e) reduced by mod p, so (A(t))−1(u) is in general not
equal to (Rx, Ry, k, e) mod n if there are overflows in the computation of u. is a quasilinear implicit function of F (i) for 1 ≤i ≤t. The white-box implementations of Challenges 226 and 227 contain this implicit imple-
mentation of E(1), with underlying encoded implementation E(1), given by the t implicit
round functions {T (1), . . . , T (t)} in Eq. (18). Moreover, E(1) is evaluated in our white-box
implementations by implicitly evaluating the encoded round functions F (i). In other words,
given the output u of the round i−1, the output v of the ith round is computed by finding
the solution of the affine system T (i)(u; v) = 0 for v. Barbu et al.
is a quasilinea
The white-
mentation of E
round function
implementatio
given the outp
the solution of
5.2.2
White
Now we turn
compute r =
As opposed to
implicit functi
The vector
is a quasilinea
{T (t+1)
1
, T (t+1)
2
T (t+1)(Rx, Ry
in values for R
The encode
function that
function of E(
where (A(t))−
matrix mod n
on input u =
remaining syst
However, t
input to E(2)
equal to (Rx, R
vector of overfl
then (A(t))−1(
affine map A(t
To deal wit
vector o and s
If the guess is
r, s will be rec
to get a candi
output the firs
need to protec
If A(t) was
would be very 539 Barbu et al. is a quasilinear implicit function of F (i) for 1 ≤i ≤t. 5.2.1
White-boxing the Scalar Multiplication (14) it is easy to see that P + Q = R holds if and only if the relations From Eq. (14) it is easy to see that P + Q = R holds if and only if the relations (Px + Qx + Rx)(Qx −Px)2 = (Qy −Py)2
(15)
(Ry + Py)(Qx −Px) = (Qy −Py)(Px −Rx)
(16) (15)
(16) (15)
(16) (16) hold. Note that these relations have degree 3 (degree 1 over the variables Rx and Ry),
while Eq. (14) has a high degree due to the inversion over Fp. g
p
Px, Py, Qx, Qy, Rx, Ry) = (IMP0, IMP1) defined by (
)
p
Thus, the function IMP(Px, Py, Qx, Qy, Rx, Ry) = (IMP0, IMP1) defined by IMP0 = (Qy −Py)2 −(Px + Qx + Rx)(Qx −Px)2
IMP1 = (Qy −Py)(Px −Rx) −(Ry + Py)(Qx −Px)
(17) (17) is a quasilinear implicit round function of ADD with degree 3, assuming none of the points
is the point at infinity and assuming the x-coordinates of the points are different. From the above implicit function of the elliptic curve addition, it is easy to derive a
quasilinear implicit function T (i) of each round function F (i). Then, we sample a linear
permutation M (i) for each round i, and by Lemma 1 the function T (i) = M (i) ◦T (i) ◦
(A(i−1))−1, (A(i))−1
(18) (18) 4The only elliptic curve additions performed in E(1) are the additions between the random points Gi,j,
and the probability that these additions involve points at infinity or points with the same x-coordinate is
negligible. 5.2.2
White-boxing the Computation of s Let o be the
vector of overflows mod p, such that u = A(t)(Rx, Ry, k, e) −po ,
(21) (21) then (A(t))−1(u) = (Rx, Ry, k, e) −pL−1
t (o) mod n, where Lt is the linear part of the
affine map A(t) (i.e., A(t)(x) = Lt(x) + c for some constant term c). To deal with this problem, we correct for the overflow mod p by guessing the overflow
vector o and setting u′ = u + po before plugging u′ into T (t+1)(u; s, r) to solve for (r, s). If the guess is correct, then u′ is equal to A(t)(Rx, Ry, k, e) over the integers, so the correct
r, s will be recovered. Therefore, we repeatedly run the last step with random guesses of o
to get a candidate signature (r, s). Then we run the verification algorithm on (r, s) and
output the first (r, s) for which the verification algorithm succeeds. Note that we do not
need to protect the verification algorithm because it does not use secret information. If A(t) was a random affine map with entries of size up to p, then guessing o correctly
would be very unlikely. Therefore, we choose the affine map A(t) with small entries. For Attacks and Designs from CHES 2021 Challenge 540 xample, we could use A(t)(Rx, Ry, k, e) =
1
0
1
2
1
1
2
0
0
1
2
1
1
2
0
1
Rx
Ry
k
e
+ c . (22) (22) With this choice, the weight of each row is four, so there are at most four overflows mod p
in each entry of u, which means o can be guessed more easily. Not all guesses are equally
likely, (e.g., o = [4, 4, 4, 4] only occurs if Rx, Ry, k, e are all quite big, which is unlikely). Rather than inefficiently guessing o ∈[0, 4]4 at random, we precompute a list of guesses
L ordered from more likely to be correct to less likely, and we iterate through the list of
guesses in that order. The white-box implementations of Challenges 226 and 227 contain the implicit function
T (t+1), which allows the implicit evaluation of E(2), together with the correction for the
overflow mod p described above and summarized in Algorithm 4. 5.2.2
White-boxing the Computation of s Note that the severe restriction on the size of the entries of A(t) makes the conversion
from Fp to Fn one of the most vulnerable points in the white-box implementation. In
particular, an attacker knowing the specifications of the design can easily recover A(t) by
exhaustive search if no additional countermeasures are used. Algorithm 4: White-box implementation of ECDSA signature algorithm for
winning challenges
Input
: 256-bit hashed message digest e
Output : the signature (r, s)
1 e ←e mod p
2 (v1, v2, v3) ←E(1)(e)
// implicit evaluation
3 for o in L do
4
(u1, u2, u3) ←(v1, v2, v3) + p · o
5
(r, s) ←E(2)(u1, u2, u3)
// implicit evaluation
6
if
VerifySignature(r, s, e) = valid then
7
return (r, s)
8
end
9 end Algorithm 4: White-box implementation of ECDSA signature algorithm for
winning challenges // implicit evaluation 5https://tigress.wtf/transformations.html
6https://github.com/CryptoExperts/whibox_contest_submission_server 5.3
Additional Countermeasures The representation of the implicit round functions as systems of multivariate polynomials
allows applying countermeasures from multivariate public-key cryptosystems. In fact,
Challenges 227 and 226 only differ in the additional countermeasures used. In particular, we considered two techniques. First, we obfuscated the components (seen
as polynomials) of the implicit round functions T (i) by multiplying them with random
polynomials in the input variables. Note that the multiplication of input variables preserves
the quasilinear property. Moreover, the image of a random polynomial is non-zero with
high probability, and multiplying an equation with a non-zero value does not change its
solution set. In the unlikely case that one of the added polynomials vanishes, the output of
the corresponding implicit function will be invalid, and no valid signature will be obtained. To prevent this extreme case, we made the first implicit round function dependent on an
initial value; if no valid signature is found, we simply repeated the whole process with a
different initial value. 541 Barbu et al. This first technique increases the degree of the implicit round functions, significantly
increasing the implementation size. Thus, for the lightweight Challenge 227 we only applied
this technique to raise the degree of the components to the total degree of the functions,
but for Challenge 226 we multiplied with polynomials of higher degree to increase the total
degrees of the implicit round functions. The final degrees are listed in Tables 3 and 4. The second technique we used was adding additional variables and components to
the implicit round functions but preserving the input-output behaviour of the underlying
encoded round functions. In particular, to avoid the bias in the most significant part of the nonce k due to the
constraint k < n (see Section 5.2.1), we duplicated the nonce variable and its equations so
that E(1) outputs an additional nonce variable k′ similarly to k, k′ = k′
1,b1 + · · · + k′
t,bt,
X
i
max(k′
i,0, k′
i,1) < n , and E(2) uses the sum of the nonce variables k + k′ as the final nonce. On top of that,
instead of e, the input L(e) is given to E(1) for some hard-coded low-degree encoding L,
and its inverse L(−1) is composed to E(2) to recover e. 5.3
Additional Countermeasures Note that this is a minor trick since
the encoding L is not merged or composed with other functions (as opposed to the other
encodings A(i)), and the computation L(e) is done in clear. Since adding additional variables and equations also introduces significant overhead
in the implementation size, we only applied the second technique to Challenge 226. In
particular, we added two variables and two equations in the implicit round functions of
E(1), and two variables and one equation in those of E(2). We also used Tigress [Col] for both challenges to obfuscate the C source code. Tigress
is an obfuscator for C language that protects programs against dynamic and static reverse
engineering attacks. We used the transformations5 Flatten (flattens the code to remove
structured flow), AntiTaintAnalysis (disrupts tools that make use of dynamic taint
analysis), AddOpaque (adds opaque predicates), EncodeLiterals (replaces integers
and strings with run-time expressions) and CleanUp (renames variables and functions). 5.4.2
Security Analysis Challenge 227 can be broken in several ways. Here, we explain how the attacks of Sect. 4
allow one to recover the secret key of Challenge 227 or why they do not work. Hooking shared libraries. During the implicit evaluation of E(2) for the valid input
u = A(t)(Rx, Ry, k, e), the affine system T (t+1)(u; s, r) is solved for r and s. By denoting
the entries of M as M =
m0
m1
m2
m3
, it is easy to see that this affine system is given by
the equations the equations (
c1s + c3r −c5 = 0
c2s + c4r −c6 = 0
(23) (23) where the coefficients ci are given by c1 = m0k,
c3 = m1,
c5 = m0e −m0dRx −m1Rx
c2 = m2k,
c4 = m3,
c6 = m2e −m2dRx −m3Rx
. (24) (24) We stress that k, e, and Rx do not appear in the clear. They are expressed as linear
combinations of the input u = A(t)(Rx, Ry, k, e) of E(2). Nevertheless, the coefficients ci
are operated in the clear during the Gaussian elimination, and the adversary can obtain
their values. Some of these coefficients are sensitive. In particular, if the attacker manages to find
c1 during the computation of two different signatures (r, s) and (r′, s′), he may solve the
following system of two equations with two unknowns (m0 and d) in Fn: (
m0(e + rd) = c1s
m0(e′ + r′d) = c1s′
. (25) (25) Therefore, recovering the value c1 = m0k mod n for two different signatures allows an
attacker to compute the private key. Finding the interesting values inside the white-box may seem difficult without a reverse
engineering step. However, the attack turns out to be easily automated on challenges
that use the GMP library, such as Challenge 227. Indeed, one of the coefficients of s, say
c1 = m0k, will be inverted modulo n during the resolution of the system, and finding this
inversion is easy when one can simply trace the calls to the function mpz_invert(). The
team TheRealIdefix was thus able to efficiently apply this attack on Challenge 227 during
the contest without any reverse engineering step. 5.4.1
Description Following the method described in Section 5.2, we built Challenge 227 (keen_ptolemy)
as a lightweight white-box implementation. The only additional countermeasures from
Section 5.3 included in Challenge 227 are the degree increase of each component to the
total degree of the corresponding vectorial function and the code obfuscation by Tigress. Challenge 227 was the winning implementation of the WhibOx contest; it achieved the
highest number of strawberries (20.39) and stood for 33 hours as the second-longest. (
)
Table 3 describes the memory complexity of Challenge 227 (after applying the additional
countermeasures) by describing {T (1), . . . , T (t)} and T (t+1), the implicit round functions
of E(1) and E(2) respectively. The number of coefficients in Table 3 denotes the maximum
number of non-zero coefficients of a quasilinear vectorial function with a given number of
input variables, components, and degrees. If each coefficient is represented with 256 bits,
{T (1), . . . , T (t)} and T (t+1) require in total roughly 4 MB. After obfuscating the code with Tigress, the size of the final C source code of Challenge
227 is 4.4 MB. In a modern personal laptop with the environment6 provided by the
competition, the size of the compiled binary is 4.42 MB, and the average running time and 542 Attacks and Designs from CHES 2021 Challenge Table 3: Information of the implicit round functions T (i) of Challenge 227. T (1)
{T (2), . . . , T (t−1)}
T (t)
T (t+1)
input variables
2+4
5+4
5+3
3+2
number of components
4
4
3
2
degree
3
3
4
2
number of coefficients
27 × 4
130 × 4
255 × 3
18 × 2 Table 3: Information of the implicit round functions T (i) of Challenge 227. RAM consumed are 0.04 seconds and 6.14 MB respectively. The code obfuscation did not
impact the running time but increase the binary size by 8% and the average RAM by 3%. 5.4.2
Security Analysis This attack may seem very specific, but multiplying the nonce with a constant may
appear as an easy way to protect the inversion step, and could very well be used by
designers. This attack shows it is not a robust countermeasure. 543 Barbu et al. Biased nonces. There exists a more generic way of breaking Challenge 227. Indeed, the
way the ephemeral key is constructed (see Sect. 5.2.1) opens the way for an attack using
lattice reduction techniques. Given that the ephemeral key k is obtained by summing 256 scalars ki,j according
to each bit of the input, one can obtain the following signatures by selecting a couple of
hashes (e0, ei), with e0 = 0 and ei = 2i :
s0
=
255
P
j=0
kj,0
!−1
(e0 + r0d) mod n
si
=
ki,1 +
255
P
j=0,j̸=i
kj,0
!−1
(ei + rid) mod n
,
(26) (26) which allows us to construct 256 equations involving only one of the ki,j: ki,1 +
255
P
j=0,j̸=i
kj,0 −
255
P
j=0
kj,0
=
s−1
i (ei + rid) −s−1
0 (e0 + r0d) mod n
ki,1 −ki,0
=
s−1
i ei −s−1
0 e0 + (s−1
i ri −s−1
0 r0)d mod n
. (27) (27) Now, the additional constraint k < n lets us estimate that each ki,j is sampled from
J0, ⌊n/256⌋K. Consequently, |ki,1 −ki,0|n = |s−1
i ei −s−1
0 e0 + (s−1
i ri −s−1
0 r0)d|n <
j n
256
k
,
(28) (28) with |y|n := min
a∈Z |y −an| to denote the distance of y ∈R to the closest integer multiple of
n. with |y|n := min
a∈Z |y −an| to denote the distance of y ∈R to the closest integer multiple of
n We recognize in Eq. (28) an instance of the Hidden Number Problem (HNP) [BV96]. Indeed, we are given many HNP inequalities of the form: |αti −ui|n <
j n
256
k
,
(29) (29) with ti = s−1
i ri −s−1
0 r0, ui = s−1
0 e0 −s−1
i ei and the hidden number α is the private key d. Solving HNP instances in the context of ECDSA given inequalities such as Eq. (29)
has been described numerous times in the literature. We refer the reader to [JSSS20]
for a more detailed description7. 7We also highlight that the authors of [JSSS20] made their code available at https://github.com/crocs-
muni/minerva. 5.4.2
Security Analysis In particular, the authors detail the reduction of the
HNP instance to a Closest Vector Problem instance in a specific lattice as well as the
construction of this lattice. Finally, we use 75 relations such as Eq. (28) (out of the 255 we can establish) to build
a lattice whose reduction allows us to recover the private key d. DCA. As explained in Sect. 4, we did not mount this side-channel attack during the
contest. With the design of Challenge 227 in hand, we can see that it would have been
unsuccessful, at least at the first order, thanks to the linear masking scheme used to protect
all the implementation. Fault injections. None of the faults injected on Challenge 227 during the contest were
exploitable. This can be explained by the presence of the signature verification that is used
to check if the guess for the overflow between E1 and E2 is correct. If a fault is induced,
the signature is rejected and recomputed. g
Of course, a reverse engineering step could be performed to get rid of this verification,
but this would be quite time-consuming. Furthermore, even without this verification, 544 Attacks and Designs from CHES 2021 Challenge the fault attack is still not trivial to perform because of the linear masking scheme. In
particular, Rx is not manipulated directly in E2. It is expressed as a linear combination of
the input A(t)(Rx, Ry, k, e), so modifying one of the shares would probably also fault e, k
or Ry, making the resulting faulty signature unexploitable. 5.5.1
Description Challenge 226 (clever_kare) was the second white-box implementation that we built
following the method described in Section 5.2 and including all the additional countermea-
sures from Section 5.3. While this challenge stood for the longest (35 hours), Challenge
226 achieved the second-highest number of strawberries (11.19) due to its higher time and
memory complexity than Challenge 227. Table 4 describes the memory complexity of {T (1), . . . , T (t)} and T (t+1) of Challenge
226 after applying the additional countermeasures. Given each coefficient as a 256-bit value,
{T (1), . . . , T (t)} and T (t+1) require in total roughly 15 MB. The impact of the additional
countermeasures on the number of equations, the degree, and the number of variables can
be seen by comparing this table with Table 3. Table 4: Information of the implicit round functions T (i) of Challenge 226. Table 4: Information of the implicit round functions T (i) of Challenge 226. T (1)
{T (2), . . . , T (t−1)}
T (t)
T (t+1)
input variables
2+6
7+6
7+5
5+2
number of components
6
6
5
2
degree
3
3
4
5
number of coefficients
37 × 6
322 × 6
854 × 5
504 × 2 The size of the final C source code of Challenge 226 is 17.54 MB, the size of the
compiled binary is 15.44 MB, and the average running time and RAM consumed are 0.15
seconds and 17.27 MB, respectively. The code obfuscation did not significantly impact the
performance of Challenge 226; the running time, the binary size, and the average RAM
increased by less than 1%. in d in order to obtain the secret key. in d in order to obtain the secret key. Therefore, this challenge can be easily broken once reverse-engineered. Nevertheless,
such an attack is quite time-consuming, and resisting TheRealIdefix’s automated attacks
on ECDSA in the white-box contest is already an achievement considering that only 5
challenges resisted these attacks during the contest. 5.5.2
Security Analysis During the WhibOx contest, the team theRealIdefix did not manage to break Challenge 226
with any of the automated attacks presented in Sect. 4.1. DCA and fault injection, which
fail to break Challenge 227, are also not applicable to Challenge 226 since it is designed to
be more secure. Moreover, the two attacks presented in Sect. 5.4.2 also fail to recover any
secret information. Hooking shared libraries. As mentioned in Sect. 5.3, Challenge 226 implements an
additional countermeasure which consists in multiplying the components of the implicit
round functions T (i) with random polynomials in the input variables. Hence, the coefficients
of s in the system T (t+1)(u; s, r) for the valid input u are no longer fixed multiples of k,
and the attack cannot be mounted anymore. Biased nonces. Likewise, the additional countermeasures implemented in Challenge 226
makes the lattice attack described in Sect. 5.4.2 fail. The additional variable k′ alone
would only reduce by 1 bit the bias observed in Eq. 29 and the attack would still be 545 Barbu et al. practical. However, without the knowledge of the encoding L introduced in this challenge
it is impossible to exhibit such a bias leading to key recovery. practical. However, without the knowledge of the encoding L introduced in this challenge
it is impossible to exhibit such a bias leading to key recovery. As explained, none of the automated attacks that the TheRealIdefix team used during
the contest were successful on Challenge 226. Nevertheless, with the design in hand, one
could easily break this challenge. Indeed, knowing that the matrix M of the last affine
encoding A(t) contains small entries, the attacker could, for example: • Compute two signatures (r1, s1) and (r2, s2) for two messages e1 and e2 and extract
the two valid E(2) inputs u1 = A(t)(v1) and u2 = A(t)(v2) from the execution. Note
that v1 −v2 contains the nonce difference κ = k1 −k2. • Compute two signatures (r1, s1) and (r2, s2) for two messages e1 and e2 and extract
the two valid E(2) inputs u1 = A(t)(v1) and u2 = A(t)(v2) from the execution. Note
that v1 −v2 contains the nonce difference κ = k1 −k2. • Find κ by exhaustive search over M; for each guess M ′, obtain a candidate v1 −v2 =
(M ′)−1(u1 −u2) and check if one of its entries κ satisfies (κG)x = r1 −r2. 5.5.2
Security Analysis • Find κ by exhaustive search over M; for each guess M ′, obtain a candidate v1 −v2 =
(M ′)−1(u1 −u2) and check if one of its entries κ satisfies (κG)x = r1 −r2. • Solve the equation s−1(e1 + r1d) −s−2(e2 + r2d) = κ
(30) (30) s−1(e1 + r1d) −s−2(e2 + r2d) = κ 6
Conclusion This work describes several attack techniques and designs used in the WhibOx 2021 contest. We explained the attack methods used by the team TheRealIdefix, which broke the most
challenges, and we showed the success of each method against all the implementations in
the contest. Fault attacks were the most efficient and effective ones; collision and lattice
attacks were slightly less efficient, and hooking succeeded against weak implementations
only. Among the white-box implementations that resisted these attacks, the one with the
highest score was Challenge 226 (clever_kare). This challenge, together with Challenge
227 (keen_ptolemy), was submitted by the team zerokey, and they obtained the second-
highest and the highest score in the contest, respectively. In this work, we described the
design methodology of these two challenges, which was inspired by the implicit white-box
framework. The large number of implementations broken by our automated attacks and the fact
that no challenge survived more than two days show that securing ECDSA in the white-box
model is a challenging problem. White-box attacks benefit from the huge progress in
side-channel and fault attacks against ECDSA implementations, but not much research
has been done on the design part. To this end, our designs provide insightful examples for
future works, and our attacks highlight the weak points future research should address. One of the main challenges specific to white-boxing ECDSA is the conversion from Fp to
Fn. While grey-box countermeasures can protect this step (e.g., with Arithmetic-to-Boolean
and Boolean-to-Arithmetic mask conversions), these techniques rely on randomness, which
is ineffective in white-box implementations. In particular, the conversion from Fp to Fn is
one of the weakest points in our designs, and further research in white-boxing the field
conversion is needed. 546 Attacks and Designs from CHES 2021 Challenge Acknowledgment The authors would like to thank the other members of the TheRealIdefix team: Yannick
Bequer, Luk Bettale, Laurent Castelnovi, Thomas Chabrier, Nicolas Debande, Roch
Lescuyer, Sarah Lopez and Nathan Reboud. Adrián Ranea is supported by a PhD
Fellowship from the Research Foundation – Flanders (FWO) under grant No. 11E1921N. Chaoyun Li is an FWO post-doctoral fellow under grant No. 1283121N. Ward Beullens is
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LNCS, pages 49–58. Springer, Heidelberg, September 2009. [Van92]
Scott Vanstone. Responses to NIST’s Proposal. Communications of the ACM,
35:50–52, 1992. [WMGP07] Brecht Wyseur, Wil Michiels, Paul Gorissen, and Bart Preneel. Cryptanalysis
of white-box DES implementations with arbitrary external encodings. In
Carlisle M. Adams, Ali Miri, and Michael J. Wiener, editors, SAC 2007,
volume 4876 of LNCS, pages 264–277. Springer, Heidelberg, August 2007. [XL09] [XL09]
Yaying Xiao and Xuejia Lai. A secure implementation of white-box aes. In
2nd International Conference on Computer Science and its Applications, pages
1–6. IEEE, 2009. 550 Attacks and Designs from CHES 2021 Challenge A
Attacks Summary Table Table 5 presents for each challenge submitted to WhibOx 2021 the successful attacks and
the value of the corresponding key. During the contest, we broke 92 out of 97 challenges. The 5 remaining challenges have been broken a posteriori. Table 5: Vulnerabilities of the various challenges. Table 5: Vulnerabilities of the various challenges. 8This challenge has not been broken by our automatic tools so we have used reverse engineering techniques. A
Attacks Summary Table g
Challenge
Hooking
Collision
Fault
Lattice
Key
3
✓
✓
✓
✓
45189C81EADEE03202BFA06EAA15831789F0C76575508A563E1A739CA37B87BE
4
✓
✓
✓
✓
22BEF7AC4C31B2B98227D95B5EB49AF23343004CF2713FED48BEC3B5B7C3D24D
8
✓
✓
✓
✓
F484955872415A32B1B5B731EA1A8C729458055C17DC5FE9C57BCB39D1A40BFE
10
✓
✓
✓
✓
32D67733DF0D0257DA78E92752494CFD5112E303BA1413388126EA33BB60AEFC
11
✓
✓
✓
✓
E7F3287D91B528D78BF19D5E62828C845E1A4027A3E1F988B62B7407EBF5CF38
12
✓
✓
773F0C0FFACB531F50FAE0987D2B8972FE1B9231BBF46859F475BAFB45257FED
13
✓
✓
034332A23341538143FDB88F314FD942501FF8B6BA6A14D5013F1FC0984924BE
15
✓
✓
3F77C51259E1C8CC48217A66998CCF3212A17120B0FCA09163E300576DFCD9E7
16
✓
23773F0BECFACB534250FAE0987D2B8969D1AFD7EF942F148746DC73A3C6B39A
32
✓
✓
✓
32D67733DF0D0257DA78E92752494FFD5112E303BA14133FF126EA33BB60AEFC
33
✓
CD9540B70C2F92B2894594CABC4E724203A615B9144C459714758BC3CAA12242
34
✓
✓
✓
70253E6587D04D7A9A30A1461A80FCD235B28FBFC11FE8534CDFCE0A341C9257
36
✓
10D7EF92F06DF6EB94F2F344085DAD51D3A550E24A4569922460F579CB5DF11A
38
✓
70C3A9F11773C8DD795FD7942B5DB448FDFA5D12E6EC387691A19B6E523AE6AE
42
✓
1BEDDC1DD79F8856BF2E1FD66EB194073D60FEC658C5D0E2C8BAE02DC72ADF65
44
✓
B519BB44EC5BF3380CB2DF555F39ED836CDBF4961E43A66C218FADB211BF468C
45
✓
✓
✓
✓
32D67733DF3D0257DA78E92752494FFD5112E303BA14133FF126EA33BB60AEFC
50
✓
7A7AA97370B1EE16D64C71C7C5BC8C9F9456FBEA603780883399D89DA43F8A15
54
✓
✓
✓
✓
32D67733DF3D0257DA78E92752494FFD5112E22222222222F126EA33F6E49790
55
✓
✓
✓
✓
00498594859849584954E92752494FFD5112E2222222EE22F126EA33F6E49790
57
✓
✓
✓
✓
7D1BBD475A8EB5AF7DDB238CD8A67F86B601E0EA101C04036849B31F96CA6083
58
✓
✓
✓
BD3026C700A75B5970807802E2B47C2A892DF85E3CE57366D335EEBABCAAE255
61
✓
✓
✓
F4DDC95A88146CF52DEC752E737F8E3FB16AE4F6B7E726068946F3B0BA0C8E95
62
✓
✓
✓
0A99EB20F9DE4DD7607288B8B766F6217FE5D2CE6DDD51C6159941066AF192ED
66
✓
✓
✓
8836AC84AA148440A20628810CA65EB038BB625841275CC11590D8F5BC7F1BAC
70
✓
✓
✓
21A35C57E23B2D23ADDA19EA30325F1B532DA645489E29E47A13E92CA1F6670C
71
✓
✓
✓
588BEED930355AF54EEBAFAA46A7D26DA378A36EF5CD15D1F876D753A395F8AF
72
✓
✓
✓
B7A9B0F7661FC9A1DEC001F2C2C9EAE08748AEB187E1247726663E3DD1AB36BF
73
✓
✓
✓
4DAA29CBD634F28137499B9557104FDD36D4D4EFDFE87EFC0D8BD03555F8497F
74
✓
✓
✓
12691AAC55A079F529FE81205DF775EF297A14CA81499BF0857643E694CF8816
76
✓
✓
✓
F5178EEC7A9779E13CE01B35C8264BF32C094B172051CA32156DC61485718318
77
✓
✓
✓
A0543814F86D1C4AF6A08094CD0246F606F7E76CEE47EC052B62328038146D93
78
✓
✓
✓
511128DCBF369E985B99D07CC1668A2D28F4BA535CF7AC7926D4C5F696C3D35F
79
✓
✓
✓
595AD4C8A0EB2FDA798BC01D322F4C5ED098A2E749004B2B54FD815215F46686
80
✓
✓
✓
8E938EA9BE9E51A28DFD30BD6EDB9D6765C1272B8F7048CE81021194759C3E52
81
✓
✓
✓
F134975C5A989635F1D9FA7469C848A953622E9DA1BED7E12455DCD2AFA070BE
84
✓
✓
✓
36A990B9F35B79934FB25C64681DE3A83FC178DC2383C585FFCFDDD7C1F6C2B7
85
✓
✓
✓
✓
AB700D75274336FD26A1FE49D400ACEAE89F0FDBFE4BDE9A70373CA693003CA8
87
✓
✓
✓
9A4D4A94A1FE0FA1C559764C85D06496BD752498E0B5A2459624211013B9A088
89
✓
✓
C80682FCB2D78B2515A70A70D17C47A8512E24A127E797C073566D54586B9482
94
✓
✓
A04B6199A1DFE39EF35F6302454D71C872771A2F02A27AB5EC8130DA226F6F90
96
✓
✓
AFAAABE59B2EBB4FE15274E4EB5D1999C0554CC2D498BC92C59A3F6CD8FE2BC0
97
✓
✓
✓
✓
0754CA8EA936675EC3F64782A14E1A75B3D357044D4B2C434C6011279D17E829
100
✓
7F58EDB783C1F3FA7FF424CF7F5DF6D4BCDCF18D8A98CE4559EC22EB17030578
101
✓
25D31D3AFF5773799ECF43DEC1882B8F05D9231697BDDA5482DE05B14FB8A63B
103
✓
✓
CC977E0748722D615B845C1B10EA554B69DFCA640440CA5C468BBEF84B8C0442
104
✓
✓
✓
638C9DFBF9F376CBB3E3B01DF27960EC53A689D2FF4DFF23D97EE5351ED4A3D0
105
✓
✓
✓
D29E9D130016D930BF830BCAD071BC6503F877FB207922A9E495CF71A79631FE Key C
H
C
Fa
La
K
3
✓
✓
✓
✓
45189C81EADEE03202BFA06EAA15831789F0C76575508A563E1A739CA37B87BE
4
✓
✓
✓
✓
22BEF7AC4C31B2B98227D95B5EB49AF23343004CF2713FED48BEC3B5B7C3D24D
8
✓
✓
✓
✓
F484955872415A32B1B5B731EA1A8C729458055C17DC5FE9C57BCB39D1A40BFE
10
✓
✓
✓
✓
32D67733DF0D0257DA78E92752494CFD5112E303BA1413388126EA33BB60AEFC
11
✓
✓
✓
✓
E7F3287D91B528D78BF19D5E62828C845E1A4027A3E1F988B62B7407EBF5CF38
12
✓
✓
773F0C0FFACB531F50FAE0987D2B8972FE1B9231BBF46859F475BAFB45257FED
13
✓
✓
034332A23341538143FDB88F314FD942501FF8B6BA6A14D5013F1FC0984924BE
15
✓
✓
3F77C51259E1C8CC48217A66998CCF3212A17120B0FCA09163E300576DFCD9E7
16
✓
23773F0BECFACB534250FAE0987D2B8969D1AFD7EF942F148746DC73A3C6B39A
32
✓
✓
✓
32D67733DF0D0257DA78E92752494FFD5112E303BA14133FF126EA33BB60AEFC
33
✓
CD9540B70C2F92B2894594CABC4E724203A615B9144C459714758BC3CAA12242
34
✓
✓
✓
70253E6587D04D7A9A30A1461A80FCD235B28FBFC11FE8534CDFCE0A341C9257
36
✓
10D7EF92F06DF6EB94F2F344085DAD51D3A550E24A4569922460F579CB5DF11A
38
✓
70C3A9F11773C8DD795FD7942B5DB448FDFA5D12E6EC387691A19B6E523AE6AE
42
✓
1BEDDC1DD79F8856BF2E1FD66EB194073D60FEC658C5D0E2C8BAE02DC72ADF65
44
✓
B519BB44EC5BF3380CB2DF555F39ED836CDBF4961E43A66C218FADB211BF468C
45
✓
✓
✓
✓
32D67733DF3D0257DA78E92752494FFD5112E303BA14133FF126EA33BB60AEFC
50
✓
7A7AA97370B1EE16D64C71C7C5BC8C9F9456FBEA603780883399D89DA43F8A15
54
✓
✓
✓
✓
32D67733DF3D0257DA78E92752494FFD5112E22222222222F126EA33F6E49790
55
✓
✓
✓
✓
00498594859849584954E92752494FFD5112E2222222EE22F126EA33F6E49790
57
✓
✓
✓
✓
7D1BBD475A8EB5AF7DDB238CD8A67F86B601E0EA101C04036849B31F96CA6083
58
✓
✓
✓
BD3026C700A75B5970807802E2B47C2A892DF85E3CE57366D335EEBABCAAE255
61
✓
✓
✓
F4DDC95A88146CF52DEC752E737F8E3FB16AE4F6B7E726068946F3B0BA0C8E95
62
✓
✓
✓
0A99EB20F9DE4DD7607288B8B766F6217FE5D2CE6DDD51C6159941066AF192ED
66
✓
✓
✓
8836AC84AA148440A20628810CA65EB038BB625841275CC11590D8F5BC7F1BAC
70
✓
✓
✓
21A35C57E23B2D23ADDA19EA30325F1B532DA645489E29E47A13E92CA1F6670C
71
✓
✓
✓
588BEED930355AF54EEBAFAA46A7D26DA378A36EF5CD15D1F876D753A395F8AF
72
✓
✓
✓
B7A9B0F7661FC9A1DEC001F2C2C9EAE08748AEB187E1247726663E3DD1AB36BF
73
✓
✓
✓
4DAA29CBD634F28137499B9557104FDD36D4D4EFDFE87EFC0D8BD03555F8497F
74
✓
✓
✓
12691AAC55A079F529FE81205DF775EF297A14CA81499BF0857643E694CF8816
76
✓
✓
✓
F5178EEC7A9779E13CE01B35C8264BF32C094B172051CA32156DC61485718318
77
✓
✓
✓
A0543814F86D1C4AF6A08094CD0246F606F7E76CEE47EC052B62328038146D93
78
✓
✓
✓
511128DCBF369E985B99D07CC1668A2D28F4BA535CF7AC7926D4C5F696C3D35F
79
✓
✓
✓
595AD4C8A0EB2FDA798BC01D322F4C5ED098A2E749004B2B54FD815215F46686
80
✓
✓
✓
8E938EA9BE9E51A28DFD30BD6EDB9D6765C1272B8F7048CE81021194759C3E52
81
✓
✓
✓
F134975C5A989635F1D9FA7469C848A953622E9DA1BED7E12455DCD2AFA070BE
84
✓
✓
✓
36A990B9F35B79934FB25C64681DE3A83FC178DC2383C585FFCFDDD7C1F6C2B7
85
✓
✓
✓
✓
AB700D75274336FD26A1FE49D400ACEAE89F0FDBFE4BDE9A70373CA693003CA8
87
✓
✓
✓
9A4D4A94A1FE0FA1C559764C85D06496BD752498E0B5A2459624211013B9A088
89
✓
✓
C80682FCB2D78B2515A70A70D17C47A8512E24A127E797C073566D54586B9482
94
✓
✓
A04B6199A1DFE39EF35F6302454D71C872771A2F02A27AB5EC8130DA226F6F90
96
✓
✓
AFAAABE59B2EBB4FE15274E4EB5D1999C0554CC2D498BC92C59A3F6CD8FE2BC0
97
✓
✓
✓
✓
0754CA8EA936675EC3F64782A14E1A75B3D357044D4B2C434C6011279D17E829
100
✓
7F58EDB783C1F3FA7FF424CF7F5DF6D4BCDCF18D8A98CE4559EC22EB17030578
101
✓
25D31D3AFF5773799ECF43DEC1882B8F05D9231697BDDA5482DE05B14FB8A63B
103
✓
✓
CC977E0748722D615B845C1B10EA554B69DFCA640440CA5C468BBEF84B8C0442
104
✓
✓
✓
638C9DFBF9F376CBB3E3B01DF27960EC53A689D2FF4DFF23D97EE5351ED4A3D0
105
✓
✓
✓
D29E9D130016D930BF830BCAD071BC6503F877FB207922A9E495CF71A79631FE 551 Barbu et al. 9Challenges 54, 55, 57, 58, 61, 62, 66, 70, 71, 72, 73, 74, 76, 77, 78, 79, 80, 81, 84, 87, 89, 94, 96, 103,
104, 105, 107, 136 and 139 are not deterministic. A
Attacks Summary Table Challenge
Hooking
Collision
Fault
Lattice
Key
107
✓
✓
✓
4E420B6AA9E9F07F19CF7ED97497871C1223BC2A68E83716575C235DE6D63E17
108
✓
60609404F0B9086D3A995AF0680D048724CF2B1AF2B33CEA8DD4AF4B62A5DDBB
114
✓
✓
✓
0000000000000000000000000000000000000000000000000000000000000005
127
✓
1144D82B9568581405D10CF8B219FF7E94E4559E0832B06056F1F87D43C75777
135
✓
✓
✓
✓
0C2A5692FE1A7F9B8EE7EB4A7CD59CD62BCE33576B3123CECBB6406837BF51F5
136
✓
✓
✓
0C2A5692FE1A7F9B8EE7EB4A7CD59CD62BCE33476B3123CECBB6406837BF51F4
139
✓
✓
✓
000000000000000000000000000000004319055358E8617B0C46353D039CDAA9
153
✓
✓
9C29EDDAEF2C2B4452052B668B83BE6365004278068884FA1AC3F6D0622875C3
157
✓
✓
✓
✓
F04DBFD1147F9D43747538C1C9256DD2BC20562F9D92B83E9AFA751299B160A4
165
✓
✓
✓
84DAF8B6620FC6669BF1EE264D1B214A4FBECACEADDFDC0DCBC89CF4B6E3232B
166
✓
✓
C746740A4A6BCBD462D9041023A0FEF5CCF0328FF80D9C50132682030D77D33C
172
✓
✓
✓
285E57F7BDDAAA6201D8870A0B9B168C7A5D8200085F62504EE3EBFCC11EF150
174
✓
✓
✓
✓
9C29EDDAEF2C2B4452052B668B83BE6365004278068884FA1AC3F6D0622875EC
185
✓
✓
✓
✓
7729EDDAEF2C2B4452052B668B83BE6365004278068884FA1AC3F6D0622875EC
187
✓
✓
✓
✓
7779EDDAEF2C2B4452052B668B83BE6365004278068884FA1AC3F6D0622875EC
192
✓
09302BDFA5313312B9A665316F7E9365DCC57DA7E21FD8612CDCD553BABB51FE
193
✓
E0FE06BE0684455EDD2F5134A3AE8B9F6852561C821672FA16606986233BF811
209
✓
6E3A09F8EC613B8A524F7608CB80B2D3C510E27506AD84FA14C3B6D018E659F7
212
✓
D663E156F036F11D4E73CC0EC09A952DEAED316947DF73EB28467EC623C5740D
2268
6F1D9093F3D5AE7C5F133659295914C9AF22E54B4ADE38CA421CA9BBD3D48A50
227
✓
✓
ADA6C6A1049825989811C9495D83681A68C67AB5E8EBDDC126CEE77056A7BB27
228
✓
EA7BA345EB9D99F54261D01AE6319B184769E5745621706D77018E0DB46DDAFA
231
✓
✓
✓
✓
8ADE24EE6413C6E408784DBB4D81D04F33238AB503CBE35C77400517EE5ABC96
235
✓
✓
✓
✓
000000000000000000000000000000000000000000000000D0FACADE0DEFACED
251
✓
✓
✓
DDE098A74086ECBB4DBA1848511BEA924145D1A9ED2EC9E64E0C5934BAAC97AE
253
✓
✓
B22DB44C9E66D567B3B2CBB3C720309D1EEAD38717017F5E79F05274F289A52C
256
✓
F1662664E7E303740C0CA3927F9870A789978DAE95892302E73C85E3993B4CC9
261
✓
✓
3266C9F6379DFDAE4AA763E8E6BA94526504CA364C482306829D4BF1E97BFF92
262
✓
A0F00DCAA5DAB169FD4DFE2186BCBCBD22631AB68BFEFF1FC19306174EAF8970
264
✓
D0EE17829A397C18074EA3888057AE815B5336773F9668E6CE4464D4B2B05F1F
267
✓
✓
✓
✓
C17536B60BCF94326A9C8CA17E0FC4EDBD76822532B350E8237CA2D8CF9C74B0
274
✓
✓
✓
✓
0080ECD2A00080ECD2A00080ECD2A00080ECD2A00080ECD2A00080ECD2A00080
283
✓
79FE8D884DC2F7440824DE79C9F7C513C2B4549631D343523C73CB8F85983A4F
299
✓
✓
✓
✓
3A0F803A874CD5B826023F2073FF200371D399E76E66B05E1241AA787B0564D6
304
✓
EE8942A527CA1A58B8A8EA369441CB8518836DDB98F6380B8008B6053BC8182C
305
✓
311EA92FBCDD3C6A29D269589A9E71F13A231FFEC85FF36B398967EC9934805E
307
✓
✓
FA3FCDE70679E7E44391F7157E2B5822F5B9B9C93ADD95C2BA90FF4B95C8A6BB
308
✓
✓
84CCCAA904CB397F41A36FF9E05D4EB6C58B8E203E02373C465B6C3F03280C82
314
✓
7E045DB89DD77BD6B2EAF23172A89A656B5084748642DB82BBAE931E737560C2
320
✓
✓
✓
D235C2B1D089F158A0AE4E7799C2DCA9985E3D44C8F243BAD8B5E1A4EB647E1B
321
✓
✓
✓
✓
BA15757E1B0DB122F349C0C50C97071A4CFFF4FD2875B4A092FBDD985E8595DE
323
✓
✓
✓
✓
C7491BBC530FFA9DDCF3E7D732536FACF04239693D549C50DDAD41931A6244C2
325
✓
✓
6902CD65AE124A45B9DD16BAEFD26D9CFFB5C291DC1E256D9CCE17BE3CF11775
327
✓
2BC6F2467C7F8DFA164EDC68DDCF65E795B8A2153182565481D8D6878D80EA81
328
✓
37170CF851A89AAFD3511234BE2B96C89B783A44D7A6C22E9A150872809F7CDF
335
✓
EC90CC12DC70E3C5A7D47B6083A988F3F6C6B2B63EB0D8991F84B19E21ACC061
3368
D2E2AE325946DDADC9A67A2DFE8EE74065D8D39968707F5D818D7B62910894EA
345
✓
3266C9F6378DFDAE4AA763E9166B131E6514CA364C482306829D4BF1E97BFF92
346
✓
5EE43950837D0ABA419FE5B586D1A7AA44DDAAC6327DADC3133F18A850211B9F Challenge
Hooking
Collision
Fault
Lattice
Key
107
✓
✓
✓
4E420B6AA9E9F07F19CF7ED97497871C1223BC2A68E83716575C235DE6D63E17
108
✓
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5EE43950837D0ABA419FE5B586D1A7AA44DDAAC6327DADC3133F18A850211B9F 552 Attacks and Designs from CHES 2021 Challenge g
,
,
,
,
,
,
,
,
,
104, 105, 107, 136 and 139 are not deterministic. B
Some Remarks on the Challenges Among the various submissions, we notice the following facts: • Challenges 15 and 16 have a very small code size, only 194 bytes! To obtain such
tiny implementations, the designers use a fixed nonce k = 1 (i.e. r = Gx) and a
private key d such that dr ≡2i mod n. In such a case, the signature of a hash e is
equal to (Gx, e + 2i). • Challenge 114 uses a very small private key, indeed d114 = 5 • Some designer teams modify a few bits only of the private key in several challenges
(cf. Challenges 174, 185 and 187 for instance). In such a case, if one implementation
is broken, then the private keys of the other challenges of the same team could be
recovered by brute force search. • Despite what is indicated in the rules (cf. Sect. 2), some challenges are not deter-
ministic9, i.e. the two signatures of the same message could be different. All these
challenges use the time() function to obtain some randomness. However, it is easy
to hook such calls and return a constant value.
|
https://openalex.org/W2741088600
|
https://www.nature.com/articles/cddis2017336.pdf
|
English
| null |
Retinoic acid-induced upregulation of miR-219 promotes the differentiation of embryonic stem cells into neural cells
|
Cell death and disease
| 2,017
|
cc-by
| 10,228
|
1College of Veterinary Medicine, Northwest A&F University, 22 Xinong Road, Yangling 712100, China and 2Key Laboratory of Animal Biotechnology, Ministry of Agriculture,
Northwest A&F University, 3 Taicheng Road, Yangling 712100, China
*Corresponding author: H Wu or Y Zhang, College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China. Tel/Fax: +86 29 87080092;
E-mail: hbwu029@nwsuaf.edu.cn or zhangy1956@sina.com
Received 22.3.17; revised 21.5.17; accepted 14.6.17; Edited by y Shi Retinoic acid-induced upregulation of miR-219
promotes the differentiation of embryonic stem cells Zhao1,2, Beibei Fu1,2, Songna Yin2, Chao Song1,2, Jingcheng Zhang1,2, Shanting Zhao1 and Yong Zhang*,1, aibo Wu*,1,2, Jiamin Zhao1,2, Beibei Fu1,2, Songna Yin2, Chao Song1,2, Jingcheng Zhang1,2, Shanting Zhao MicroRNAs (miRNAs) regulate critical cell processes, such as apoptosis, proliferation, and development. However, the role of
miRNAs in embryonic stem cell (ESC) neural differentiation induced by retinoic acid (RA) and factors that govern neural directional
differentiation remain poorly understood. In this study, we demonstrated that miR-219 is sufficient in promoting mouse ESCs to
undergo neural differentiation. We discovered that Foxj3 and Zbtb18, two target genes of miR-219, are not able to determine the
process of RA-induced differentiation, however they prevent ESCs from differentiating into neural cells. We identified four
downstream genes, namely, Olig1, Zic5, Erbb2, and Olig2, which are essential to the gene interaction networks for neural
differentiation. These data explain the mechanism of RA-induced neural differentiation of mESCs on the basis of miRNAs and
support the crucial role of miR-219 in neurodevelopment. pp
p
Cell Death and Disease (2017) 8, e2953; doi:10.1038/cddis.2017.336; published online 27 July 2017 MicroRNAs (miRNAs) are a class of non-coding, single-
stranded, and 22–25-nucleotide RNA molecules that nega-
tively regulate gene expression in plants and animals.1
MiRNAs mainly pair with the 3′-untranslated regions (UTRs)
of a target gene and consequently degrade the target mRNA
or inhibit translation. MiRNAs are involved in the regulation of
embryonic
development,
apoptosis,
and
proliferation.2–7
Moreover, some miRNAs, as oncogenes or tumor suppressor
genes, are closely related to tumor formation.8–11 we demonstrated that miR-219 can promote embryonic stem
cells (ESCs) to differentiate into neural cells, and Foxj3 and
Zbtb18, as targets of miR-219, are dominant factors in neural
differentiation. These results provide a theoretical basis for RA
application in ESC research and make a contribution to the
understanding of neural regulatory networks. Citation: Cell Death and Disease (2017) 8, e2953; doi:10.1038/cddis.2017.336
Official journal of the Cell Death Differentiation Association Citation: Cell Death and Disease (2017) 8, e2953; doi:10.1038/cddis.2017.336
Official journal of the Cell Death Differentiation Association OPEN OPEN www.nature.com/cddis Results All-trans retinoic acid (RA), a metabolic compound derived
from Vitamin A, is important in cellular differentiation and
neurogenesis.12–17 RA is the most used morphogen for
producing neural progenitor cells and neurons from pluripotent
stem cells in vitro.18–21 The action of RA on differentiation is
probably mediated by RA receptors (RARs) in the nucleus. This action is done through the binding of the receptors to DNA
sequences located in the promoter regions of RA-responsive
genes.17,22,23 Furthermore, the molecular signaling pathways
involved in neuronal differentiation are complex. The specific
mechanism of RA-induced neural differentiation in pluripotent
stem cells is not well characterized, and the role of miRNAs in
this process remains unknown.24–26 RA-induced miRNA expression changes in ESCs. RA is
widely used to induce neurogenesis.18–20 We determined
whether or not miRNAs are involved in RA-induced neural
directional
differentiation
by
performing
an
array-based
miRNA profiling on mouse J1 ESCs (gene expression
omnibus (GEO) accession number: GSE54145; all tested
miRNAs sorted by names are listed in Supplementary Data
s03). The results showed that 43 miRNAs were upregulated
and 281 miRNAs were downregulated after 48 h of RA
treatment
in
ESCs. Of
the
43
upregulated
miRNAs
(Supplementary Table S1), 18 exhibited more than a five-
fold change, and five other miRNAs that are well known to be
functionally related with development were then confirmed by
qRT-PCR.32–34 Consistent with the microarray findings, the
18 changed miRNAs were upregulated by RA treatment at
varying degrees(Figure 1a). Three of them, namely, miR-10a-
5p, miR-219-5p, and miR-219-2-3p, showed highly significant
fold changes. As previously reported, RA-induced differentia-
tion of mESCs is powerful and irreversible, and the first
36–48 h of induction is critical.12 Consequently, the expres-
sion levels of these three significantly changed miRNAs were
detected
at
different
time
points
during
RA
induction Forkhead box J3 (Foxj3) belongs to a gene family of
transcription
factors
that
regulate
skeletal
muscle
and
peripheral artery developments.27,28 Meanwhile, zinc-finger
and BTB domain containing 18 (Zbtb18), also known as
Zfp238, is involved in skeletal muscle differentiation and
myogenesis.29,30 When combined with PDGFRα and Sox6,
Foxj3 and Zbtb18 can control oligodendrocyte differentiation
and myelination.31 However, the roles of Foxj3 and Zbtb18 in
stem cell differentiation are poorly understood. In this study, MiR-219 promotes neural differentiation
H Wu et al Figure 1
MiRNA expression changes in J1 ESCs induced by RA. Results (a) J1 ESCs were treated with RA for 48 h, and qRT-PCR was performed to determine the relative
expression levels of 23 miRNAs. U6 served as the internal control. (b–d) Relative expression levels of miR-10-5p (b), miR-219 (c), and miR-219-2-3p (d) at different time points
after RA treatment on ESCs. All data are presented as mean ± S.D. and derived from three independent experiments
MiR-219 promotes neural differentiation
H Wu et al 2 2 Figure 1
MiRNA expression changes in J1 ESCs induced by RA. (a) J1 ESCs were treated with RA for 48 h, and qRT-PCR was performed to determine the relative
expression levels of 23 miRNAs. U6 served as the internal control. (b–d) Relative expression levels of miR-10-5p (b), miR-219 (c), and miR-219-2-3p (d) at different time points
after RA treatment on ESCs. All data are presented as mean ± S.D. and derived from three independent experiments Foxj3 and Zbtb18 are the targets of miR-219. To investi-
gate the underlying mechanism, we examined the potential
targets of miR-219 by searching the PicTar, miRanda, and
targetScan databases. Among the candidate targets, the
3′-UTR of mouse Foxj3 and Zbtb18 contains putative regions
that match the miR-219 seed sequence, which is conserved
in humans and rats (Figure 3a). To confirm the predicted
results, the 3′-UTRs of Foxj3 and Zbtb18 containing the
putative
regions were
amplified
and
inserted
into
the
psicheck-2 vector. They were then transfected to NIH/3T3
fibroblast cell line for dual luciferase reporter (DLR) assays. As shown in Figure 3b, the miR-219 mimics dramatically
suppressed the activities of wild-type (WT) 3′-UTRs of Foxj3
and Zbtb18. By contrast, the double mutation type (MUT)
group (site 1+2 mut) with mutated seed sequences was
unaffected. qRT-PCR and western blot were performed to
examine the mRNA and protein levels of Foxj3 and Zbtb18 in
ESCs transfected with miR-219 mimics or inhibitors. The
results
showed
that
the
miR-219
mimics
considerably
decreased the protein levels of Foxj3 and Zbtb18 rather than
the mRNA levels of these genes (Figures 3c–e). Thus,
miR-219 regulates the expression levels of Foxj3 and Zbtb18
at the post-transcriptional level. These results indicated that
Foxj3 and Zbtb18 are the targets of miR-219. (Figures 1b–d). The results showed that miR-219 was
dramatically and highly expressed during the initial 48 h,
and its expression decreased gradually over time (Figure 1c). Results (b, c) ESCs were transfected with
miR-219 mimics for 48 h; the relative levels of Oct4, Nestin, and Map2 were detected through qRT-PCR (b) and western blot (c). (d) Immunofluorescence shows the abundance of
Oct4, Nestin, and Map2, as well as the morphologies of the ESCs after transfection with miR-219 mimics for 48 h. (e, f) ESCs were pretreated with RA for 48 h and transfected
with miR-219 inhibitors. The relative levels of Oct4, Nestin, and Map2 were detected using qRT-PCR (e) and western blot (f). (g) Immunofluorescence shows the abundance of
Oct4, Nestin, and Map2, as well as the morphologies of the ESCs pretreated with RA for 48 h and transfected with miR-219 inhibitors. **Po0.01; NS, no significance Figure 2
MiR-219 mediates ESCs to differentiate into neural cells. (a) Confirmation of the effects of miR-219 mimics and inhibitors. (b, c) ESCs were transfected with
miR-219 mimics for 48 h; the relative levels of Oct4, Nestin, and Map2 were detected through qRT-PCR (b) and western blot (c). (d) Immunofluorescence shows the abundance of
Oct4, Nestin, and Map2, as well as the morphologies of the ESCs after transfection with miR-219 mimics for 48 h. (e, f) ESCs were pretreated with RA for 48 h and transfected
with miR-219 inhibitors. The relative levels of Oct4, Nestin, and Map2 were detected using qRT-PCR (e) and western blot (f). (g) Immunofluorescence shows the abundance of
Oct4, Nestin, and Map2, as well as the morphologies of the ESCs pretreated with RA for 48 h and transfected with miR-219 inhibitors. **Po0.01; NS, no significance and Zbtb18 are involved in neural differentiation. Foxj3 and
Zbtb18 were transiently transfected to ESCs, and the relative
abundance of Nestin was detected. As expected, the Foxj3 or
Zbtb18 disrupted the upregulation of Nestin after the miR-219
mimics treatment. Particularly, the synergistic effect of Foxj3
and Zbtb18 returned Nestin expression to basal levels
(Figures 4a and b; Supplementary Figures S1F–K). Knock-
down experiments were then conducted with small interfering
RNA (siRNA) to verify the results. The results showed that
Nestin expression was upregulated from 3.5- to 4.5-fold when Foxj3 or Zbtb18 was knocked down, and knockdown of both
Foxj3 and Zbtb18 at one time intensified neural differentiation
(Supplementary Figures 4C, D). These results suggested that
Foxj3 and Zbtb18 prevent the differentiation of ESCs into
neural cells. Results This result was verified in D3 and B6 ESCs (Supplementary
Figure S1A), suggesting that miR-219 plays an important role
in RA-induced ESC differentiation. MiR-219 mediates ESCs to differentiate into neural
cells. Next, miR-219 mimics and inhibitors were used to
investigate whether or not miR-219 is involved in RA-induced
differentiation. The validity of miR-219 mimics and inhibitors
were verified in Figure 2a. As expected, the mESCs
differentiated into neural-type cells 48 h after transfection of
miR-219 mimics. The stem cell marker Oct4 was reduced
considerably, whereas Nestin (a marker for neural stem
cells), and Map2 and Tuj1 (markers for neural cells) were
increased (Figures 2b–d). Furthermore, the ESCs transfected
with miR-219 inhibitors resisted the RA-induced neural
differentiation. As shown in Figures 2e–g, the miR-219
inhibitors blocked the RA-induced upregulation of Nestin,
Map2, and Tuj1. The ESCs were differentiated after RA
treatment regardless of the presence of miR-219 inhibitors,
as characterized by the decreased Oct4 and loss of tight
colony morphology (Figure 2g). Interestingly, the miR-219
inhibitors prevented ESCs from differentiating in a neural
directional manner (Figures 2e–g; Supplementary Figures
S1B–E). These findings suggested that miR-219 mediates
ESCs to differentiate into neural cells. Foxj3 and Zbtb18 prevent ESCs from differentiating into
neural cells. MiR-219 promotes the neural differentiation
ESCs
(Figures
2e–g),
targeting
Foxj3
and
Zbtb18
(Figures 3b–d). We then investigated whether or not Foxj3 Cell Death and Disease MiR-219 promotes neural differentiation
H Wu et al 3 Figure 2
MiR-219 mediates ESCs to differentiate into neural cells. (a) Confirmation of the effects of miR-219 mimics and inhibitors. (b, c) ESCs were transfected with
miR-219 mimics for 48 h; the relative levels of Oct4, Nestin, and Map2 were detected through qRT-PCR (b) and western blot (c). (d) Immunofluorescence shows the abundance of
Oct4, Nestin, and Map2, as well as the morphologies of the ESCs after transfection with miR-219 mimics for 48 h. (e, f) ESCs were pretreated with RA for 48 h and transfected
with miR-219 inhibitors. The relative levels of Oct4, Nestin, and Map2 were detected using qRT-PCR (e) and western blot (f). (g) Immunofluorescence shows the abundance of
Oct4, Nestin, and Map2, as well as the morphologies of the ESCs pretreated with RA for 48 h and transfected with miR-219 inhibitors. **Po0.01; NS, no significance Figure 2
MiR-219 mediates ESCs to differentiate into neural cells. (a) Confirmation of the effects of miR-219 mimics and inhibitors. Results The ES cell lines that stably expressed Foxj3
and Zbtb18 were produced by pCDH-Puro-Foxj3/Zbtb18
lentivirus to investigate the functional roles of Foxj3 and
Zbtb18 in neural differentiation. The resulting cell lines were
used for in vitro differentiation under RA treatment. Compared
with normal ESCs, Foxj3/Zbtb18-overexpressing (OE) ESCs Cell Death and Disease MiR-219 promotes neural differentiation
H Wu et al 4 ure 3
Foxj3 and Zbtb18 are the targets of miR-219. (a) 3′-UTR analysis of Foxj3 and Zbtb18 containing putative regions that match the seed sequence of miR-219. (b) A
after NIH/3T3 fibroblast cells were transfected with miR-219 mimics, luciferase reporter constructs containing WT or MUT-type UTRs were transfected as indicated. Ce
es were harvested for DLR assays. (c–e) MiR-219 mimics or inhibitors were transfected to ESCs. After 48 h, cells were harvested for qRT-PCR (c) and western blot (d)
t the relative levels of Foxj3 and Zbtb18. The relative intensities of the protein bands (e) were quantified with Image J software and calculated using the samples normalize
actin. All data are presented as mean ± S.D. and derived from three independent experiments. Scale bars, 30 μm. **Po0.01 igure 3
Foxj3 and Zbtb18 are the targets of miR-219. (a) 3′-UTR analysis of Foxj3 and Zbtb18 containing putative regions that match the seed sequence of miR-219. (b
4 h after NIH/3T3 fibroblast cells were transfected with miR 219 mimics luciferase reporter constructs containing WT or MUTtype UTRs were transfected as indicated Figure 3
Foxj3 and Zbtb18 are the targets of miR-219. (a) 3′-UTR analysis of Foxj3 and Zbtb18 containing putative regions that match the seed sequence of miR-219. (b) At
24 h after NIH/3T3 fibroblast cells were transfected with miR-219 mimics, luciferase reporter constructs containing WT or MUT-type UTRs were transfected as indicated. Cell
lysates were harvested for DLR assays. (c–e) MiR-219 mimics or inhibitors were transfected to ESCs. After 48 h, cells were harvested for qRT-PCR (c) and western blot (d) to
detect the relative levels of Foxj3 and Zbtb18. The relative intensities of the protein bands (e) were quantified with Image J software and calculated using the samples normalized
to β-actin. All data are presented as mean ± S.D. and derived from three independent experiments. Scale bars, 30 μm. **Po0.01 differentiated but not in a neural directional manner, as
characterized by morphology and the expression levels of
neural markers Tuj1 and NeuN (Figure 4e). Cell Death and Disease Results Foxj3 and Zbtb18 (Supplementary Table S2, pretreated Foxj3/
Zbtb18-ESC 5.56% versus pretreated ESC 38.9%; represen-
tative
neural-like
tissues
are
shown
in
Supplementary
Figure S2). Moreover, the Foxj3/Zbtb18-ESCs pretreated with
RA
retained
the
ability
to
form
epidermis-like
tissues
(Figure 4f), suggesting that Foxj3 and Zbtb18 affected
neuroectodermal development rather than the development
of the entire ectoderm. In addition, the Foxj3/Zbtb18-OE ESCs
generated less ectoderm-specific tissues (21.1% versus
52.2%) and more mesoderm-specific (24.4% versus 13.3%)
and
endoderm-specific
(28.9%
versus
15.6%)
tissues
(Supplementary Table S2) compared with RA-pretreated Teratoma formation tests in nude mice were performed to
confirm
this finding. The
Foxj3/Zbtb18-OE
ESCs were
pretreated with RA for 48 h and injected into nude mice to
generate teratomas. The teratomas were harvested 14 days
later for further examination. The hematoxylin/eosin- (H&E)
stained samples showed that the RA-pretreated ESCs tend to
form neuronal-like tissues (Supplementary Table S2, pre-
treated 38.9% versus untreated 12.2%), and the neuronal
differentiation potential of ESCs was mostly suppressed by Cell Death and Disease MiR-219 promotes neural differentiation
H Wu et al 5 Figure 4
Foxj3 and Zbtb18 prevent ESCs from differentiating into neural cells. (a, b) ESCs were pretreated with miR-219 mimics for 24 h and followed by transfection of
pCMV-Foxj3 or pCMV-Zbtb18 as indicated. After 48 h, the relative level of Nestin was detected through qPCR (a) and western blot (b). Actin served as the loading control. (c, d)
ESCs were pretreated with miR-219 mimics for 24 h and followed by transfection of Foxj3-siRNA or Zbtb18-siRNA. After 48 h, the relative level of Nestin was detected through
qPCR (c) and western blot (d). (e) Foxj3/Zbtb18-OE ESCs were induced by RA for 10 days. The morphology was observed and photographed. Scale bars: 100 μm. (f) Control
ESCs or Foxj3/Zbtb18-OE ESCs were pretreated with RA for 48 h and injected into nude mice to generate teratomas. The differentiations of the three germ layers were analyzed
through H&E staining. Scale bars: 100 μm. **Po0.01; NS, no significance Figure 4
Foxj3 and Zbtb18 prevent ESCs from differentiating into neural cells. (a, b) ESCs were pretreated with miR-219 mimics for 24 h and followed by transfection of
pCMV-Foxj3 or pCMV-Zbtb18 as indicated. After 48 h, the relative level of Nestin was detected through qPCR (a) and western blot (b). Actin served as the loading control. Results This finding was supported by the immunohistochemistry
analysis results. In the WTembryos, Oct4 was restricted in the
posterior region, and Nestin was expressed in the anterior
region because of forebrain formation (Figures 5B, a and b). However, Oct4 disappeared and Nestin was detected in the
entire ectoderm of the miR-219-injected embryos (Figures 5B,
c and d). These results indicated that miR-219 induces neural
development in the mouse embryos. On the contrast, embryos
injected with Foxj3/Zbtb18 mRNAs formed egg cylinders at
E6.5 (Figures 5A, e and f), but 48.8% (40/82) of the sectioned
embryos showed quick degeneration and various abnormal
phenotypes, 23.2% (19/82) of the embryos were resorbed
completely or left a trace of residual pyknotic tissue by E7.5
(Figure 5B, e and f; Supplementary Figure S4). The extremely
high rate of mutants in the miR-219- and Foxj3/Zbtb18-
injected embryos implied that the normal expression of Foxj3/
Zbtb18 is crucial in early embryonic development in mice. (
pp
y
g
pp
y
)
To locate the core regulatory genes involved in neural
regulatory networks, gene co-expression networks were
constructed with the overlapping genes in the Foxj3-ES and
Zbtb18-ES (Figure 6a). Core regulatory factors were then
identified on the basis of the degree of differences between the
control and Foxj3/Zbtb18-ES groups. The genes with a
difference degree of more than eight were selected. Five
genes, namely, Olig1, Zic5, Erbb2, Numbl, and Olig2, met the
specification in the Zbtb18-ES group. Meanwhile, eight genes,
namely, Olig1, Shank1, Erbb2, Zic5, Smarcd3, Ina, Chd7, and
Olig2, met the specifications in the Foxj3-ES group. In
addition, the following genes were present in both groups:
Olig1, Zic5, Erbb2, and Olig2 (Figures 6b and c). We then
performed qRT-PCR to validate the relationships of these
genes with miR-219, Foxj3, and Zbtb18. As expected, Olig1,
Zic5, Erbb2, and Olig2 were upregulated two-fold to five-fold
when the ESCs were induced by RA treatment or miR-219
mimics, whereas they were dramatically inhibited when Foxj3
or Zbtb18 was overexpressed (Figure 6d). These findings
were consistent with the RNA-seq results. To understand the
roles of Foxj3, Zbtb18, and the four genes involved in
transcription factor networks during RA-dependent neural Olig1 and Olig2 are important in miR-219-mediated
neural differentiation. The total mRNAs of Foxj3-ES and
Zbtb18-ES were extracted and used for RNA-seq and
transcriptome analyses to understand the specific mechan-
ism by which Foxj3 and Zbtb18 control neural differentiation
of ESCs. Results (c, d)
ESCs were pretreated with miR-219 mimics for 24 h and followed by transfection of Foxj3-siRNA or Zbtb18-siRNA. After 48 h, the relative level of Nestin was detected through
qPCR (c) and western blot (d). (e) Foxj3/Zbtb18-OE ESCs were induced by RA for 10 days. The morphology was observed and photographed. Scale bars: 100 μm. (f) Control
ESCs or Foxj3/Zbtb18-OE ESCs were pretreated with RA for 48 h and injected into nude mice to generate teratomas. The differentiations of the three germ layers were analyzed
through H&E staining. Scale bars: 100 μm. **Po0.01; NS, no significance of miR-219 and Foxj3/Zbtb18 on developing embryos between
egg-cylinder and primitive streak stages. MiR-219 agomirs or
Foxj3/Zbtb18 mRNAs were injected into the zygote cytoplasm. Microinjected embryos that reached the blastocyst stage were
transferred to pseudopregnant female recipients. The Oct4
and Nestin expression patterns in the serially sectioned E6.5–
E7.5 embryos were then examined by immunohistochemistry
analysis (Figure 5). At E6.5, Nestin expression in the miR-219-
injected embryos occurred earlier than the control group
(Figures 5A,b and d). At E7.5, the differences between the
control and miR-219-injected embryos became more appar-
ent. Approximately 27.8% (15/54) of the miR-219-injected ESCs. We then tested the expression levels of mesoderm-
specific (Brachyury, Flk1) and endoderm-specific (Sox17,
Foxa2) markers in ESCs treated with RA or miR-219
antagomirs. The results showed that the increase in the
expression levels of the mesoderm- and endoderm-specific
markers in ESCs transfected with miR-219 antagomirs were
larger than that in the RA-treated cells (Supplementary
Figure S3). These data indicated that RA-induced ESCs tend
to differentiate into mesodermal and endodermal cells upon
miR-219 inhibition. To elucidate the role of miR-219-Foxj3/Zbtb18 in the
regulation of neural induction in vivo, we examined the effects Cell Death and Disease MiR-219 promotes neural differentiation
H Wu et al 6 Figure 5
MiR-219 induces neural development in mouse embryos. MiR-219 agomirs, Foxj3/Zbtb18 mRNAs, and their corresponding negative controls were microinjected
into the cytoplasm of zygotes as indicated. The expression patterns of Oct4 and Nestin in the serially sectioned E6.5 and E7.5 embryos were examined by immunohistochemistry
analysis. (A) Immunohistochemistry analysis of the E6.5 embryos. Representative images are shown. Black dashed frames in E6.5 embryos indicate the embryonic region. (B)
Immunohistochemistry (a–d) and H&E staining (e, f) analysis of E7.5 embryos. Results a and b show the representative control embryo and indicate how the embryonic region is
organized and how the ectodermal and endodermal tissues can be distinguished. c and d show the representative abnormal miR-219-injected embryo, which exhibits a severe
overdevelopment in its anterior region. (e, f) show representative abnormal Foxj3/Zbtb18 mRNA-injected embryo with disorganized embryonic region, apparent arrested
development, or resorption. Full images of serially sectioned embryos are shown in Supplementary Figure S4. a, anterior; ac, amniotic cavity; al, allantois; d, distal; exc,
exocoelomic cavity; p, posterior; px, proximal. Scale bars, 200 μm Figure 5
MiR-219 induces neural development in mouse embryos. MiR-219 agomirs, Foxj3/Zbtb18 mRNAs, and their corresponding negative controls were microinjected
into the cytoplasm of zygotes as indicated. The expression patterns of Oct4 and Nestin in the serially sectioned E6.5 and E7.5 embryos were examined by immunohistochemistry
analysis. (A) Immunohistochemistry analysis of the E6.5 embryos. Representative images are shown. Black dashed frames in E6.5 embryos indicate the embryonic region. (B)
Immunohistochemistry (a–d) and H&E staining (e, f) analysis of E7.5 embryos. a and b show the representative control embryo and indicate how the embryonic region is
organized and how the ectodermal and endodermal tissues can be distinguished. c and d show the representative abnormal miR-219-injected embryo, which exhibits a severe
overdevelopment in its anterior region. (e, f) show representative abnormal Foxj3/Zbtb18 mRNA-injected embryo with disorganized embryonic region, apparent arrested
development, or resorption. Full images of serially sectioned embryos are shown in Supplementary Figure S4. a, anterior; ac, amniotic cavity; al, allantois; d, distal; exc,
exocoelomic cavity; p, posterior; px, proximal. Scale bars, 200 μm differentiation, and thus the genes located in the four
overlapping
biological
processes
(i.e.,
nervous
system
development, central nervous system development, neuron
fate commitment, and forebrain development) of the enriched
GO terms were selected for analysis. A total of 37 candidate
genes in the Foxj3-ES group and 39 candidate genes in the
Zbtb18-ES group were present. Between these groups, 17
genes overlapped, namely Arid1a, Camk2b, Chd7, Erbb2,
Glis2, Gnao1, Id2, Ina, Map1s, Ndn, Numbl, Olig1, Olig2,
Shank1, Smarcd3, Zic2, and Zic5. We hypothesized that
these genes contribute to RA-mediated neural differentiation. Their expression levels were verified through qRT-PCR
(Supplementary Figure S5C; Supplementary Table S3). embryos were resorbed or had no significant progress in their
development, and 38.9% (21/54) of the embryos showed
varying degrees of overdevelopment in the anterior region. Results Compared with control, 1331 genes were differen-
tially expressed (Po0.05) by two-fold in the Foxj3-ESCs (557
were underexpressed and 774 were overexpressed), and
1175 genes were differentially expressed (Po0.05) by two-
fold in Zbtb18-ESCs (548 were underexpressed and 627
were overexpressed). Gene ontology (GO) analysis indicated
that 53 GO terms in Foxj3-ES and 54 GO terms in Zbtb18-ES
were enriched (Po0.01; Supplementary Figures S5A, B). Foxj3 and Zbtb18 work synergistically to control neural Cell Death and Disease MiR-219 promotes neural differentiation
H Wu et al Figure 6
Olig1, Zic5, Erbb2, and Olig2 participate in neural regulatory networks. (a) Co-expression networks of differentially expressed genes in the control, Foxj3-ES, and
Zbtb18-ES groups. The overlapping genes were selected to construct gene co-expression networks. Sixteen genes located in the four focused GO terms are arrow-shaped. Solid
lines, positively corrected; dashed lines, negatively corrected. (b, c) The genes with difference degrees above eight between Foxj3-ES (b) or Zbtb18-ES (c) and control ESCs. (d)
The relative levels of Olig1, Zic5, Erbb2, and Olig2 in the ESCs were detected through qRT-PCR. The ESCs were treated or transfected with RA, pCMV-Foxj3, pCMV-Zbtb18, or
miR-219 for 48 h. (e–g) Relative levels of Nestin and Map2 were detected through qRT-PCR (e) and western blot (f) in ESCs transfected with Olig1-, Zic5-, Erbb2-, or Olig2-
encoding plasmids for 48 h. Relative intensities of protein bands (g) were quantified by using Image J software and calculated by using the samples normalized to β-actin. All data
are presented as mean ± S.D. and derived from three independent experiments. *Po0.05; **Po0.01; NS, no significance Figure 6
Olig1, Zic5, Erbb2, and Olig2 participate in neural regulatory networks. (a) Co-expression networks of differentially expressed genes in the control, Foxj3-ES, and
Zbtb18-ES groups. The overlapping genes were selected to construct gene co-expression networks. Sixteen genes located in the four focused GO terms are arrow-shaped. Solid
lines, positively corrected; dashed lines, negatively corrected. (b, c) The genes with difference degrees above eight between Foxj3-ES (b) or Zbtb18-ES (c) and control ESCs. (d)
The relative levels of Olig1, Zic5, Erbb2, and Olig2 in the ESCs were detected through qRT-PCR. The ESCs were treated or transfected with RA, pCMV-Foxj3, pCMV-Zbtb18, or
miR-219 for 48 h. (e–g) Relative levels of Nestin and Map2 were detected through qRT-PCR (e) and western blot (f) in ESCs transfected with Olig1-, Zic5-, Erbb2-, or Olig2-
encoding plasmids for 48 h. Discussion these four factors are involved in neural regulatory networks. Notably, only Olig1 and Olig2 facilitate the expression levels of
Map2 and Tuj1 (neural cell markers) in the ESCs (Figures
6e–g). We hypothesized that Olig1 and Olig2 are the most
dominant downstream factors of miR-219-mediated neural
differentiation. Olig1/2 overexpression and knockdown were
performed to demonstrate the involvement of Olig1 and Olig2
in the downstream of the RA-miR-219-Foxj3/Zbtb18 pathway. The results showed that Nestin, Map2, and Tuj1 dramatically
increased in ESCs with overexpressed Olig1/Olig2, and the
levels of neural differentiation were approximate to RA
treatment (Figures 7a and b). Moreover, Olig1/2-knockdown
ESCs cultured in the presence of RA or transfected with
miR-219 mimics failed to differentiate into neural cells, as
characterized by the reduction of Oct4 and absence of
significant increase in neural cell markers (Map2 and Tuj1)
(Figures 7c–e). These results indicated that Olig1 and Olig2
are the main factors regulated by miR-219 in RA-induced
neural differentiation. these four factors are involved in neural regulatory networks. Notably, only Olig1 and Olig2 facilitate the expression levels of
Map2 and Tuj1 (neural cell markers) in the ESCs (Figures
6e–g). We hypothesized that Olig1 and Olig2 are the most
dominant downstream factors of miR-219-mediated neural
differentiation. Olig1/2 overexpression and knockdown were
performed to demonstrate the involvement of Olig1 and Olig2
in the downstream of the RA-miR-219-Foxj3/Zbtb18 pathway. The results showed that Nestin, Map2, and Tuj1 dramatically
increased in ESCs with overexpressed Olig1/Olig2, and the
levels of neural differentiation were approximate to RA
treatment (Figures 7a and b). Moreover, Olig1/2-knockdown
ESCs cultured in the presence of RA or transfected with
miR-219 mimics failed to differentiate into neural cells, as
characterized by the reduction of Oct4 and absence of
significant increase in neural cell markers (Map2 and Tuj1)
(Figures 7c–e). These results indicated that Olig1 and Olig2
are the main factors regulated by miR-219 in RA-induced
neural differentiation. RA can induce various types of cells in a concentration-
dependent manner. For example, a high RA concentration
increases the rate of neural differentiation, whereas a low RA
concentration
induces
cardiomyocyte
differentiation
of
mESCS.35 Okada et al.36 reported that RA concentration
regulates dorsoventral identity, that is, high RA concentration
induces a dorsal phenotype, and low RA concentrations
induces a ventral phenotype during in vitro mESC differentia-
tion. Results Relative intensities of protein bands (g) were quantified by using Image J software and calculated by using the samples normalized to β-actin. All data
are presented as mean ± S.D. and derived from three independent experiments. *Po0.05; **Po0.01; NS, no significance differentiation, we examined the relative expression levels of
these genes in the first 10 days of RA-induced differentiation
(Supplementary Figure S6). The results showed that Foxj3
and Zbtb18 exhibited a similar expression pattern, that is, their
expression levels rapidly declined in the first 2 days, and the
temporary increased, and finally returned to a relatively low
level during neural differentiation (Supplementary Figures
S6A, B). However, the expression levels of Olig1 and Olig2
progressively increased and a sudden rise was observed around
day
7
after
RA-induced
neural
differentiation
(Supplementary Figures S6C, F). This effect may be attributed
to the presence of glial cells and neurons on that day. Furthermore, we evaluated whether these four genes are
involved in the neural differentiation. As shown in Figures 6e–
g, ESCs were differentiated into neural-type cells 48 h after
expression of plasmids that encode Olig1, Zic5, Erbb2, or
Olig2 were transfected, as characterized by the dramatic Oct4
reduction and Nestin increase. These results suggested that Cell Death and Disease MiR-219 promotes neural differentiation
H Wu et al 8 8 Figure 7
Olig1 and Olig2 are critical to miR-219-mediated neural differentiation. (a, b) Relative levels of Nestin, Map2, and Tuj1 were detected through qRT-PCR (a) and
western blot (b) in ESCs transfected with Olig1/Olig2-encoding plasmids for 48 h. (c, d) ESCs were transfected with siRNAs that were targeted to Olig1/Olig2 for 12 h and treated
with RA or miR-219 mimics for another 36 h. Relative levels of Nestin, Map2, and Tuj1 were detected through qRT-PCR (c) and western blot. (e) Immunofluorescence shows the
abundance of Oct4, Nestin, and Map2, as well as the morphologies of ESCs transfected with Olig1/Olig2 or miR-291 mimics. **Po0.01; NS, no significance Figure 7
Olig1 and Olig2 are critical to miR-219-mediated neural differentiation. (a, b) Relative levels of Nestin, Map2, and Tuj1 were detected through qRT-PCR (a) a
western blot (b) in ESCs transfected with Olig1/Olig2-encoding plasmids for 48 h. (c, d) ESCs were transfected with siRNAs that were targeted to Olig1/Olig2 for 12 h and treat
with RA or miR-219 mimics for another 36 h. Results Relative levels of Nestin, Map2, and Tuj1 were detected through qRT-PCR (c) and western blot. (e) Immunofluorescence shows t
abundance of Oct4, Nestin, and Map2, as well as the morphologies of ESCs transfected with Olig1/Olig2 or miR-291 mimics. **Po0.01; NS, no significance Figure 7
Olig1 and Olig2 are critical to miR-219-mediated neural differentiation. (a, b) Relative levels of Nestin, Map2, and Tuj1 were detected through qRT-PCR (a) and
western blot (b) in ESCs transfected with Olig1/Olig2-encoding plasmids for 48 h. (c, d) ESCs were transfected with siRNAs that were targeted to Olig1/Olig2 for 12 h and treated
with RA or miR-219 mimics for another 36 h. Relative levels of Nestin, Map2, and Tuj1 were detected through qRT-PCR (c) and western blot. (e) Immunofluorescence shows the
abundance of Oct4, Nestin, and Map2, as well as the morphologies of ESCs transfected with Olig1/Olig2 or miR-291 mimics. **Po0.01; NS, no significance Discussion The most familiar mechanism
is the binding of RA to RAR, which is bound to a DNA region
called the RA response elements and affects the binding of
other transcription factors.39–41 Moreover, RA acts through the
Hox genes, which ultimately control the anterior and posterior
patterns in early developmental stages.13 RA also affects the
changes in the epigenetic marks on histones and creates a
heritable change in chromatin responsiveness.42–44 Kashyap
et al.45 reported that coincident with the RA-induced transcrip-
tional activation of the Hoxa and Hoxb cluster genes, the
epigenomic configuration of these clusters is rapidly remo-
deled by H3K4me3 and acH3. Notably, RA treatment
stabilizes p53 in human ESCs, and p53 activates the
expression levels of miR-34a and miR-145, which then
repress the stem cell factors Oct4, Klf4, Lin28a, and Sox2
and accelerate ESC differentiation.46 These findings sug-
gested that miRNAs are important in the regulation of RA-
associated differentiation of ESCs. Recent studies have suggested that miR-219 regulates NPC
proliferation and differentiation.49,50 However, the specific role
of miR-219 in RA-associated ESC differentiation is not
elucidated. In this study, we found that miR-219 is sufficient
in promoting neural differentiation by targeting Foxj3 and
Zbtb18. On the basis of the results of the RNA-seq performed
on Foxj3-ESCs and Zbtb18-ESCs, we constructed three gene
co-expression networks and identified 17 core genes that are
functionally related to neurodevelopment. Interestingly, 16 of
these genes were upregulated during neural differentiation,
showing that most factors that dominate neural differentiation
can be induced. We then demonstrated that Foxj3 and Zbtb18
are the molecular switches in the neurodevelopment system. Furthermore, we identified four critical genes by constructing
gene co-expression networks. Two of them, namely, Olig1 and
Olig2 might be the most essential elements in neural
differentiation of ESCs. Olig1/2-knockdown ESCs cultured in
the presence of RA or transfected with miR-219 mimics failed
to differentiate into neural cells, as characterized by the
absence of increase of neural cell markers (Map2 and Tuj1). Notably, Nestin (neural stem cell marker) appeared in the
Olig1/2-knockdown ESCs treated with RA or miR-219 mimics. This finding may be attributed to other factors or mechanisms
involved in the miR-219-mediated neural differentiation apart
from Olig1/2. However, this hypothesis requires validation
through further experiments. Moreover, the mechanism of how
RA upregulates miR-219 is not well understood. Discussion We speculate
that histone demethylases accelerate DNA demethylation at
the promoter region of miR-219, thereby increasing the
expression level of miR-219 and facilitating neural differentia-
tion. In addition to epigenetic modifications, other transcription
factors can possibly mediate the effects of RA on miR-219
upregulation.51 Thus the involvement of histone demethylases
or other transcription factors with the RA and miR-219
pathway is interesting for further investigation. To this end, we characterized the expression of miRNAs in
mESCs with RA treatment for 48 h. Our previous studies
showed that ESCs start to differentiate irreversibly after RA
pretreatment for 48 h and continued to do so even after RA
withdrawal.12 Therefore, miRNAs with high expression levels
during the early stage of RA induction may be critical in RA-
induced neural directional differentiation of mESCs. We
performed most of the experiments in J1 ESCs to investigate
the miR-219-dependent neural regulatory networks, which
was later confirmed in D3 and B6 ESCs and in C57BL/6 mice,
to ensure that this finding is not a particular characteristic of a
single cell line. MiR-219 is necessary in promoting oligodendrocyte differ-
entiation, and partially rescues oligodendrocyte differentiation
defects caused by total miRNA loss.31 Kocerha et al.47
reported that miR-219 is important in the expression of
behavioral
aberrations
associated
with
NMDA
receptor
hypofunction. Santa-Maria et al.48 found that miR-219 down-
regulation promotes neurodegeneration by targeting Tau. Overall, we discovered that functionally important gene
expression changes mediated by miRNA contribute to
RA-induced differentiation. We demonstrated that Foxj3 and
Zbtb18, two target genes of miR-219, are the main controls in
neural differentiation of ESCs (Figure 8). Our findings illustrate Figure 8
Schematic representation of RA-induced neural differentiation of ESCs. RA treatment led to upregulation of miR-219, which targeted Foxj3 and Zbtb18. Downregulation of Foxj3 and Zbtb18 resulted in the activation of Olig1, Zic5, Erbb2, and Olig2. The neural regulatory networks were triggered, and the neural directional
differentiation of ESCs was determined. TF, transcription factor Figure 8
Schematic representation of RA-induced neural differentiation of ESCs. RA treatment led to upregulation of miR-219, which targeted Foxj3 and Zbtb18. Downregulation of Foxj3 and Zbtb18 resulted in the activation of Olig1, Zic5, Erbb2, and Olig2. The neural regulatory networks were triggered, and the neural directional
differentiation of ESCs was determined. TF, transcription factor Figure 8
Schematic representation of RA-induced neural differentiation of ESCs. RA treatment led to upregulation of miR-219, which targeted Foxj3 and Zbtb18. Discussion A similar phenomenon was observed in human pluripo-
tent stem cells, in which low-dose RA increased hematopoietic
progenitor cell generation, whereas low-dose RA abrogated
blood cell generation in vitro.37 Here, we found that 1 μM RA is
sufficient to induce mESCs that cultured without any stromal
cell line, neural culture medium, or neural growth factors
undergo neural differentiation. During early embryonic development, RA can facilitate
embryonic positioning along the embryonic axis by acting as Cell Death and Disease MiR-219 promotes neural differentiation
H Wu et al MiR-219 promotes neural differentiation
H Wu et al 9 an intercellular signaling molecule that guides the develop-
ment of the posterior portion of the embryo.18,38 Efforts have
been undertaken to uncover the mechanism of the effects of
RA in embryonic development. The most familiar mechanism
is the binding of RA to RAR, which is bound to a DNA region
called the RA response elements and affects the binding of
other transcription factors.39–41 Moreover, RA acts through the
Hox genes, which ultimately control the anterior and posterior
patterns in early developmental stages.13 RA also affects the
changes in the epigenetic marks on histones and creates a
heritable change in chromatin responsiveness.42–44 Kashyap
et al.45 reported that coincident with the RA-induced transcrip-
tional activation of the Hoxa and Hoxb cluster genes, the
epigenomic configuration of these clusters is rapidly remo-
deled by H3K4me3 and acH3. Notably, RA treatment
stabilizes p53 in human ESCs, and p53 activates the
expression levels of miR-34a and miR-145, which then
repress the stem cell factors Oct4, Klf4, Lin28a, and Sox2
and accelerate ESC differentiation.46 These findings sug-
gested that miRNAs are important in the regulation of RA-
associated differentiation of ESCs. an intercellular signaling molecule that guides the develop-
ment of the posterior portion of the embryo.18,38 Efforts have
been undertaken to uncover the mechanism of the effects of
RA in embryonic development. Materials and Methods
Ethi
t t
t Thi
t Double-stranded mmu-
miR-219-5p (miRBase accession number MIMAT0000664, 5'-UGAUUGUCCA
AACGCAAUUCU-3') mimics, single-stranded mmu-miR-219-5p inhibitors and their
corresponding negative controls were purchased from GenePharma (GenePharma,
Shanghai, China). Negative control siRNA and siRNAs that target Foxj3, Zbtb18,
Olig1, and Olig2 were chemically synthesized by Ribobio (Guangzhou, Guangdong,
China). The knockdown efficiencies of siRNAs were verified by qRT-PCR and western
blot (Supplementary Figure S8). The miRNAs and siRNAs were transfected with
Lipofectamine RNAiMAX reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA)
following the manufacturer’s instructions. RA (all-trans-Retinoic acid, product number:
R2625) was purchased from Sigma-Aldrich (St. Louis, MO, USA). A stock solution of
1 mM RA was prepared in absolute ethanol, stored at −20 °C, and protected from
light. The working concentration of RA was 1 μM. Culture medium and RA were
changed daily and ESCs were passaged every 2–3 days at a 1 : 3–1 : 5 ratio
according to cell density. A direct induction system without any stromal cell line, neural
culture medium, or neural growth factors as neural inducer was performed to render
the normal cultured ESCs a perfect control. RNA-seq and data analysis. Total RNA was extracted from Foxj3-ESCs or
Zbtb18-ESCs by Trizol reagent (Invitrogen) separately. Then, the RNA samples
were sent to Novel Bioinformatics company (Shanghai, China) for RNA-seq. The
RNA quality was checked by Bioanalyzer 2200 (Agilent Technologies, Santa
Clara, CA, USA) and kept at −80 °C. The RNA with RIN48.0 is right for cDNA
library construction. RNA libraries were prepared for sequencing using IonProton. The cDNA libraries were processed for
the proton sequencing process
according to the commercially available protocols. Data were submitted to the
GEO archive. The accession number is GSE61748. All tested genes sorted by fold
changes are listed in Supplementary Data s04-s06. Pathway analysis was used to
find out the significant pathway of the differential genes according to KEGG
database. Fisher’s exact test was calculated to select the significant pathway,
and the threshold of significance was defined by P-value and false discovery
rate (FDR).53 GO analysis was performed to facilitate elucidating the biological implications of
unique genes in the significant or representative profiles of the differentially expressed
gene in the experiment. GO annotations were downloaded from NCBI (http://www. ncbi.nlm.nih.gov/), UniProt (http://www.uniprot.org/), and the GO (http://www. geneontology.org/). Fisher’s exact test was applied to identify the significant GO
categories and FDR was used to correct the P-values. Construction of plasmids. Materials and Methods
Ethi
t t
t Thi
t Materials and Methods
Ethic statement. This study was carried out in strict accordance with the
Guidelines for the Care and Use of Animals of Northwest A&F University. All animal
experimental procedures were approved by the Animal Care Commission of the
College of Veterinary Medicine, Northwest A&F University. C57BL/6 mice were
purchased from Xi’an Jiao-tong University, China. Five-week-old female athymic
nude mice were purchased from The Fourth Military Medical University. Every effort
was made to minimize animal pain, suffering, and distress, and reduce the number
of animals used. Cell culture, transient transfection, and treatment. J1, R1, and D3
mESCs were purchased from American Type Culture Collection (ATCC, Manassas,
VA, USA), and grown adherent to plastic plates (Corning Costar, Cambridge, MA,
USA) coated with matrigel (Geltrex, Thermo Fisher Scientific, San Jose, CA, USA)
and maintained under a feeder-free and serum-free system. The ESC culture
medium was composed of Knockout Dulbecco's modified Eagle's medium
(Knockout DMEM, Thermo Fisher Scientific), 15% (v/v) Knockout Serum
Replacement (Thermo Fisher Scientific), 1 × non-essential amino acids (Thermo
Fisher Scientific), 100 μM β-mercaptoethanol (Millipore, Bedford, MA, USA), 2 mM
glutamine (Thermo Fisher Scientific), 50 units/ml penicillin, 50 μg/ml streptomycin,
and 1000 units/ml LIF (ESGRO, Millipore). The growth condition of mESCs was
consistent throughout this study. Western blot analysis. Western blot analysis was performed as previously
described.52 Blots were probed with 1/1000 rabbit anti-Oct4 (#2788), 1/1000 rabbit
anti-MAP2 (#4542) (Cell Signaling Technology, Beverly, MA, USA), 1/500 rabbit anti-
Foxj3 (#SAB2100844), 1/500 rabbit anti-Zbtb18 (#SAB2103436), 1/1000 mouse
anti-Nestin (#MAB5326), 1/1000 rabbit anti-Brachyury (#B8436), 1/2000 mouse
anti-β-actin (#A5441) (Sigma-Aldrich), 1/1000 goat anti-Flk1 (VEGF receptor 2,
#AF644; R&D Systems, Minneapolis, MN, USA), 1/1000 mouse anti-Sox17
(#ab192453), 1/1000 rabbit anti-Foxa2 (#ab108422) (Abcam, Cambridge, MA,
USA), and 1/1000 mouse anti-Tuj1 (neuronal class III β-tubulin, #MMS-435P;
Covance, Princeton, NJ, USA). Immunoblots were revealed by autograph using
SuperSignal west pico substrate (Thermo Fisher Scientific). The relative intensity of
the protein bands was quantified using Image J software (NIH, Bethesda, MD, USA)
and calculated by samples normalized to the controls. All data were presented as
mean ± S.D. and derived from three independent experiments. Plasmids were transfected with Xfect Transfection Reagent (Clontech, Palo Alto,
CA, USA) following the manufacturer’s instructions. The transfection efficiency was
monitored by a backbone vector with an extra eGFP element and calculated by flow
cytometry. The average transfection efficiency was ~ 75%–85%, and the
representative result is shown in Supplementary Figure S7. Discussion Downregulation of Foxj3 and Zbtb18 resulted in the activation of Olig1, Zic5, Erbb2, and Olig2. The neural regulatory networks were triggered, and the neural directional
differentiation of ESCs was determined. TF, transcription factor Figure 8
Schematic representation of RA-induced neural differentiation of ESCs. RA treatment led to upregulation of miR-219, which targeted Foxj3 and Zbtb18. Downregulation of Foxj3 and Zbtb18 resulted in the activation of Olig1, Zic5, Erbb2, and Olig2. The neural regulatory networks were triggered, and the neural directional
differentiation of ESCs was determined. TF, transcription factor Cell Death and Disease MiR-219 promotes neural differentiation
H Wu et al 10 the mechanism of miRNA-mediated neural differentiation of
ESCs, as well as the plasticity and dynamic nature of the gene
regulatory networks during neural differentiation. However, the
Foxj3 and Zbtb18 regulatory activities dependent on other
transcription factors remain unclear. The specific mechanism
of Foxj3 and Zbtb18 for determining the fate of neural
differentiation and the functional roles of Foxj3 and Zbtb18 in
multiple biological processes requires further investigation. the mechanism of miRNA-mediated neural differentiation of
ESCs, as well as the plasticity and dynamic nature of the gene
regulatory networks during neural differentiation. However, the
Foxj3 and Zbtb18 regulatory activities dependent on other
transcription factors remain unclear. The specific mechanism
of Foxj3 and Zbtb18 for determining the fate of neural
differentiation and the functional roles of Foxj3 and Zbtb18 in
multiple biological processes requires further investigation. Luciferase assays. Luciferase assays were performed using the DLR Assay
System (Promega) as previously described.52 In brief, ESCs were cotransfected with
a luciferase reporter construct and internal control plasmid pRL-SV40 (Promega). Cells were lysed 24 h after transfection or treatment, and relative luciferase activities
were measured by firefly luciferase luminescence divided by Renilla luciferase
luminescence. Luciferase assays. Luciferase assays were performed using the DLR Assay
System (Promega) as previously described.52 In brief, ESCs were cotransfected with
a luciferase reporter construct and internal control plasmid pRL-SV40 (Promega). Cells were lysed 24 h after transfection or treatment, and relative luciferase activities
were measured by firefly luciferase luminescence divided by Renilla luciferase
luminescence. Real-time qRT-PCR. We used qRT-PCR to analyze miRNA expression. Total
RNA samples (including small RNA molecules) were isolated using mirVana miRNA
Isolation Kit (Ambion, Austin, TX, USA). Purified RNA was reverse-transcribed using a
miScript II RT Kit (Qiagen, Hilden, Germany). Discussion The expression of mature miRNAs was
quantified using a miScript SYBR Green PCR Kit, which contained 10× miScript
Universal Primer (Qiagen), and was performed according to the manufacturer’s
instructions. Quantization of U6 was performed to normalize miRNA expression levels. We also used qRT-PCR to analyze gene expression. Total RNA samples were
isolated using Trizol reagent (Invitrogen). Purified RNA was reverse-transcribed using
a SYBR PrimeScript RT–PCR Kit (Takara, Otsu, Shiga, Japan). The expression of
mRNAs was quantified using a SYBR Premix ExTaq II Kit (Takara). Real-time PCR
was performed on an ABI StepOnePlus PCR system (Applied Biosystems, Foster
City, CA, USA), and results were normalized to β-actin mRNA levels. Data were
analyzed using the 2 ΔΔCt method. Primer sequences used for qPCR are listed in
Supplementary Tables S6 and S7. PUBLISHER’S NOTE Teratoma formation, H&E staining, and scoring method. The
ESCs with stable expressions of Foxj3/Zbtb18 were pretreated with RA for 48 h, and
injected to nude mice to generate teratomas. The differentiations of the three germ
layers were analyzed by H&E staining. In brief, teratomas were fixed with 10%
buffered formaldehyde for more than 24 h, embedded in paraffin, sectioned, and
stained with H&E according to the standard procedure. To evaluate the
potential of three germ layer differentiation, we generated nine teratomas from
the control ESCs, RA-pretreated ESCs, and RA-pretreated Foxj3/Zbtb18-OE ESCs,
respectively. We obtained 10 slices from each teratoma, and each slice was
observed by three operators. Individual germ layer-specific tissue scores were
added to calculate the germ layer score. The scores of the nine teratomas
were added to determine the percentages of three germ layer differentiations
of the control ESCs, RA-pretreated ESCs, or RA-pretreated Foxj3/Zbtb18-OE
ESCs. The scoring of all the slices was performed by the same operators for
consistency. Springer Nature remains neutral with regard to jurisdictional claims in published maps
and institutional affiliations. 1. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 116:
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zygotes under a Zeiss Axio Observer Z1 fluorescence microscope (Carl Zeiss,
Jena, Germany) equipped with an Eppendorf Transfer-Man NK2 micromanipulator
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t Full-length coding sequences of Foxj3 (NCBI
accession
number
NM_172699)
and
Zbtb18
(NCBI
accession
number
NM_001012330) were amplified from J1 cDNA. These fragments were inserted
into pCMV-HA (Clontech; verified in Supplementary Figure S8), pCDH-MCS-T2A-
Puro-MSCV (System Biosciences, Palo Alto, CA, USA), and pCAG-EGFP (kindly
provided by Dr Wei Zhang) vectors. The 3′-UTR of Foxj3 (1084 bp in length. Located at bases 2041–3124 refer to NM_172699.3; –34 to +1050 if consider base
2075 as the start site of UTR) and Zbtb18 (936 bp in length. Located at bases
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the start site of UTR) were amplified from J1 cDNA and inserted into the psicheck-2
vector (Promega, Madison, WI, USA) through standard molecular cloning methods
and confirmed by sequencing. All the primers used for plasmid construction are
listed in Supplementary Tables S4 and S5. Gene co-expression networks were presented to find the relations among genes. Gene co-expression networks were built according to the normalized expression
values of genes selected from genes in significant GO terms. For each pair of genes,
we calculate the Pearson correlation and choose the significant correlation pairs
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numbers one node has to the other.55 While considering genes in different networks,
core regulatory factors were determined by the degree differences (Dif degree)
between control ESCs and Foxj3/Zbtb18-ES. K-cores were also introduced to simplify
the graph topology analysis. K-core means that at least K other genes are
connected to a node.56,57 Cell Death and Disease MiR-219 promotes neural differentiation
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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
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Acad Sci USA 2009; 106: 3507–3512. r The Author(s) 2017 Supplementary Information accompanies this paper on Cell Death and Disease website (http://www.nature.com/cddis) Cell Death and Disease
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English
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Suppression Substractive Hybridization and NGS Reveal Differential Transcriptome Expression Profiles in Wayfaring Tree (Viburnum lantana L.) Treated with Ozone
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Frontiers in plant science
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cc-by
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ORIGINAL RESEARCH
published: 01 June 2016
doi: 10.3389/fpls.2016.00713 Elena Gottardini 1, Antonella Cristofori 1, Elisa Pellegrini 2*, Nicola La Porta 1, 3, 4,
Cristina Nali 2, Paolo Baldi 5 and Gaurav Sablok 1, 6 Elena Gottardini 1, Antonella Cristofori 1, Elisa Pellegrini 2*, Nicola La Porta 1, 3, 4,
Cristina Nali 2, Paolo Baldi 5 and Gaurav Sablok 1, 6 1 Fondazione Edmund Mach, Sustainable Agro-Ecosystems and Bioresources Department, Research and Innovation Centre,
Trento, Italy, 2 Department of Agriculture, Food and Environment, University of Pisa, Pisa, Italy, 3 MOUNTFOR Project Centre,
European Forest Institute, Trento, Italy, 4 Consiglio Nazionale delle Ricerche, Istituto per la Valorizzazione del Legno e delle
Specie Arboree, Florence, Italy, 5 Fondazione Edmund Mach, Genomics and Biology of Fruit Crops Department, Research
and Innovation Centre, Trento, Italy, 6 Plant Functional Biology and Climate Change Cluster (C3), University of Technology
Sydney, Sydney, NSW, Australia Edited by: Edited by:
Mohammad Anwar Hossain,
Bangladesh Agricultural University,
Bangladesh Tropospheric ozone (O3) is a global air pollutant that causes high economic damages
by decreasing plant productivity. It enters the leaves through the stomata, generates
reactive oxygen species, which subsequent decrease in photosynthesis, plant growth,
and biomass accumulation. In order to identify genes that are important for conferring
O3 tolerance or sensitivity to plants, a suppression subtractive hybridization analysis was
performed on the very sensitive woody shrub, Viburnum lantana, exposed to chronic
O
−
3 treatment (60 ppb, 5 h d 1 for 45 consecutive days). Transcript profiling and relative
expression assessment were carried out in asymptomatic leaves, after 15 days of O3
exposure. At the end of the experiment symptoms were observed on all treated leaves
and plants, with an injured leaf area per plant accounting for 16.7% of the total surface. Cloned genes were sequenced by 454-pyrosequencing and transcript profiling and
relative expression assessment were carried out on sequenced reads. A total of 38,800
and 12,495 high quality reads obtained in control and O3-treated libraries, respectively
(average length of 319 ± 156.7 and 255 ± 107.4 bp). The Ensembl transcriptome
yielded a total of 1241 unigenes with a total sequence length of 389,126 bp and an
average length size of 389 bp (guanine-cytosine content = 49.9%). mRNA abundance
was measured by reads per kilobase per million and 41 and 37 ensembl unigenes
showed up- and down-regulation respectively. Unigenes functionally associated to
photosynthesis and carbon utilization were repressed, demonstrating the deleterious
effect of O3 exposure. Unigenes functionally associated to heat-shock proteins and
glutathione were concurrently induced, suggesting the role of thylakoid-localized proteins
and antioxidant-detoxification pathways as an effective strategy for responding to O3. Gene Ontology analysis documented a differential expression of co-regulated transcripts Mohammad Anwar Hossain,
Bangladesh Agricultural University,
Bangladesh Reviewed by:
Golam Jalal Ahammed,
Zhejiang University, China
Marcelo Falsarella Carazzolle,
State University of Campinas, Brazil *Correspondence:
Elisa Pellegrini
elisa.pellegrini@for.unipi.it Specialty section:
This article was submitted to
Plant Physiology,
a section of the journal
Frontiers in Plant Science Received: 18 December 2015
Accepted: 09 May 2016
Published: 01 June 2016 Suppression Substractive
Hybridization and NGS Reveal
Differential Transcriptome
Expression Profiles in Wayfaring Tree
(Viburnum lantana L.) Treated with
Ozone Elena Gottardini 1, Antonella Cristofori 1, Elisa Pellegrini 2*, Nicola La Porta 1, 3, 4,
Cristina Nali 2, Paolo Baldi 5 and Gaurav Sablok 1, 6 June 2016 | Volume 7 | Article 713 INTRODUCTION respect, it is important to categorize acute vs. chronic exposures,
respectively in short- and long-time treatments (Miller, 2011). Ozone (O3) is a gas naturally present in both troposphere and
stratosphere. Particularly, tropospheric O3 results from a series of
complex photochemical reactions involving primary pollutants,
such as nitrogen oxides (NOx), volatile organic compounds
(VOC) and carbon monoxide (CO) mainly generated by
human activities (Jenkin and Clemitshaw, 2000). O3-producing
photochemical reactions are favored by high temperatures and
elevated light intensities (Cristofanelli and Bonasoni, 2009). During summertime, the Mediterranean basin is characterized
by specific meteorological conditions (i.e., sunny, hot, and dry
climate) that enhance photochemical O3 formation (Millàn et al.,
2000). At the mid-latitudes of the Northern Hemisphere, O3
concentrations have more than doubled over the last decades
(Monks et al., 2015). Especially in Europe, a general trend toward
a decline in peak concentrations has been observed, taking into
account the implementation of European air pollution policies
reducing precursor emissions. On the other hand, Dawnay
and Mills (2009) documented a concomitant increase in O3
background concentrations, due to the rise in anthropogenic
emissions on a global scale. The ambient O3 concentrations have
a marked impact not only on human health (Yang and Omaye,
2009; Norval et al., 2011), but also on natural ecosystems, crop
productivity (yield and quality), manufactures and works of art
(Cass et al., 1989). For these reasons, it is important to understand
the regulatory behavior of O3 induced stress in plants (e.g., Braun
et al., 2014; Doring et al., 2014a; González-Fernández et al.,
2014). Taking into account the literature, a bio-molecular approach
might enable a better understanding of oxidative stress-plant
interactions. Current knowledge concerning specific molecular
alterations caused by O3 at the transcriptomic level is limited
(Heath, 2008; Kanter et al., 2013) and has been primarily
elucidated (i) in model plants, like Arabidopsis thaliana
(Hirayama and Shinozaki, 2010) and Medicago truncatula
(Puckette et al., 2009, 2012); (ii) in crops, like Pisum sativum
(Savenstrand et al., 2000), Oryza sativa (Frei, 2015; Sarkar et al.,
2015); and (iii) in herbaceous annual plants, like Centaurea
jacea (Francini et al., 2008), Melissa officinalis (Doring et al.,
2014b). Moreover, interactions occurring between O3 exposure
and changes in the expression profile of several genes have been
described in some woody species. Olbrich et al. INTRODUCTION (2010) studied transcriptional responses in
juvenile Fagus sylvatica saplings fumigated with a double
concentration of ambient O3 over 3 years by conducting
microarray hybridization. Recently, the effects of O3 (twice
ambient concentration) on F. sylvatica were investigated
performing
a
large-scale
protein
analysis
based
on
2-D
Fluorescence Difference Gel Electrophoresis (2-DE DIGE)
(Kerner et al., 2014). Rizzo et al. (2007) identified the
differential expression of genes induced by an episodic O3
treatment (150 ppb for 5 h) in two poplar clones with
different O3-sensitivity, performing Suppression Subtractive
Hybridization (SSH). Nathaniel et al. (2011) investigated the
transcriptional and genetics O3 responsiveness (chronic and
acute treatment) in two divergent Populus species. Using an
inbred F2 mapping population derived from these two species,
they mapped quantitative trait loci (QTLs) associated with
O3 response, and examined segregation of the transcriptional
response to O3 and co-localized genes showing divergent
responses between tolerant and sensitive genotypes. Tuomainen
et al. (1996) studied the acute O3-induced reactions at
biochemical and transcriptomic levels in two Betula clones
differing in O3-sensitivity, whereas Zinser et al. (2000) focalized
on Pinus sylvestris. Similarly, Kontunen-Soppela et al. (2010)
revealed the patterns of gene expression in Betula papyrifera
leaves exposed to twice ambient O3 concentration using
microarray analyses. After entering into the leaf via the open stomata, O3 interacts
immediately with biological molecules (like bio-membranes and
enzymes) and releases reactive oxygen species (ROS), thereby
triggering an oxidative burst (Jaspers et al., 2005). Plants deploy
several response mechanisms, some of which are universally
conserved among species (Whaley et al., 2015). Through a
signaling cascade (Vainonen and Kangasjärvi, 2015), O3 affects
primarily biological processes involved in plant productivity,
such as regulation of structural and chemical components
of photosynthesis (Pellegrini et al., 2015), respiration and
transpiration (Heath, 2008). The toxicology of this pollutant is
complex: many factors such as species, provenance, genotype
and leaf age together with environmental, nutritional, and health
conditions, play a key role in determining the overall plant
response (Manninen et al., 2009). Moreover, contrasting results
may be caused by different O3 concentrations as well as by
different spatial and temporal scales of this pollutant. Citation: Gottardini E, Cristofori A, Pellegrini E,
La Porta N, Nali C, Baldi P and
Sablok G (2016) Suppression
Substractive Hybridization and NGS
Reveal Differential Transcriptome
Expression Profiles in Wayfaring Tree
(Viburnum lantana L.) Treated with
Ozone. Front. Plant Sci. 7:713. doi: 10.3389/fpls.2016.00713 June 2016 | Volume 7 | Article 713 Frontiers in Plant Science | www.frontiersin.org Gottardini et al. Ozone-Molecular Response of Wayfaring for several functional categories, including specific transcription factors (MYB and
WRKY). This study demonstrates that a complex sequence of events takes place in the
cells at intracellular and membrane level following O3 exposure and elucidates the effects
of this oxidative stress on the transcriptional machinery of the non-model plant species
V. lantana, with the final aim to provide the molecular supportive knowledge for the use
of this plant as O3-bioindicator. Keywords:
SSH,
gene
ontology,
photosynthesis,
detoxification,
HSP20-like
chaperone,
PCR-selectTM,
lipoxygenase activity Frontiers in Plant Science | www.frontiersin.org Cultural Practices, Plant Material, and
Ozone Exposure One-year-old agamically reproduced saplings of V. lantana were
grown for 1 month in plastic pots containing a mix of steam
sterilized medium soil and peat (1:1) in a controlled environment
facility (steady temperature of 20 ± 1◦C, relative humidity
(RH) of 85 ± 5% and photon flux density at plant height of
530 µmol photon m−2 s−1 provided by incandescent lamps,
following a 14 h photoperiod). A sub-sample of 18 uniform
plants were selected when they were ca. 35 cm tall (ca. 30 fully
expanded leaves), and were placed in a controlled fumigation
environment facility under the same climatic conditions as the
growth chamber. Nine plants were exposed to 60 ± 13 ppb of
O3 (1 ppb = 1.96 µg m−3, at 20◦C and 101.325 kPa) for 45
consecutive days (5 h d−1, in form of a square wave between
9:00 a.m. and 2:00 p.m.). At the same time, nine control plants
were exposed to charcoal-filtered air. The entire methodology
was performed according to Lorenzini et al. (1994). Leaf samples
(n = 5) were collected from three treated and three control plants
after 15 days of fumigation (before the onset of foliar symptoms),
immediately frozen in liquid nitrogen (N2) and kept at −80◦C for
RNA extraction. INTRODUCTION In this In this study, we focused on wayfaring tree (Viburnum lantana
L.), a common deciduous shrub species, widespread in most
part of Europe, North Africa, North America and temperate
Asia, and well-known for its sensitivity to O3 (Novak et al., Frontiers in Plant Science | www.frontiersin.org June 2016 | Volume 7 | Article 713 Frontiers in Plant Science | www.frontiersin.org 2 Ozone-Molecular Response of Wayfaring Gottardini et al. was added. After 30 min on ice, the RNA was selectively pelleted
by centrifugation (21,000 × g for 20 min at 4◦C) and re-
suspended in 500 µl of SSTE buffer [10 mM Tris-HCl (pH
8.0), 1 mM EDTA (pH 8), 1% SDS, 1 M NaCl], pre-heated at
65◦C. An equal volume of chloroform:isoamyl alcohol (24:1 v/v)
was added, centrifuged (11,000 × g for 10 min at 4◦C) and
the supernatant was transferred to a new tube and precipitated
with one volume of cold isopropanol. The pellet was washed
with ethanol (70%), air-dried and re-suspended in DEPC-treated
water. Messenger RNA was isolated using GenEluteTM mRNA
Miniprep kit (Sigma-Aldrich, St. Louis, MO, USA) according to
the manufacturer’s instructions. SSH was performed using PCR-
selectTMcDNA subtraction kit (Clontech Laboratories, Mountain
View, CA, USA) following the procedure described in the
user manual. Forward and reverse subtractions were performed
using control and O3-treated leaves and PCR products were
subsequently sequenced using 454-pyrosequencing (GS FLX+
System, Roche Diagnostics GmbH, Penzberg, Germany). Raw
reads obtained from the present study are recorded in the
European Nucleotide Archive (ENA) under the project number
PRJEB9317. 2003; Calatayud et al., 2010). Wayfaring tree sensitivity has been
already assessed in terms of morphological (i.e. foliar symptoms)
and physiolgical traits in order to evaluate its potential as bio-
indicator (Gottardini et al., 2010, 2014a,b). Although a series
of studies has been conducted on Viburnum spp. (Clement and
Donoghue, 2012), this report represents the first attempt to assess
the O3 sensitivity of this species at molecular level. Specifically,
paired SSH and 454-pyrosequencing analyses were performed in
plants exposed to near-ambient O3 concentrations in controlled
environmental conditions. Data collected in this study may be
useful to better understand results obtained in natural field
conditions. Sequence Cleaning, Processing, and
Functional Annotation Sequencing reads, obtained from the two libraries (induced
and repressed), were cleaned, removing adapter and primer
sequences and trimming low-quality ends. We further removed
reads shorter than 100 bp and having an average Phred
equivalent quality score lower than 15 bp. After read cleaning,
homo-polymer stretches (polyA/T) were estimated and were
subsequently masked using an in-house PERL script. Masking of
the polyA/T was done to increase the sensitivity and specificity
in the assembly. For the creation of the Ensembl transcriptome,
all the cleaned reads of the induced and repressed libraries were
concatenated and a single Ensembl transcriptome was built using
MIRA (Chevreux et al., 2004) and CAP3, an overlap layout
consensus assembly approach (Huang and Madan, 1999) with an
overlap percentage identity cut-offof 97%, to avoid the formation
of the spurious assemblies. Resulting contigs and singletons were
clustered into representative set of unigenes for each library. All
the repetitive reads that were falling into the mega-hub during the
MIRA assembly were discarded so as to ensure the correctness
and the accuracy of the assembled unigenes. Symptom Assessment Each marked leaf was scored in percent of O3-damaged surface
(5%-classes) by in-hand examination with a 10x hand lens and
symptoms identified as reported by Gottardini et al. (2014a). For
each plant, the mean percentage of injured leaf area of the marked
leaves per date was calculated. Following unigene assembly, unigenes in each library were
subjected to BLASTx with an E-value cut offof 1E-5 against
NCBI database available from http://www.ncbi.nlm.nih.gov. All
the unigenes were translated into six possible translational frames
using the sixpack package of the EMBOSS available from http://
emboss.sourceforge.net/ and the putative open-reading frames
(ORF) were extracted using the Getorf of the EMBOSS package. All the translated frames were queried for the identification of
the InterPro domains and the longest frame with no internal
stop codon and with assigned InterPro domain was kept as
an assigned functional domain to that respective unigene. FastAnnotator (Chen T. W. et al., 2012) and PLAZA version 2.5
(Van Bel et al., 2013) were run to identify functional annotations Read Generation and De novo Assembly
for Plants Exposed to O3 Stress associated with the unigenes. Gene Ontology (GO) was derived
for each unigenes, slimmed using the plant GO slim (available
from http://www.geneontology.org) and classified according to
biological and molecular functions and cellular localization. Transcription factors were identified using the PlantTFcat (Dai
et al., 2013). p
3
A total of 43,815 and 13,610 high quality reads were generated in
induced and repressed libraries, respectively (Table 1). Following
sequencing, reads were filtered as described in the Materials
and Methods section, resulting in a total of 38,800 and 12,495
clean reads (Table 1). The mean read length for the induced
and repressed library was 319 ± 156.7 and 255 ± 107.4 bp,
respectively (data not shown). 454-assembly using MIRA of the
Ensembl transcriptome yielded a total of 1238 unigenes with a
total sequence length of 389,126 bp and an average length size
of 389 bp (GC = 49.9%, Table 1). ORF predictions revealed
a total of 161 (13.0%) sequences with a proper start codon
and a total of 574 sequences (46.3%) with proper stop codons. Interestingly, a low number of unigenes (73, 5.9%) with predicted
ORF contain frameshifts, which indicates a good quality of the
assembly. Identified frameshifts were corrected using FrameDP
(Gouzy et al., 2009). Expression Assessment using Read
Mapping Back to Assembled Ensemble
Transcriptome To
evaluate
the
expression
levels
of
the
unigenes
in
induced/repressed libraries, we mapped the individual library
reads back to the ensembl transcriptome and reads per kilobase
per million (RPKM) was calculated as expression estimate:
RPKM (A) = 1,000,000 × C × 1000)/(N × L), where A is defined
as the expression of the unigene, C corresponds to the reads that
align uniquely to the unigene, N refers to total number of reads
that uniquely aligned to all genes, and L refers to the length of
gene A. Transcript mapping was performed using BWA-SW
algorithm, as implemented in Burrows-Wheeler Aligner [BWA,
available from http://bio-bwa.sourceforge.net/; Li and Durbin
(2009)]. Expression values were further analyzed to identify the
transcripts whose expression was significantly up- or down-
regulated during the O3 treatment, using the log2 fold change
(RPKM induced/RPKM repressed), as previously described in
Kanter et al. (2013). To identify the functionally and statistically
enriched biological pathways and GOs in the up- and down-
regulated unigenes, those showing up- and down-regulation in
Ensembl unigenes were analyzed using KOBAS, with A. thaliana
as a background dataset. All the identified biological pathways
and GOs were statistically evaluated using the hypergeometric
test/Fisher’s exact test followed by Benjamini and Hochberg FDR
correction [P < 0.001 (Mao et al., 2005; Wu et al., 2006; Xie et al.,
2011)]. Functional Classification for Plants
Exposed to O3 Functional classification of the assembled unigenes indicates
the putative functional changes occurring at gene level in plants
subjected to O3. The Ensembl transcriptome was annotated by
performing stringent BLASTx searches (E-value threshold, 1E-5)
against NCBI and PLAZA version 2.5. Functional annotation
of the Ensembl transcriptome (1,238 unigenes) revealed a
total of 744 transcript sequences (60.1%), with an assigned
GO category and a total of 772 transcript sequences (62.4%),
with an assigned InterPro domain, respectively (Table 1 and
Supplementary Table 1
and
Supplementary
Datasheet
1). The results of BLASTx (E-value threshold, 1E-5) searches
against NCBI database and PLAZA version 2.5 (Van Bel
et al., 2013) resembled those reported for whole genome
expressed genes and tissue-specific cDNA extracts in other plant
species (Legrand et al., 2007, 2010; Hao et al., 2015). Based on
GO-slim annotations, Ensembl unigenes were classified into
three ontological categories: cellular component, biological RNA Extraction and PCR-Select for Library
Creation Total RNA was extracted from leaves using the protocol
described by Gambino et al. (2008). Frozen leaves were ground
to fine powder using a pre-chilled mortar and a pestle. Five
milliliter of extraction buffer [2% CTAB, 2.5% PVP-40, 2 M NaCl,
100 mM Tris-HCl (pH 8.0), 25 mM EDTA (pH 8.0), and 2%
β-mercaptoethanol, added just before use] were heated at 65◦C
and added to 1 g of ground tissue. After 10 min of incubation
at 65◦C, two independent extractions were performed, using
chloroform:isoamyl alcohol (24:1 v/v). The supernatant was
transferred to a new tube and LiCl (3 M final concentration) Frontiers in Plant Science | www.frontiersin.org June 2016 | Volume 7 | Article 713 3 Ozone-Molecular Response of Wayfaring Gottardini et al. Frontiers in Plant Science | www.frontiersin.org Expression Estimates and Functional
Profiling of Up- and Down-Regulated
Unigenes FIGURE 1 | Functional classification of Viburnum lantana ensembl
unigenes within cellular component (A), biological process (B), and
molecular function (C) categories. g
Expression abundance for the unigenes was estimated by
mapping the reads from the individual library to the assembled
Ensemble transcriptome and was assessed as number of reads
per RPKM. According to Kanter et al. (2013), prior to linking
the expression estimates to biological functions, unigenes having
a RPKM lower than 7 were discarded to avoid the false
interpretation of the log2 fold change values. A total of 78 (6.3%)
Ensembl unigenes showed a variation in expression values after
O3-treatment. In particular, 41 ensembl unigenes showed an up-
regulation average [log2 (RPKM induced/RPKM repressed) of 3.7
(2.1 SE) ranged between 0.4 and 8.7 (Table 2)] and the other
37 showed a down-regulation [log2 (RPKM induced/RPKM
repressed) ranged between –0.1 and –8.7 (Table 3)]. Functional
ontology and the assigned InterPro domain of the up- and down-
regulated unigenes (30 and 29, respectively) are tabulated in
the Supplementary Table 2. We observed induction of unigenes
(EnsVib0416 and EnsVib0041), which are functionally associated
to heat-shock proteins (IPR008978, HSP20-like chaperone;
IPR002068, Heat shock protein Hsp20; Supplementary Table 2). The expression levels of HSPs (IPR008978 and IPR002068)
in terms of RPKM for the induced library were 58,328.8
and 12,510.56 (vs. 140.4 and 212.3, for the repressed library
respectively), which clearly indicate a strong induction of
HSPs in O3-treated plants [log2 fold 8.7 and 5.9, respectively
(Table 2)]. The observed results are in accordance with previous
observations reported by Eckey-Kaltenbach et al. (1997) in
Petroselinum crispum seedlings exposed to O3 (200 ppb, 10 h)
and post-cultivated in pollutant-free air, using a Northern
blot analysis. Recently, Al-Whaibi (2011) reviewed the role of
HSPs demonstrating their function as molecular chaperones,
regulating the (i) localization, (ii) degradation, (iii) accumulation, FIGURE 1 | Functional classification of Viburnum lantana ensembl
unigenes within cellular component (A), biological process (B), and
molecular function (C) categories. and (iv) folding of proteins during their synthesis. Taking into
account these studies, HSPs can be considered as the first line of
defense against O3 (Haslbeck and Vierling, 2015). Using a proteomic approach, Torres et al. (2007) documented
that expression levels of HSPs were strongly increased in
O3-stressed young leaves of maize (200 ppb, 3 h) and that
their functions were correlated with glycolysis, photosynthesis,
antioxidant-, and pathogen-related defense. Visible Foliar Symptoms y
p
After 15 days of O3 fumigation leaves did not show any visible
symptoms. Thirty days from the beginning of the exposure
[AOT40 = 3000 ppb h; AOT40: ozone Accumulated Over a
Threshold of 40 ppb, sensu de Leeuw and van Zantwoort (1997)],
fully expanded leaves from O3-fumigated plants showed several
minute (Ø 1-2 mm) roundish dark-blackish necrosis located
in the interveinal area of the adaxial surface. Symptoms were
observed on all the examined leaves (n = 30) and plants (n = 6),
with an injured leaf area per plant accounting for 4.2% (SE 1.4%;
range 1–20%). At the end of the experiment (AOT40 4500 ppb
h, 45 days from the beginning of exposure), the injured area was
16.7% (SE 2.47%; range 5–60%) of the total surface. Visible foliar
injury has been used in many field experiments as an indicator of
the response of V. lantana to O3 exposure (e.g., Gottardini et al.,
2010). Fully expanded leaves showed symptoms similar to those
previously reported in seedlings from OTC experiments (Novak
et al., 2005, 2008) and in plants grown in natural conditions
(Gottardini et al., 2014a,b). TABLE 1 | Summary of RNA sequencing and de novo assembly to
construct the gene set of Viburnum lantana plants exposed to O3
treatment (60 ppb of O3, 5 h d−1 for 15 consecutive days). Induced
Repressed
Ensembl
Initial sequencing reads
43815
13610
57425
Cleaned reads
38800
12495
51295
Unigenes
543
705
1238
Total length of Unigenes (bp)
137438
254585
389126
N50 stats (pb):
277
434
389
Total GC count (pb):
68866
126700
194216
GC (%):
50.1
49.8
49.9
GO categories
262 (48.3%)
481 (68.2%)
744 (60.1%)
Functional protein domains
295 (54.3%)
476 (67.5%)
772 (62.4%)
Number of the sequencing reads, clustering details, and the unigene details. GC,
guanine-cytosine content; GO, gene ontology. TABLE 1 | Summary of RNA sequencing and de novo assembly to
construct the gene set of Viburnum lantana plants exposed to O3
treatment (60 ppb of O3, 5 h d−1 for 15 consecutive days). Frontiers in Plant Science | www.frontiersin.org June 2016 | Volume 7 | Article 713 4 Ozone-Molecular Response of Wayfaring Gottardini et al. process, and molecular function (Figure 1). Within the cellular
component category, 21 GO slims were identified including
cell, membrane, thylakoid, and other apparatus (Figure 1A). Visible Foliar Symptoms Cell, cellular component, intracellular, and cytoplasm were
the most represented slims in terms of number of genes (393,
393, 323, and 231, respectively). On the other hand, 42 GO
slims were recognized within the biological process category,
such as translation, reproduction, transport, photosynthesis,
cell death, and other functions (Figure 1B). Among all these
slims, metabolic and cellular processes were the most frequent
ones (502 and 419 genes, respectively). Aside from GO cellular
processes, the response to stress was the next most abundant GO
slim (110 gene numbers) after biological and biosynthetic process
(400 and 154, respectively). It comprised responses to different
stresses, including abiotic and endogenous stimuli (93 and 46
genes, respectively, Figure 1B). Within the molecular function
category, 24 GO slims were observed including protein binding,
transcription regulator activity, carbohydrate binding, hydrolase
activity,
receptor
activity,
and
other
enzymatic
function
(Figure 1C). Among all of these slims, binding and catalytic
activity appeared more frequently (415 and 380, respectively). FIGURE 1 | Functional classification of Viburnum lantana ensembl
unigenes within cellular component (A), biological process (B), and
molecular function (C) categories. Frontiers in Plant Science | www.frontiersin.org Expression Estimates and Functional
Profiling of Up- and Down-Regulated
Unigenes It is worthwhile
to mention that HSPs (especially HSP20 superfamily) play a
pivotal role in protecting the photosynthetic machinery against
damage caused by photo-oxidative stress (Lee et al., 2000). Although this property has been demonstrated during heat stress,
we can suppose that the induction of HSPs by O3 treatment
could (i) increase the resistance of photosynthetic machinery to
photoinhibition, and (ii) affect stress tolerance. June 2016 | Volume 7 | Article 713 Frontiers in Plant Science | www.frontiersin.org 5 Ozone-Molecular Response of Wayfaring Ozone-Molecular Response of Wayfaring Gottardini et al. TABLE 2 | Expression log2(RPKM induced/RPKM repressed) of the up-regulated Ensembl unigenes having homologous gene families acco
PLAZA version 2.5 and associated Interpro domains. Ensembl Unigene Homologous Gene Families Associated InterPro domains
Log2 (RPKM induced/RP
EnsVib0002
HOM000181
IPR001024 IPR008976 IPR001246 IPR000907 IPR013819
7.1
EnsVib0022
HOM000082
IPR009038 IPR000348
2.4
EnsVib0041
HOM000926
IPR008978 IPR002068
5.9
EnsVib0046
HOM005074
IPR008892
1.0
EnsVib0120
–
–
0.7
EnsVib0128
HOM000463
IPR002912
3.6
EnsVib0140
HOM000058
IPR013128 IPR000668 IPR013201
2.2
EnsVib0155
HOM000058
IPR013128 IPR000668 IPR013201
1.4
EnsVib0159
HOM002664
IPR002020 IPR016142 IPR016141
5.5
EnsVib0185
HOM001028
IPR017936 IPR012335 IPR000866 IPR012336
0.4
EnsVib0225
–
–
2.5
EnsVib0241
HOM000826
IPR012677 IPR000504
5.1
EnsVib0243
HOM000826
IPR012677 IPR000504
6.5
EnsVib0251
–
–
5.6
EnsVib0267
HOM002675
IPR000772 IPR001574 IPR017989 IPR016139 IPR016138 IPR008997
5.3
EnsVib0271
–
–
1.2
EnsVib0273
HOM000085
IPR012269 IPR000425
4.0
EnsVib0283
–
–
2.5
EnsVib0308
–
–
4.5
EnsVib0314
HOM003594
IPR004662 IPR011148 IPR001057 IPR001048
3.6
EnsVib0316
–
–
1.0
EnsVib0332
–
–
4.3
EnsVib0341
HOM000031
IPR000109 IPR016196
5.0
EnsVib0359
–
–
4.7
EnsVib0376
HOM000016
IPR002213
5.9
EnsVib0383
HOM000438
IPR007608
4.5
EnsVib0387
HOM000533
IPR008580
4.7
EnsVib0402
–
–
5.9
EnsVib0416
HOM000926
IPR008978 IPR002068
8.7
EnsVib0419
HOM000451
IPR005016
4.9
EnsVib0421
HOM002452
IPR003823
1.4
EnsVib0469
HOM000198
IPR012335 IPR002109 IPR012336 IPR014756 IPR003172
1.0
EnsVib0492
HOM004549
IPR011990 IPR006597
3.4
EnsVib0505
HOM000025
IPR003959 IPR005936 IPR003593 IPR000642
5.1
EnsVib0516
HOM000393
IPR012335 IPR010987 IPR004046 IPR012336 IPR004045 IPR017933
5.3
EnsVib0535
HOM001845
IPR008962 IPR002110 IPR000535 IPR020683
0.5
EnsVib0765
HOM005953
IPR014946
3.5
EnsVib0767
–
–
3.3
EnsVib0812
HOM000507
IPR016040 IPR001891 IPR012301 IPR012302
1.5
EnsVib0830
HOM000080
IPR014778 IPR012287 IPR017930 IPR006447 IPR009057
4.9
EnsVib0869
HOM000033
IPR013525 IPR017871 IPR003593 IPR003439
1.5
RPKM, kilo base of exon model per million mapped reads. We
observed
a
log2
fold
up-regulation
(RPKM
(Maccarrone et al., 1992). Recent RNA-seq based tran TABLE 2 | Expression log2(RPKM induced/RPKM repressed) of the up-regulated Ensembl unigenes having homologous gene families according to
PLAZA version 2.5 and associated Interpro domains. Expression Estimates and Functional
Profiling of Up- and Down-Regulated
Unigenes Ensembl Unigene Homologous Gene Families Associated InterPro domains
Log2(RPKM induced/RPK
EnsVib0016
HOM000635
IPR020568 IPR000754 IPR014721
−4.6
EnsVib0032
HOM005074
IPR008892
−1.3
EnsVib0035
HOM000181
IPR001024 IPR008976 IPR001246 IPR000907 IPR013819
−2.2
EnsVib0036
HOM000181
IPR001024 IPR008976 IPR001246 IPR000907 IPR013819
−8.7
EnsVib0039
HOM000950
IPR011032 IPR013149 IPR013154 IPR016040 IPR002085 IPR020843
−0.1
EnsVib0071
–
–
−1.6
EnsVib0080
HOM004197
IPR006311 IPR008797
−5.7
EnsVib0083
HOM000339
IPR020478 IPR010979 IPR001892 IPR001965 IPR011011 IPR017956
−3.2
EnsVib0085
HOM001274
IPR003959
−1.3
EnsVib0139
HOM000013
IPR018957 IPR001841
−0.1
EnsVib0148
HOM000250
IPR001395
−0.6
EnsVib0162
HOM000005
IPR001128 IPR017973 IPR002401
−6.0
EnsVib0178
HOM000181
IPR001024 IPR008976 IPR001246 IPR000907 IPR013819
−5.0
EnsVib0199
HOM001274
IPR003959
−5.4
EnsVib0212
HOM004197
IPR006311 IPR008797
−3.8
EnsVib0217
–
–
−0.2
EnsVib0244
HOM000648
IPR013785 IPR000741
−3.6
EnsVib0266
HOM001274
IPR003959
−4.4
EnsVib0284
HOM003875
IPR009346
−1.6
EnsVib0313
HOM000858
IPR000894
−5.3
EnsVib0435
HOM000051
IPR016040 IPR002347 IPR002198
−0.8
EnsVib0445
HOM005189
IPR017498
−6.1
EnsVib0462
HOM000934
IPR013845 IPR000876 IPR013843 IPR005824 IPR002942
−2.2
EnsVib0476
HOM001646
IPR020568 IPR000851 IPR013810 IPR005324 IPR014721 IPR018192
−1.1
EnsVib0499
–
–
−1.4
EnsVib0501
HOM001714
IPR006214
−4.5
EnsVib0509
–
–
−5.1
EnsVib0512
HOM002153
IPR008991 IPR018259 IPR001147
−8.5
EnsVib0518
HOM000034
IPR012334 IPR000070 IPR006501 IPR011050
−7.2
EnsVib0638
HOM002856
IPR006082
−1.2
EnsVib0688
HOM000502
IPR018957 IPR001650 IPR001841 IPR014021 IPR014001 IPR000330
−0.6
EnsVib0735
HOM003079
IPR003095 IPR015609 IPR001623
−2.6
EnsVib0746
HOM000011
IPR005123
−2.6
EnsVib0792
–
–
−2.6
EnsVib0899
–
–
−0.6
EnsVib0984
–
–
−1.3
EnsVib1062
–
–
−1.6
RPKM, kilo base of exon model per million mapped reads. TABLE 3 | Expression log2(RPKM induced/RPKM repressed) of the down-regulated Ensembl unigenes having homologous gene families according to
PLAZA version 2.5 and associated Interpro domains. RPKM, kilo base of exon model per million mapped reads. A. thaliana ecotype Columbia (Col-0) plants exposed to O3
(300 ppb, 6 h) after 3 h from the beginning of the treatment. Antioxidant enzymatic activities were expected to rise during
a situation that leaded to increased oxidative stress. Results
indicate that GSH-dependent detoxification pathways were
induced by O3. Specifically, the expression level of GSH in terms
of RPKM for the induced library was 1,198.5 (vs. 312.6 for the
repressed library), which suggests an involvement of disulphide
bridges in redox-control process. This is in agreement with the
results reported by D’Haese et al. (2006) for A. thaliana plants
exposed to an episodic O3 treatment (150 ppb, 8 h). has been implicated in membrane alteration and could mediate
the O3-effect. Expression Estimates and Functional
Profiling of Up- and Down-Regulated
Unigenes TABLE 2 | Expression log2(RPKM induced/RPKM repressed) of the up-regulated Ensembl unigenes having homologous gene families according to
PLAZA version 2.5 and associated Interpro domains. Ensembl Unigene Homologous Gene Families Associated InterPro domains RPKM, kilo base of exon model per million mapped reads. We
observed
a
log2
fold
up-regulation
(RPKM
induced/RPKM repressed = 7.1) of a unigene (EnsVib0002)
functionally associated to lipoxygenase activity (GO:0016165,
Supplementary Table 2). This is supported by similar findings in
Lens culinaris seedlings exposed to O3 flux after 30 min from the
beginning of the treatment (Maccarrone et al., 1997). In soybean
seedlings, O3 up-regulated the lipoxygenase gene and its activity,
with a concomitant enhanced membrane lipid peroxidation (Maccarrone et al., 1992). Recent RNA-seq based transcriptomics
indicated an increased expression of genes involved in lipid
metabolic process in two soybean varieties exposed to an
episodic O3 treatment [25–75 ppb, 4 h (Whaley et al., 2015)]. Plant lipoxygenases, which use molecular oxygen to produce
hydroperoxides from unsaturated fatty acids, play a key role in
(i) growth and development, (ii) senescence, and (iii) responses
to biotic and abiotic stresses. In particular, lipoxygenase activity June 2016 | Volume 7 | Article 713 Frontiers in Plant Science | www.frontiersin.org 6 Ozone-Molecular Response of Wayfaring Gottardini et al. TABLE 3 | Expression log2(RPKM induced/RPKM repressed) of the down-regulated Ensembl unigenes having homologous gene families ac
PLAZA version 2.5 and associated Interpro domains. Frontiers in Plant Science | www.frontiersin.org Transcription Factors Thirty-four families of transcription factors were observed for
Ensembl unigenes (Supplementary Table 3). Myeloblast (MYB)-
factors were previously described to be associated with a
diverse array of cellular responses, including plant secondary
metabolism as well as biotic and abiotic tolerance (Kwon et al.,
2013) and were observed in the induced library. MYB proteins
responded at the transcriptional level to O3 stress in V. lantana
(EnsVib0607_ORF+3, IPR001005, and IPR009057 domains). MYB transcription factors could have repressing effects on
genes involved in the biosynthesis of phenylpropanoids (Bender
and Fink, 1998), flavonoids (Borevitz et al., 2000), auxin and
consequently have an effect on the maintenance of cell wall
development, cuticle formation, and lipid metabolism. Few
studies based on the evaluation of plant responses to drought,
salt, and UV stress (Hemm et al., 2001; Golldack et al., 2014) have
reported the functional role of MYBs. To identify the functional enriched terms and pathways
and to approximate the coverage of sequenced and assembled
unigenes, we compared the identified 78 unigenes, whose
expression altered during the O3 stress in V. lantana to
A. thaliana genome predicted coding sequences (CDS) using
KOBAS (E-value threshold, 1E-08; Supplementary Figure 1). Interestingly, 45 out of 78 unigenes showed putative functional
orthologs in A. thaliana (Table 4). GO enrichment was observed
according to the observed functionally enriched linoleate 13S-
lipoxygenase activity (GO:0016165, P = 1.86E-07, corrected
P = 1.59E-05), which might indicate the conversion of the
linoleate into 13-HPODE, responsible for the activation of the
lipid peroxidation process. This suggests that O3 treatments
may induce deleterious effects on (i) integrity, (ii) conformation,
and (iii) transport capacity of membranes in V. lantana,
as reported in other species (Yan et al., 2010; Pellegrini
et al., 2011). Dynamics of gas exchange results strongly
altered as confirmed by the enrichment of GO categories
related to thylakoid (GO:0009579, GO:0009535, GO:0055035,
GO:0044436, GO:0009534, GO:0031976). It confirms that O3
exposure, by free-radical production, induces alterations in the (i) In our study, WRKY-factors were found, which have been
previously reported to be important components in the complex
signaling processes during plant stress responses (Dong et al.,
2003; Zhang et al., 2015). Far less information is available to
understand the function of WRKY proteins in abiotic stress. Some studies demonstrated that the expression of many WRKY
genes is greatly and rapidly induced in response to wounding,
temperature, nutrient deficiency, drought, and salinity (Chen
L. et al., 2012). Expression Estimates and Functional
Profiling of Up- and Down-Regulated
Unigenes It is worthwhile to mention that lipid oxidation is
a double-edged event due to (i) its damaging effects on lipids and
membrane components and (ii) a putative beneficial role in the
signaling pathway [e.g., jasmonic acid formation, Vaultier and
Jolivet (2015)]. An
induction
of
unigenes
(EnsVib0516)
functionally
associated with glutathione (GSH) was observed with GO GSH
binding (GO:0043295), GSH transferase (GO:0004364), and GSH
peroxidase activities (GO:0004602, Supplementary Table 2),
according with the earlier reports by Tosti et al. (2006) in June 2016 | Volume 7 | Article 713 Frontiers in Plant Science | www.frontiersin.org 7 Ozone-Molecular Response of Wayfaring Gottardini et al. A repression of unigenes (EnsVib0080 and EnsVib0212)
functionally associated to photosynthetic process with GO:
photosystem (GO:0009521) and photosynthesis (GO:0015979,
Supplementary Table 2) was found. The expression levels of
photosynthetic process in terms of RPKM for the induced
library were 95.8 and 368.2 (vs. 4,909.0 and 5,013.0, for the
repressed library respectively), clearly suggesting a decrease in
photosynthetic performance in O3-treated plants [log2 (RPKM
induced/RPKM repressed) = −5.8 and −3.8, respectively
(Table 3)]. Similarly, Kontunen-Soppela et al. (2010) reported
a decreased expression of photosynthesis- and carbon fixation-
related genes in B. papyrifera plants exposed to O3 alone
(2x ambient O3 concentration) or in combination with CO2
(target 550 ppm), during the growing season since 1998. A
previous microarray analysis indicated that many genes involved
in photosynthesis were down-regulated in Populus tremuloides
plants (clone 216) subjected to chronic O3 fumigation (1.5x
ambient O3 concentration for 5 consecutive years; Gupta et al.,
2005). photosynthetic apparatus, (ii) content/pattern of thylakoid, and
(iii) functional state of chloroplast membranes. Particularly, O3-
derivative molecular species induce changes in gene expression
responsible for rearrangements in the thylakoid architecture
of V. lantana leaves limiting the damage of PSII activity (as
a compensatory mechanism for the inhibiting photosynthetic
effects) and, generally, counteracting the oxidative stress. Using RNA-seq, Liu et al. (2015) indicated the abundance
and enrichment of the differentially expressed genes involved
in the thylakoid, plastid part, chloroplast, and plastids envelope
in Reaumuria soongorica leaves subjected to UV-B radiation. Additionally, previous immunological studies documented that
O3 treatment could affect energy transfer processes by inducing
alterations in thylakoid membrane proteins (Tognini et al., 1997;
Ranieri et al., 2000). Following O3 treatment, a complex sequence
of events takes place in the cells of V. Expression Estimates and Functional
Profiling of Up- and Down-Regulated
Unigenes lantana (at intracellular
and membrane level), altering key biological processes [such as
metabolism, protein fate (folding, modification, and destination)
and transports] and molecular functions (catalytic and hydrolase
activities). This suggests that plants react to O3 changing
metabolic processes (for example lipid catabolism, sugar and
amino acid metabolism) that are both used (i) directly, as
alternative sources of energy, (nitrogen and carbon skeletons)
and (ii) indirectly, as substrates of secondary metabolite
modifications. According to Heath (2008), we can conclude that
O3 induces deep changes in the expression of genes responsible
for biochemical adjustments and metabolic shifts. Oxidation reactions would be expected to reduce net
photosynthesis predisposing plants to the inhibition of PSII
electron transport (Pellegrini, 2014) and possibly, accelerating
the onset of cell senescence (Pellegrini et al., 2015). In the
present study, impaired photosynthesis was seen as a down-
regulation of PSII oxygen-evolving complex (OEC) PsbQ genes
(regulators for the biogenesis of optically active PSII). According
to Gururani et al. (2015), the down-regulation of OEC is an
efficient and dynamic feedback mechanism to (i) reduce/regulate
the generation of reactive oxygen radicals in PSII (favoring
electron donation by non-water electron donors with a high rate
constant) and (ii) to provide protection against photodamage
in response to abiotic stress. In our case, the easy accessibility
of non-water electrons from antioxidant molecules and the
concomitant increase of GSH-related genes confirm that plants
use a specific set of active mechanisms for ROS scavenging during
O3 exposure. Transcription Factors WRKY proteins respond to O3 stress at the
transcriptional level in V. lantana plants (EnsVib0769_ORF-
1, IPR003657 domain) and WRKY transcription factors could
have inducing effects on genes involved in O3 perception/signal June 2016 | Volume 7 | Article 713 Frontiers in Plant Science | www.frontiersin.org 8 Ozone-Molecular Response of Wayfaring Gottardini et al. TABLE 4 | Statistical analysis on functional homologs (GO categories) between Viburnum lantana and Arabidopsis thaliana ontology assignment and
enrichment analysis using hypergeometric test/Fisher’s exact test and corrected values after Benjamini and Hochberg FDR correction. lysis using hypergeometric test/Fisher’s exact test and corrected values after Benjamini and Hochberg FDR correction. m
Gene Ontology
P-value
Corrected P-value
membrane
GO:0034357
3.63E-11
2.02E-08
GO:0009579
3.94E-11
2.02E-08
koid membrane
GO:0009535
1.65E-10
3.37E-08
membrane
GO:0055035
1.73E-10
3.37E-08
iccation
GO:0009269
1.79E-10
3.37E-08
GO:0044436
1.97E-10
3.37E-08
ane
GO:0042651
3.28E-10
4.80E-08
koid
GO:0009534
9.10E-10
1.04E-07
GO:0031976
9.10E-10
1.04E-07
mpartment
GO:0031984
1.02E-09
1.04E-07
c process
GO:0009106
1.62E-07
1.51E-05
oxygenase activity
GO:0016165
1.86E-07
1.59E-05
bivore
GO:0080027
4.34E-07
3.43E-05
process
GO:0006546
6.30E-07
4.61E-05
no acid catabolic process
GO:0009071
7.82E-07
5.34E-05
c process
GO:0006544
9.61E-07
6.16E-05
no acid metabolic process
GO:0009069
1.42E-06
7.68E-05
oenzyme metabolic process
GO:0006733
1.42E-06
7.68E-05
n transport
GO:0070838
2.16E-06
1.11E-04
c cation transport
GO:0072511
2.34E-06
1.14E-04
ope
GO:0009941
2.61E-06
1.21E-04
acid biosynthetic process
GO:0006636
3.40E-06
1.45E-04
acid metabolic process
GO:0033559
3.40E-06
1.45E-04
GO:0009526
4.11E-06
1.65E-04
meostasis
GO:0030003
5.25E-06
2.07E-04
ostasis
GO:0006873
7.37E-06
2.07E-04
activity
GO:0016702
7.40E-06
2.07E-04
ma
GO:0009570
8.42E-06
2.98E-04
etic process
GO:0031408
8.85E-06
3.02E-04
homeostasis
GO:0055082
1.02E-05
3.36E-04
nthetic process
GO:0009108
1.07E-05
3.34E-04
GO:0009532
1.13E-05
3.46E-04
c process
GO:0006766
1.22E-05
3.46E-04
c process
GO:0031407
1.23E-05
3.46E-04
bolic process
GO:0006732
1.25E-05
3.46E-04
metabolic process
GO:0000096
1.72E-05
4.65E-04
asis
GO:0019725
1.79E-05
4.70E-04
GO:0031975
1.96E-05
4.90E-04
pe
GO:0031967
1.96E-05
4.90E-04
GO:0044434
2.02E-05
4.93E-04
perature stimulus
GO:0009266
2.08E-05
4.95E-04
GO:0015979
2.23E-05
5.19E-04
id biosynthetic process
GO:0008652
2.30E-05
5.24E-04
GO:0044435
2.66E-05
5.78E-04
sis
GO:0055080
2.71E-05
5.78E-04
s with P < 0.001 are showed (45 on the total 78 found for Viburnum lantana). Only GO categories with P < 0.001 are showed (45 on the total 78 found for Viburnum lantana). DNA binding domain). Similar findings were reported by Tosti
et al. (2006) in Col-0 Arabidopsis plants 3 h after the beginning of
O3 treatment. SSH analysis indicated that WRKY genes may be
involved in redox regulation in two poplar hybrid clones exposed DNA binding domain). Similar findings were reported by Tosti
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14-321 Calatayud, V., Marco, F., Cervero, J., Sanchez-Pena, G., and Jose Sanz, M. (2010). Contrasting ozone sensitivity in related evergreen and deciduous shrubs. Environ. Pollut. 158, 3580–3587. doi: 10.1016/j.envpol.2010.08.013 Cass, G. R., Druzik, J. R., Grosjean, D., Nazaroff, W. W., Whitmore, P. M., and
Wittman, C. L. (1989). Protection of Works of Art from Photochemical Smog. Transcription Factors (2006) in Col-0 Arabidopsis plants 3 h after the beginning of
O3 treatment. SSH analysis indicated that WRKY genes may be
involved in redox regulation in two poplar hybrid clones exposed transduction pathway and in redox regulation. This result
suggests that WRKY-factors could act as redox-responsive
sequences and, consequently, as promoter elements specific for
redox regulation (since they possess a redox-sensitive zinc-finger June 2016 | Volume 7 | Article 713 Frontiers in Plant Science | www.frontiersin.org 9 Ozone-Molecular Response of Wayfaring Gottardini et al. to an episodic O3 treatment (Rizzo et al., 2007). Similarly,
Mahalingam et al. (2003) documented an over-representation of
WRKY motifs in the promoter region of genes up-regulated by
an episodic O3 exposure (350 ppb, 6 h) in Col-0 Arabidopsis
plants. Xu et al. (2015) observed that four WRKY-transcriptional
factors genes were highly induced by O3 treatment (350 ppb, 2
h) in Col-0 Arabidopsis plants. Furthermore, the expression gene
profile after O3 fumigation was similar to that of tomato after
Botrytis cinerea (a fungus) (Journot-Catalino et al., 2006) and
Pseudomonas syringae (a bacterium) infections (Birkenbihl et al.,
2012), suggesting that (i) O3 resembles a biotic elicitor and (ii)
stress-regulated genes represent a general stress response. the putative characterization of identified unigenes in V. lantana
and (ii) better understand the response to O3 exposure in a
non-model species. SUPPLEMENTARY MATERIAL This is the first study on differentially expressed genes after
O3 treatment in V. lantana plants. A large number of genes
involved in signaling/transcription, stress/defense, and protein
metabolism showed significant differences in expression of plants
exposed to chronic O3 treatment, suggesting that complex
molecular alterations occurred. By GO slims and pathways
enrichment of the co-regulated genes, it could be demonstrated
that following O3 exposure, a complex sequence of events takes
place in the cells at intracellular and membrane level, altering
a series of biological processes [such as metabolism, protein
fate (folding, post-translational modification, and destination)
and transport] and molecular functions (catalytic and hydrolase
activities). Specifically, the down-regulation of genes associated
to photosynthesis demonstrates the deleterious effects of O3. Up-regulation of genes involved in antioxidant-detoxification
pathway and thylakoid-localized proteins may be an effective
strategy of defense against O3. This research can be considered
as an useful basis to (i) generate the functional resources for The Supplementary Material for this article can be found
online at: http://journal.frontiersin.org/article/10.3389/fpls.2016. 00713 Supplementary Figure 1 | Statistical pathway enrichment of the
differentially expressed genes against Arabidopsis thaliana. Supplementary Figure 1 | Statistical pathway enrichment of the
differentially expressed genes against Arabidopsis thaliana. Supplementary Table 1 | Table showing the GO and associated InterPro
annotations with ensembl transcripts showing the up- and
down-regulations during the ozone treatment. Supplementary Table 2 | Detailed classification of expression log2(RPKM
induced/RPKM repressed) of the up- and down-regulated Ensembl
unigenes having homologous gene families according to PLAZA version
2.5 and associated InterPro domains. Abbreviations: RPKM: kilo base of exon
model per million mapped reads. Detailed Classification of the associated
functional categories associated with the expressed transcripts in ozone
treatment. Supplementary Table 3 | Table showing the family and associated InterPro
domains of ensembl transcription factors. AUTHOR CONTRIBUTIONS The work presented here was carried out in collaboration among
all authors. NL and CN defined the research theme and obtained
funding. EG, AC, EP, PB, and GS designed methods, carried
out laboratory experiments, and analyzed the data. AC, EP, and
GS co-designed experiments, discussed analyses, interpreted the
results, and wrote the paper. All authors have contributed to
discuss the results and implications of the work and to comment
on the manuscript at all stages before approvation. REFERENCES doi:
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3054.2000.110206.x Copyright © 2016 Gottardini, Cristofori, Pellegrini, La Porta, Nali, Baldi and Sablok. This is an open-access article distributed under the terms of the Creative Commons
Attribution License (CC BY). The use, distribution or reproduction in other forums
is permitted, provided the original author(s) or licensor are credited and that the
original publication in this journal is cited, in accordance with accepted academic
practice. No use, distribution or reproduction is permitted which does not comply
with these terms. Rizzo, M., Bernardi, R., Salvini, M., Nali, C., Lorenzini, G., and Durante, M. (2007). Identification of differentially expressed genes induced by ozone stress
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specific variations in antioxidative defense system, genome and proteome of June 2016 | Volume 7 | Article 713 Frontiers in Plant Science | www.frontiersin.org 12
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Review of: "Factors Influencing Access to Credit for Rural People in Ethiopia"
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Qeios, CC-BY 4.0 · Review, July 11, 2023
Review of: "Factors Influencing Access to Credit for Rural
People in Ethiopia"
Enyang Besong Susan
Potential competing interests: No potential competing interests to declare.
Thank you for inviting me to review the manuscrip 'Factors Influencing Access to Credit For Rural People in Ethopia".
Below our my remarks and suggestions for the authors.
The Abstract is well structured.
The introduction in my opinion is too long and does not present the theoretical underpinnings and contributions of the
paper . I will suggest the authors focus on highlighting the novelty of their project. looking at the manuscript it appears
the authors are simply summarizing what has been done. In addition the authors suggest prior studies focus on
agriculture, however they have made no effort to distinguish their work from those of prior researchers.
The authors have not clearly established the research question(s) they seek to answer. so far they said they want to
close the gap by ‘examining the important socio-economic, demographic, institutional, and other factors that influence
access to credit for rural individuals in the Bilate Zuria district, located in the Sidama region of Ethiopia’. It is not clear if
the gap is the lack of study in the Zuria district since the literature they present suggest rural credit constrians have
been well documented in Africa at large and Ethopia in particular.
The result and discussion section is too hard to read and understand. I will suggest the authors use sub-headings.
From their conceptual framework, the indicators were grouped into four categories. Thus there should be four main
headings and each variable in each category should have a sub-title. This will improve the structure and readability of
the manuscript. Also, the authors should clearly state what column represent what variable in “Table 4”
In the abstract, the authors mention policy implication. However, there are no policy recommendations in the
conclusion. I suggest the authors highlight the theoretical and policy implications of their findings in the section titled
conclusion.
The citation style is not well presented in most instances e.g ‘Samuel Semma Waje, 2020’ I think should be “Waje
2020”. The authors should check and ensure their citation is standardize.
There are also some typo errors which I suggest 'Grammarly" can quickly correct.
Qeios ID: 7J2A0K · https://doi.org/10.32388/7J2A0K
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English
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Progress in the R ecosystem for representing and handling spatial data
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Journal of geographical systems
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cc-by
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| ERROR: type should be string, got "https://doi.org/10.1007/s10109-020-00336-0\nJournal of Geographical Systems (2021) 23:515–546 https://doi.org/10.1007/s10109-020-00336-0\nJournal of Geographical Systems (2021) 23:515–546 ORIGINAL ARTICLE Abstract Twenty years have passed since Bivand and Gebhardt (J Geogr Syst 2(3):307–317, \n2000. https://doi.org/10.1007/PL00011460) indicated that there was a good match \nbetween the then nascent open-source R programming language and environment \nand the needs of researchers analysing spatial data. Recalling the development of \nclasses for spatial data presented in book form in Bivand et al. (Applied spatial data \nanalysis with R. Springer, New York, 2008, Applied spatial data analysis with R, \n2nd edn. Springer, New York, 2013), it is important to present the progress now \noccurring in representation of spatial data, and possible consequences for spatial \ndata handling and the statistical analysis of spatial data. Beyond this, it is imperative \nto discuss the relationships between R-spatial software and the larger open-source \ngeospatial software community on whose work R packages crucially depend. Keywords Spatial data analysis · Open-source software · R programming language JEL Classification C00 · C88 · R15 JEL Classification C00 · C88 · R15 JEL Classification C00 · C88 · R15 Progress in the R ecosystem for representing and handling \nspatial data Roger S. Bivand1 Received: 9 October 2019 / Accepted: 8 September 2020 \n© The Author(s) 2020\n/ Published online: 16 October 2020 1\t\nDepartment of Economics, Norwegian School of Economics, Helleveien 30, 5045 Bergen, \nNorway *\t Roger S. Bivand \n\t\nroger.bivand@nhh.no *\t Roger S. Bivand \n\t\nroger.bivand@nhh.no\n1\t\nDepartment of Economics, Norwegian School of Economics, Helleveien 30, 5045 Bergen, \nNorway 1 Introduction While Bivand and Gebhardt (2000) did provide an introduction to R as a statistical \nprogramming language and to why one might choose to use a scripted language like \nR (or Python), this article is both retrospective and prospective. It is possible that \nthose approaching the choice of tools for spatial analysis and for handling spatial \ndata will find the following less than inviting; in that case, perusal of early chapters \nof Lovelace et al. (2019) will provide useful context. Two further pointers include \nthe fact that R and most R add-on packages are open-source software and so with-\nout licence fees or other such restrictions. The second pointer is that all scripting 012341 456789)\n3 012341 456789)\n3 516 R. S. Bivand languages provide the structures needed for reproducible research, and open-source \nsoftware gives the interested researcher the means to run the scripts needed to repli-\ncate results without cost, given access to adequate hardware. Hence, an overview of \nthe development of the use of R for handling spatial data can cast light on how and \nwhy steps fashioning today’s software were taken. Of course, an overview of the use \nof R for analysing spatial data would also be tempting, but, with about 900 R pack-\nages using spatial data handling classes and objects, would far exceed the bounds of \na single article. The R statistical programming language and environment has been used for han-\ndling and analysing spatial data since its inception, partly building on its heritage \nfrom S and S-Plus. When the conceptualization of spatial data was introduced in \nthe sp package (Pebesma and Bivand 2005, 2020, on the Comprehensive R Archive \nNetwork (CRAN) since 2005), it was expected that some packages would adopt its \nclasses. Some years later, adoption rates had picked up strongly, as had use of the \nsp-based packages rgdal (Bivand et al. 2020, on CRAN since 2003) for input/output \nand rgeos (Bivand and Rundel 2020, on CRAN since 2011) for geometric manipula-\ntion of vector data. Packages depending on sp classes continue to require support as more mod-\nern data representations have been introduced in the sf (Pebesma 2018, 2020a, on \nCRAN since 2016) and stars (Pebesma 2020c, on CRAN since 2018) packages. 1 3 1 Introduction The sf package provides the data reading and writing, and geometry manipulation \nfunctionalities found in rgdal and rgeos, and an alternative class representation \nfor vector data based on the Simple Features standard (Herring 2011; ISO 2004). The stars package adds facilities for handling spatial and spatio-temporal raster and \nvector data, building in part on work with the spacetime package (Pebesma 2012, \n2020b, on CRAN since 2010) and on raster handling in the raster package (Hij-\nmans 2020a, on CRAN since 2010). The raster package will not be discussed in this \nreview, mostly because information in the Github “rspatial” organization (https://\ngithub.com/rspatial) repositories suggests that development is in flux and that raster \nis becoming a new package called terra (Hijmans 2020b). Discussion here will con-\ncentrate on packages developed and maintained by the Github “r-spatial” organiza-\ntion (https://github.com/r-spatial) of which I am a member. Newer visualization packages, such as tmap (Tennekes 2018, 2020, on CRAN \nsince 2014), mapview (Appelhans et al. 2020, on CRAN since 2015) and cartog-\nraphy (Giraud and Lambert 2016, 2017, 2020, on CRAN since 2015), give broader \nscope for data exploration and communication. Modelling and analysis packages \ndemonstrate the considerable range of implementations now available and are often \nsupported with additional code provided as supplementary material to journal arti-\ncles, for example in the Journal of Statistical Software spatial statistics special issue \n(Pebesma et al. 2015). The availability of software and scripts provides a helpful \nmechanism supporting reproducible research and hands-on reviewing in which \nreaders can read the code and scripts used in calculating the results presented in \npublished work (see, for example, Sawicka et al. 2018; Lovelace and Ellison 2018; \nEvangelista and Beskow 2018, in one issue of the R Journal). Some packages are used by others in turn forming dependency trees; because of \nthese dependencies, we can speak of an ecosystem. Class representations of data 1 3 Progress in the R ecosystem for representing and handling spatial… 517 are central, with the data frame conceptualization shaping much of the whole R \necosystem. For the modelling infrastructure to perform correctly, the relationships \nbetween objects containing data and the formula representations of models are cru-\ncial. Because both sp and sf provide similar interfaces for the data= arguments for \nmodel fitting functions by behaving as data.frame objects, transition from sp \nto sf representations is convenient by design. 1 Introduction The spdep package (Bivand 2020b, \non CRAN since 2002) for exploratory spatial data analysis and the new spatialreg \npackage (Bivand and Piras 2019, on CRAN since 2019) for spatial regression (split \nout of spdep) have been adapted to accommodate geometries held in sf classes, so \nboth approaches are viable. Other packages, such as mapview, tmap or stplanr \n(Lovelace et al. 2020) have been revised to permit use with both sp and sf objects. In \norder to retain backward compatibility, other central packages may choose to handle \nthe coexistence of sp and sf classes in the same way. Developers of new packages should choose to use sf and stars rather than sp, \nrgdal and rgeos (and terra rather than raster), but existing packages are free to \nadapt, or indeed to stay with sp, rgdal and rgeos, hoping that they may continue to \nbe maintained. Naturally, contributions to maintenance and development from those \nusing software on which one’s work depends are among the “prices” to be “paid” for \nopen-source software, so “hope” may involve commitment to take responsibility for \nmaintenance. If a maintainer is unable to continue in service, software like R add-on \npackages is termed “orphaned”, but may be adopted. This occurred very recently \nwith an R linear programming package lpSolve, which has been adopted by Gábor \nCsárdi to widespread relief and gratitude. Because there is no corporation tasked \nwith maintaining most R add-on packages, their future use has to depend on the user \ncommunity. One key reason why sf is much easier to maintain is that it was written using the \nRcpp package (Eddelbuettel et al. 2011, 2020; Eddelbuettel 2013; Eddelbuettel and \nBalamuta 2018, on CRAN since 2008) to interface C++ code in GDAL and now \nPROJ (both C++11), and GEOS code through the C API, whereas rgdal and rgeos \nhave more fragile handcrafted interfaces originally written for C99 and C++98. Maintaining Rcpp/C++11 interfaces is very much easier than older adaptations not \nas well understood by younger developers. However, the choice of C++ interfaces is \nnot necessarily robust (Kalibera 2019). Since there is a viable alternative to rgdal and rgeos, a fallback in the future for \nsp-dependent packages will be to use sf for reading and writing data, and geometry \nmanipulation, and to coerce to sp classes before passing to existing modelling code \nif sp classes are needed. 1 Introduction Maintainers of actively developed packages using sp vec-\ntor classes intensively and that are also impacted by changes in coordinate reference \nsystems (Sect. 4) are advised to consider transitioning to sf, as substantial revisions \nwill be needed anyway. In this review and prospect, the progress seen over the last 20 years will be \ndescribed, together with the unexpected consequences it engendered. The emer-\ngence of new technologies and standards has also led to the desirability of re-imple-\nmentation of data representations and analysis functionality, some of which is now \nin place and which will be described. Changes also impact the open-source librar-\nies on which core spatial functionality is built, leading to challenges in ensuring 1 3 3 3 518 R. S. Bivand backward compatibility. This will be shown using the example of coordinate refer-\nence systems. In terms of positionality, much of what follows will be presented from \nthe point of view of the author, documented as far as possible from email exchanges \nand similar sources. It is more than likely that other participants in the development \nof the R-spatial community may recall things differently, and of course, I acknowl-\nedge that this description is bound to be partial. 1 Typically plastic boxes containing static software licences attached to a computer port, often the paral-\nlel port otherwise used for printers in the pre-USB era. 2 \u0007Spatial data classes in the sp package CRAN listings show tripack (Renka and Geb-\nhardt 2020) and akima (Akima and Gebhardt 2020)—both with non-open-source 1 3 Progress in the R ecosystem for representing and handling spatial… 519 licences—available from August 1998 ported by Albrecht Gebhardt; ash and sgeo-\nstat (Majure and Gebhardt 2016) followed in April 1999. The spatial package was \navailable as part of MASS (the software supporting the four editions of Venables \nand Ripley 2002), also ported in part by Albrecht Gebhardt. In the earliest period, \nCRAN administrators helped practically with porting and publication. Albrecht and \nI presented an overview of possibilities of using R for research and teaching in spa-\ntial analysis and statistics in August 1998, subsequently published in this journal as \nBivand and Gebhardt (2000). Rowlingson and Diggle (1993) describe the S-PLUS version of splancs (Rowl-\ningson and Diggle 2017) for point pattern analysis. I had contacted Barry Rowl-\ningson in 1997, but only moved forward with porting as R’s package mechanism \nmatured. In September 1998, I wrote to him: “It wasn’t at all difficult to get things \nrunning, which I think is a result of your coding, thank you!” However, I added this \nspeculation: “An issue I have thought about a little is whether at some stage Albrecht \nand I wouldn’t integrate or harmonize the points and pairs objects in splancs, spa-\ntial and sgeostat—they aren’t the same, but for users maybe they ought to appear to \nbe so”. This concern with class representations for geographical data turned out to \nbe fruitful. A further step was to link GRASS and R, described in Bivand (2000), and fol-\nlowed up at several meetings and working closely with Markus Neteler. The inter-\nface has evolved, and its current status is presented by Lovelace et al. (2019, chap-\nter 9). A consequence of this work was that the CRAN team suggested that I attend a \nmeeting in Vienna in early 2001 to talk about the GRASS GIS interface. The meet-\ning gave unique insights into the dynamics of R development, and very valuable \ncontacts. Later the same year Luc Anselin and Serge Rey asked me to take part in a \nworkshop in Santa Barbara, which again led to many fruitful new contacts; my talk \neventually appeared as Bivand (2006), but the contacts made at the workshop were \nvery useful. 2 \u0007Spatial data classes in the sp package In the early and mid-1990s, those of us who were teaching courses in spatial analysis \nbeyond the direct application of geographical information systems (GIS) found the \npaucity of software limiting. In institutions with funding for site licences for GIS, it \nwas possible to write or share scripts for Arc/Info (in AML), ArcView (in Avenue) \nor later in Visual Basic for ArcGIS. If site licences and associated dongles1 used \nin the field were a problem (including problems for students involved in fieldwork \nin research projects), there were few alternatives, but opportunities were discussed \non mailing lists. One of these was the AI-Geostats listserve/mailing list started by \nGregoire Dubois in 1995; AI meant Arc/Info. Another community gathered around \nGRASS GIS and its transition to open-source development; it hosted, among other \nthings, src.contrib and src.garden directory trees with analysis tools (see \ncode stored in the https://github.com/OSGeo/grass-legacy repository). From late 1996, the R programming language and environment began to be seen \nas an alternative for teaching and research involving spatial analysis by a few people \nincluding the author and Albrecht Gebhardt. R uses much of the syntax of S, then \navailable commercially as S-Plus, but was and remains free to install, use and extend \nunder the GNU General Public License (GPL). In addition, it could be installed \nportably across multiple operating systems, including Windows and Apple Mac OS. At about the same time, the S-Plus SpatialStats module was published (Kaluzny \net al. 1998), and a meeting occurred in Leicester in which many of those looking \nfor solutions took part. (My contribution was published in the meeting special issue \nBivand 1998.) Much of the porting of S code to R for spatial statistics was begun by Albrecht \nGebhardt as soon as the R package mechanism matured. The library() func-\ntion was upgraded in R 0.60 published in December 1997, and the Comprehensive \nR Archive Network was operating in January 1998. An exchange between Albre-\ncht Gebhardt and Thomas Lumley on the R-beta mailing list (https://stat.ethz.ch/\npipermail/r-help/1997-November/001882.html) shows that the package mecha-\nnism was not yet working predictably before 0.60 for contributed packages. Since \nteachers moving courses from S to R needed access to the S libraries previously \nused, porting was a crucial step. 2 \u0007Spatial data classes in the sp package Further progress during the intervening 4 years in the use of R in spa-\ntial econometrics was reported in Bivand (2002), building on Bivand and Gebhardt \n(2000), but preceding the release of the spdep package. During the second half of 2002, it seemed relevant to propose a spatial statis-\ntics paper session at the next Vienna meeting to be held in March 2003 (known as \nDistributed Statistical Computing (DSC) and led to useR! meetings), together with \na workshop to discuss classes for spatial data. I had reached out to Edzer Pebesma \nas an author of the stand-alone open-source program gstat (Pebesma and Wes-\nseling 1998); it turned out that he had just been approached to wrap the program for \nS-Plus. He saw the potential of the workshop immediately, and in November 2002 \nwrote in an email: “I wonder whether I should start writing S classes. I’m afraid \nI should”. Virgilio Gómez-Rubio had been developing two spatial packages, RAr-\ncInfo (Gómez-Rubio and López-Quílez 2005; Gómez-Rubio 2011) and DCluster \n(Gómez-Rubio et al. 2005, 2015), and was committed to participating. Although he \ncould not get to the workshop, Nicholas Lewin-Koh wrote in March 2003 that: “I \nwas looking over all the DSC material, especially the spatial stuff. I did notice, after \nlooking through peoples’ packages that there is a lot of duplication of effort. My \nsuggestion is that we set up a repository for spatial packages similar to the Biocon-\nductor mode, where we have a base spatial package that has S-4-based methods and 1 3 3 520 R. S. Bivand classes that are efficient and general.” Straight after the workshop, a collaborative \nrepository for the development of software using SourceForge was established, and \nthe R-sig-geo mailing list (still with over 3600 subscribers) was created to facilitate \ninteraction. classes that are efficient and general.” Straight after the workshop, a collaborative \nrepository for the development of software using SourceForge was established, and \nthe R-sig-geo mailing list (still with over 3600 subscribers) was created to facilitate \ninteraction. So the mandate for the development of the sp package emerged in discussions \nbetween interested contributors before, during and especially following the 2003 \nVienna workshop; most of us met at Pörtschach am Wörthersee in October 2003 \nat a meeting organized by Albrecht Gebhardt. 2 \u0007Spatial data classes in the sp package Coding meetings were organized by \nBarry Rowlingson in Lancaster in November 2004 and by Virgilio Gómez-Rubio \nin Valencia in May 2005, at both of which the class definitions and implementa-\ntions were stress-tested and often changed radically; the package was first published \non CRAN in April 2005. The underlying model adopted was for S4 (new style) \nclasses to be used, for \"Spatial\" objects, whether raster or vector, to behave like \n\"data.frame\" objects, and for visualization methods to make it easy to show the \nobjects. Package developers could choose whether they would use sp classes and \nmethods directly, or rather use those classes for functionality that they did not pro-\nvide themselves. The spatstat package (Baddeley and Turner 2005; Baddeley et al. 2015, 2020) was an early example of such object conversion (known as coercion in \nS and R) to and from sp classes and classes, with the coercion methods published in \nthe maptools package (Bivand and Lewin-Koh 2020).i Reading and writing ESRI Shapefiles had been possible using the maptools \npackage (Bivand and Lewin-Koh 2020) available from CRAN since August 2003, \nbut rgdal, on CRAN from November 2003, interfacing the external GDAL library \n(Warmerdam 2008) and first written by Tim Keitt, initially only supported accessing \nand reading raster data. Further code contributions by Barry Rowlingson for han-\ndling projections using the external PROJ.4 library and the vector drivers in the then \nOGR part of GDAL were folded into rgdal, permitting reading vector and raster \ndata into sp-objects and writing from sp-objects. For vector data, it became possible \nto project coordinates, and in addition to transform them where datum specifications \nwere available. Until 2019, the interfaces to GDAL and PROJ had been relatively \nstable, and upstream changes had not led to breaking changes for users of pack-\nages using sp classes or rgdal functionalities, although they have involved signifi-\ncant maintenance effort. The final part of the framework for spatial vector data han-\ndling was the addition of the rgeos package interfacing the external GEOS library \nin 2011, thanks to Colin Rundell’s 2010 Google Summer of Coding project. The \nrgeos package provided vector topological predicates and operations typically found \nin GIS such as intersection; note that by this time, both GDAL and GEOS used the \nSimple Features vector representation internally. By the publication of Bivand et al. 1 3 readxl\nwithr\nleaflet\ntidyr\nglue\nhtmlwidgets\nrjson\nstringi\nshiny\nrlang\ncrayon\nrvest\ncovr\nDT\ntibble\ndplyr\ntidyverse\nbroom\ntidyselect\nmaps\nhttr\nxml2\nassertthat\nhtmltools\nknitr\nrmarkdown\ndigest\nR6\nspelling\npurrr\nmagrittr\ncurl\nlubridate\nRCurl\nreadr\nsf\ntestthat\ndata.table\nXML\ndevtools\nrstudioapi\nkableExtra\nforeign\ntinytest\nroxygen2\nstringr\nyaml\njsonlite\nvdiffr\nshinyjs readxl\nwithr\nleaflet\ntidyr\nglue\nhtmlwidgets\nrjson\nstringi\nshiny\nrlang\ncrayon\nrvest\ncovr\nDT\ntibble\ndplyr\ntidyverse\nbroom\ntidyselect\nmaps\nhttr\nxml2\nassertthat\nhtmltools\nknitr\nrmarkdown\ndigest\nR6\nspelling\npurrr\nmagrittr\ncurl\nlubridate\nRCurl\nreadr\nsf\ntestthat\ndata.table\nXML\ndevtools\nrstudioapi\nkableExtra\nforeign\ntinytest\nroxygen2\nstringr\nyaml\njsonlite\nvdiffr\nshinyjs geosphere\npedometrics\ndeldir\nstabs\nakima\nspacetime\nmaptools\nspData\nFRK\nspam\nRStoolbox\nbiomod2graph4lg\nRandomFields\nspatialreg\ndismo\nINLA\nspatstat\nSSDM\nsp\ngeoR\nENMTools\nCARBayes\nGSIF\ngstat\nmuHVT\nBiodiversityR\nrnaturalearth\necospat\nfields\nspdep\nagricolae\nrgeos\nsplancs\nadehabitatLT\nrdwd\nrasterVis\ntree\nrgdal\ntrajectories\nspatialEco\ninlabru\nraster\nrworldmap\ngdistance\nmapdata\nRgoogleMaps\nCSTools\nplotKML\nsnowfall Fig. 1 Wordclouds of CRAN and BioConductor package dependencies, August 2020, left panel: cluster \n6 (pagerank range 0.002196–0.000070), right panel: cluster 2 (pagerank range 0.022419–0.000390, sf = \n0.000972) Fig. 1 Wordclouds of CRAN and BioConductor package dependencies, August 2020, left panel: cluster \n6 (pagerank range 0.002196–0.000070), right panel: cluster 2 (pagerank range 0.022419–0.000390, sf = \n0.000972) among the cumulated packages held on CRAN and those published by the Biocon-\nductor project. The left panel shows cluster 6, the spatial cluster with sp having a \npagerank of 0.002196, while the right panel shows cluster 2, which is dominated by \npackages developed by RStudio, a commercial company. The sf package is in cluster \n2, with a pagerank score of 0.000972, most likely in that cluster because it itself uses \nmany of the functionalities of RStudio packages. The two word clouds are scaled by \nthe largest pagerank of included packages, so the scales differ by almost an order of \nmagnitude. 2 https://github.com/rwinlib/gdal2, https://github.com/rwinlib/gdal3. 2 \u0007Spatial data classes in the sp package (2008), a few packages not written or main-\ntained by the book authors and their nearest collaborators had begun to use sp \nclasses. By the publication of the second edition (Bivand et al. 2013), we had seen \nthat the number of packages depending on sp, importing from and suggesting it (in \nCRAN terminology for levels of dependency) had grown strongly. In late 2014, de \nVries (2014) looked at CRAN package clusters from a page rank graph and found a \nclear spatial cluster that we had not expected. Figure 1 shows word clouds with char-\nacter sizes proportional to pagerank scores for two clusters found in August 2020 1 3 Progress in the R ecosystem for representing and handling spatial… 521 3 \u0007Spatial data classes in the sf and stars packages The raster package complemented sp for handling raster objects and their inter-\nactions with vector objects. It added to input/output using GDAL through rgdal, \nand better access to NetCDF files. It may be mentioned in passing that thanks to \nhelp from CRAN administrators and especially Brian Ripley, CRAN binary builds \nof rgdal for Windows and Apple Mac OSX became available from 2006, but with \na limited set of vector and raster drivers. Support from CRAN adminstrators and \nthe maintainers of the Github rwinlib/gdal2 and rwinlib/gdal3 repositories2 remains \ncentral to making packages available to users who are not able to install R source \npackages themselves, particularly linking to external libraries. Initially, raster was \nwritten in R using functionalities in sp and rgdal with rgeos coming later. It used a \nfeature of GDAL raster drivers permitting the successive reading of subsets of ras-\nters by row and column, allowing the processing of much larger objects than could \nbe held in memory. In addition, the concepts of bricks and stacks of rasters were \nintroduced, diverging somewhat from the sp treatment of raster bands as stacked \ncolumn vectors in a data frame. 1 3 522 R. S. Bivand As raster evolved, two other packages emerged raising issues with the ways in \nwhich spatial objects had been conceptualized in sp. The rgeos package used the C \napplication programming interface (API) to the C++ GEOS library, which is itself a \ntranslation of the Java Topology Suite (JTS). While the GDAL vector drivers did use \nthe standard Simple Features representation of vector geometries, it was not strongly \nenforced. This laxity now seems most closely associated with the use of ESRI Shape \nfiles as a de facto file standard for representation, in which some Simple Features \nare not consistently representable.3 Both JTS and GEOS required a Simple Features \ncompliant representation and led to the need for curious and fragile adaptations. For \nexample, these affected the representation of sp \"Polygons\" objects, which were \noriginally conceptualized after the Shapefile specification: ring direction determined \nwhether a ring was exterior or interior (a hole), but no guidance was given to show \nwhich exterior ring holes might belong to. As R provides a way to add a character \nstring comment to any object, such comments were added to each \"Polygons\" \nobject encoding the necessary information. 3 \u0007Spatial data classes in the sf and stars packages In this way, GEOS functionality could \nbe used, but the fragility of vector representation in sp was made obvious.f Another package affecting thinking about representation was spacetime, stacking \ncolumns for regular spatio-temporal objects with space varying faster than time. So \na single earth observation band observed repeatedly would be stored in a single col-\numn in a data frame, rather than in the arguably more robust form of a four-dimen-\nsional array, with the band taking one position on the final dimension. The second \nedition of Bivand et al. (2013) took up all of these issues in one way or another, but \nafter completing a spatial statistics special issue of the Journal of Statistical Soft-\nware (Pebesma et al. 2015), it was time to begin fresh implementations of classes for \nspatial data. 3 For recent examples see https://github.com/OSGeo/gdal/issues/1787 and https://github.com/r-spatial/\nsf/issues/1121. Progress in the R ecosystem for representing and handling spatial… 523 > df <- data.frame(a=letters[1:3], b=1:3)\n> df$c <- list(d=1, e=\"1\", f=TRUE)\n> str(df)\n’data.frame’: 3 obs. of\n3 variables:\n$ a: chr\n\"a\" \"b\" \"c\"\n$ b: int\n1 2 3\n$ c:List of 3\n..$ d: num 1\n..$ e: chr \"1\"\n..$ f: logi TRUE > df <- data.frame(a=letters[1:3], b=1:3)\n> df$c <- list(d=1, e=\"1\", f=TRUE)\n> str(df)\n’data.frame’: 3 obs. of\n3 variables:\n$ a: chr\n\"a\" \"b\" \"c\"\n$ b: int\n1 2 3\n$ c:List of 3\n..$ d: num 1\n..$ e: chr \"1\"\n..$ f: logi TRUE ’data.frame’: 3 obs. of\n3 variables: $ a: chr\n\"a\" \"b\" \"c\" $ b: int\n1 2 3 $ c:List of 3 ..$ d: num 1 ..$ e: chr \"1\" ..$ f: logi TRUE ..$ f: logi TRUE At useR! in 2016, list columns were declared “tidy”, using examples including \nthe difficulty of encoding polygon interior rings in non-list columns. The decision \nto accommodate “tidy” workflows as well as base-R workflows had already been \nmade, as at least some users only know how to use “tidy” workflows. Pebesma \n(2018) showed the status of the sf towards the end of 2017, with a geometry list \ncolumn containing R wrappers around objects adhering to Simple Features speci-\nfication definitions. Note also that from R 4.0.0, data.frame() does not con-\nvert character columns to factor as it did previously (Hornik 2020); column \n\"a\" is character in R 4 or later, and factor before R 4. 3.1 \u0007Simple Features in R It was clear that vector representations needed urgent attention, so the sf package \nwas begun, aiming to implement the most frequently used parts of the specification \n(ISO 2004; Kralidis 2008; Herring 2011). Development was supported by a grant \nfrom the then newly started R Consortium, which brings together R developers and \nindustry members. A key breakthrough came at the useR! 2016 conference, follow-\ning an earlier decision to re-base vector objects on data frames, rather than as in sp \nto embed a data frame inside a spatial object. Although data frame objects in S and \nR have always been able to take list columns as valid columns, such list columns \nwere not seen as “tidy” (Wickham 2014): 3 Progress in the R ecosystem for representing and handling spatial… > library(sf) The feature geometries are stored in numeric vectors, matrices or lists of \nmatrices and may also be subject to arithmetic operations. Features are held in \nthe \"XY\" class if two-dimensional, or \"XYZ\", \"XYM\" or \"XYZM\" if such coor-\ndinates are available (\"Z\" is usually treated as height and \"M\" as some meas-\nure, perhaps accuracy; both need to have specified units); all single features are \n\"sfg\" (Simple Features geometry) objects, with arithmetic and other operators: > pt1 <- st_point(c(1,3))\n> pt2 <- pt1 + 1\n> pt3 <- pt2 + 1\n> str(pt3)\n’XY’ num [1:2] 3 5 > pt1 <- st_point(c(1,3))\n> pt2 <- pt1 + 1\n> pt3 <- pt2 + 1\n> str(pt3)\n’XY’ num [1:2] 3 5 Geometries may be represented as “Well-Known Text” (WKT): > st_as_text(pt3)\n[1] \"POINT (3 5)\" > st_as_text(pt3)\n[1] \"POINT (3 5)\" > st_as_text(pt3)\n[1] \"POINT (3 5)\" > st_as_text(pt3)\n[1] \"POINT (3 5)\" or as “Well-Known Binary” (WKB) as in database “binary large objects” \n(BLOBs), resolving the problem of representation when working with GDAL vector \ndrivers and functions, and with GEOS predicates and topological operations: 1 3 3 524 R. S. Bivand _\n_\ny(p\n)\n[1] 01 01 00 00 00 00 00 00 00 00 00 08 40 00 00 00 00 00 00 14 40 A column of Simple Features geometries (\"sfc\") is constructed as a list of \"sfg\" \nobjects, which do not have to belong to the same Simple Features category; here, we \nassign the Web Mercator CRS to indicate that the points are projected to the plane, with \nposition measured in metres: > pt_sfc <- st_as_sfc(list(pt1, pt2, pt3), crs=3857)\n> str(pt_sfc)\nsfc_POINT of length 3; first list element:\n’XY’ num [1:2] 1 3 When sf was written, the units package was available (Pebesma et al. > library(sf) 2016, 2020) \nand could utilize the metric of the coordinates given in the declared CRS, so here inter-\npoint distances are measured in metres: > st_distance(pt_sfc)\nUnits: [m]\n[,1]\n[,2]\n[,3]\n[1,] 0.000000 1.414214 2.828427\n[2,] 1.414214 0.000000 1.414214\n[3,] 2.828427 1.414214 0.000000 [3,] 2.828427 1.414214 0.000000 If we re-specify the points as geographical coordinates in decimal degrees on the \nWGS84 ellipsoid, the distances will be given as metres, but measured over the ellipsoid: > pt_sfc1 <- st_as_sfc(list(pt1, pt2, pt3), crs=4326)\n> st_distance(pt_sfc1)\nUnits: [m]\n[,1]\n[,2]\n[,3]\n[1,]\n0.0 156759.1 313424.7\n[2,] 156759.1\n0.0 156665.6\n[3,] 313424.7 156665.6\n0.0 The most recent R Consortium grant covers the extension of analysis and data handling \nto global data representation; Pebesma and Dunnington (2020) present a roadmap for the \nuse of the s2 library for operations on geographical coordinates and its integration in sf. > mat <- matrix(0, 3, 3)\n> mat[1,2] <- mat[2,1] <- s2::s2_distance(st_as_text(pt1), st_as_text(pt2))\n> mat[1,3] <- mat[3,1] <- s2::s2_distance(st_as_text(pt1), st_as_text(pt3))\n> mat[2,3] <- mat[3,2] <- s2::s2_distance(st_as_text(pt2), st_as_text(pt3))\n> mat\n[,1]\n[,2]\n[,3]\n[1,]\n0.0 157106.0 314116.3\n[2,] 157106.0\n0.0 157010.4\n[3,] 314116.3 157010.4\n0.0 1 3 1 3 Progress in the R ecosystem for representing and handling spatial… 525 Finally, an \"sfc\" object, a geometry column, can be added to a data.frame \nobject using st_geometry(), which sets a number of attributes on the object and \ndefines it as also being an \"sf\" object (the \"agr\" attribute if populated shows \nhow observations on non-geometry columns should be understood): > st_geometry(df) <- pt_sfc\n> str(df)\nClasses ‘sf’ and ’data.frame’: 3 obs. of\n4 variables:\n$ a\n: chr\n\"a\" \"b\" \"c\"\n$ b\n: int\n1 2 3\n$ c\n:List of 3\n..$ d: num 1\n..$ e: chr \"1\"\n..$ f: logi TRUE\n$ geometry:sfc_POINT of length 3; first list element:\n’XY’ num\n1 3\n- attr(*, \"sf_column\")= chr \"geometry\"\n- attr(*, \"agr\")= Factor w/ 3 levels \"constant\",\"aggregate\",..: NA NA NA\n..- attr(*, \"names\")= chr [1:3] \"a\" \"b\" \"c\" Classes ‘sf’ and ’data.frame’: 3 obs. 4 util=\"ogr2ogr\", options=\"-nlt CONVERT_TO_LINEAR\".\n5 https://www.ogc.org/standards/geopackage. > library(sf) of\n4 variables: $ a\n: chr\n\"a\" \"b\" \"c\" :List of 3 c\n:List of 3 ..$ d: num 1 ..$ e: chr \"1\" ..$ f: logi TRUE - attr(*, \"agr\")= Factor w/ 3 levels \"constant\",\"aggregate\",..: NA NA NA\n..- attr(*, \"names\")= chr [1:3] \"a\" \"b\" \"c\" The sf package implements all of the Simple Features geometry categories, but \nsome geometries need be converted to be used inside R, with, for example, the gdal_\nutils() function to convert curve geometries in an input file to linear geometries.4 \nMany of the functions in the sf package begin with st_ as a reference to the same \nusage in PostGIS, where the letters were intended to symbolize “spatial type”.i The vector file creation and reading functionality found in rgdal is also avail-\nable in sf, with substantial improvements with regard to creating spatial database \ntables and reading from databases. Writing a GeoPackage5 is as easy or easier than \nin rgdal, as the layer= and driver= arguments can be inferred from the given \nfile name. List columns cannot be written, because R list columns are not bound to \nbe consistently of the same type. > tf <- tempfile(fileext=\".gpkg\")\n> st_write(df, dsn=tf, quiet=TRUE)\nWarning message:\nIn clean_columns(as.data.frame(obj), factorsAsCharacter) :\nDropping column(s) c of class(es) list > tf <- tempfile(fileext=\".gpkg\")\n> st_write(df, dsn=tf, quiet=TRUE)\nWarning message:\nIn clean_columns(as.data.frame(obj), factorsAsCharacter) :\nDropping column(s) c of class(es) list > tf <- tempfile(fileext=\".gpkg\")\n> st_write(df, dsn=tf, quiet=TRUE)\nWarning message:\nIn clean_columns(as.data.frame(obj), factorsAsCharacter) :\nDropping column(s) c of class(es) list The standard reading method is st_read, providing a similar functionality to \nthat in rgdal, with a number of differences related to character string encoding that \nwill cease to matter when users migrate to modern formats such as GeoPackage. 3 R. S. > library(sf) Bivand 526 > df1 <- st_read(dsn=tf, quiet=TRUE)\n> df1\nSimple feature collection with 3 features and 2 fields\ngeometry type:\nPOINT\ndimension:\nXY\nbbox:\nxmin: 1 ymin: 3 xmax: 3 ymax: 5\nprojected CRS:\nWGS 84 / Pseudo-Mercator\na b\ngeom\n1 a 1 POINT (1 3)\n2 b 2 POINT (2 4)\n3 c 3 POINT (3 5) In a “tidy” workflow, read_sf() can be used, returning a \"tibble-sf\" \ninheriting from a \"tbl_df\" object rather than from a data frame, and not con-\nverting character string columns into \"factor\" categorical variables by default \n(before R 4.0.0): > read_sf(dsn=tf) %>% dplyr::filter(a == \"c\")\nSimple feature collection with 1 feature and 2 fields\ngeometry type:\nPOINT\ndimension:\nXY\nbbox:\nxmin: 3 ymin: 5 xmax: 3 ymax: 5\nprojected CRS:\nWGS 84 / Pseudo-Mercator\n# A tibble: 1 x 3\na\nb\ngeom\n* <chr> <int> <POINT [m]>\n1 c\n3\n(3 5) sf also integrates GEOS topological predicates and operations into the same \nframework, replacing the rgeos package for access to GEOS functionality. The pre-\ncision and scale defaults differ between sf and rgeos slightly; both remain fragile \nwith respect to invalid geometries, of which there are many in circulation. From \nGEOS 3.8, both rgeos and sf offer functions to create valid geometries from invalid \nones; prior to GEOS 3.8, a function in lwgeom was used. Most recently, measure-\nments and operations on non-planar geometries have been moved from lwgeom to s2 \n(Dunnington et al. 2020; Pebesma and Dunnington 2020). Native support for units \nin sf objects carries through to measurements on outputs of topological operations: > buf_df1 <- st_buffer(df1, dist=0.3)\n> st_area(buf_df1)\nUnits: [m^2]\n[1] 0.2826142 0.2826142 0.2826142\n> plot(st_geometry(buf_df1))\n> plot(st_geometry(df1), add=TRUE, pch=4) 1 3 1 3 3 Progress in the R ecosystem for representing and handling spatial… 527 Fig. 2 Three points and three buffers plotted with geometry-specific plot methods for \"sfc\" objects Fig. 2 Three points and three buffers plotted with geometry-specific plot methods for \"sfc\" objects The sf package provides simple base graphics plotting methods. Those for just \nthe \"sfc\" column retrieved by st_geometry() do not take over the layout of \nthe graphics device, but for the \"sf\" object, they do, showing multiple non-geom-\netry columns in separate displays. Figure 2 shows a simple plot of the three buffer \npolygons overplotted with the three points. 3.2 \u0007Raster representations Like sf, the stars package for scalable, spatio-temporal tidy arrays was supported \nby an R Consortium grant. Spatio-temporal arrays were seen as an alternative way \nof representing multivariate spatio-temporal data from the choices made in the spa-\ncetime package, where a two-dimensional data frame contained stacked observation \npositions in space within stacked time points or intervals. The proposed arrays might \ncollapse to a raster layer if only one variable was chosen for one time point or inter-\nval. More important, the development of the package was extended to accommodate \na backend for earth data processing in which the data are retrieved and re-sampled \nas needed from servers, most often cloud-based servers. In most cases, these would \nbe raster geometries, but the array representation also handles irregular geometries \nthrough time. The R Consortium support was chiefly used to let contributors meet to \nmake progress on concepts and implementation. This example only covers a multiband raster taken from a Landsat 7 view of a small \npart of the Brazilian coast. In the first part, a GeoTIFF file is read into memory, using \nthree array dimensions, two in planar space, the third across six bands: 1 3 1 3 R. S. Bivand 528 > library(stars)\n> fn <- system.file(\"tif/L7_ETMs.tif\", package = \"stars\")\n> L7 <- read_stars(fn)\n> L7\nstars object with 3 dimensions and 1 attribute\nattribute(s): stars object with 3 dimensions and 1 attribute\nattribute(s): L7_ETMs.tif\nMin. :\n1.00\n1st Qu.: 54.00\nMedian : 69.00\nMean\n: 68.91\n3rd Qu.: 86.00\nMax. :255.00\ndimension(s):\ns\ne\nu\nl\na\nv\nt\nn\ni\no\np\ns\ny\ns\nf\ne\nr\na\nt\nl\ne\nd\nt\ne\ns\nf\nf\no\no\nt\nm\no\nr\nf\nx\n1 349\n288776\n28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [x]\ny\n1 352 9120761 -28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [y]\nL\nL\nU\nN\nA\nN\nA\nN\nA\nN\nA\nN\n6\n1\nd\nn\na\nb L7_ETMs.tif\nMin. :\n1.00\n1st Qu.: 54.00\nMedian : 69.00\nMean\n: 68.91\n3rd Qu.: 86.00\nMax. :255.00\n( ) dimension(s):\ns\ne\nu\nl\na\nv\nt\nn\ni\no\np\ns\ny\ns\nf\ne\nr\na\nt\nl\ne\nd\nt\ne\ns\nf\nf\no\no\nt\nm\no\nr\nf\nx\n1 349\n288776\n28.5 UTM Zone 25, Southern Hem... 3.2 \u0007Raster representations FALSE\nNULL [x]\ny\n1 352 9120761 -28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [y]\nL\nL\nU\nN\nA\nN\nA\nN\nA\nN\nA\nN\n6\n1\nd\nn\na\nb The bands can be operated on arithmetically, for example to generate a new object \ncontaining values of the normalized difference vegetation index through a function \napplied across the x and y spatial dimensions, using the st_apply abstraction: > ndvi <- function(x) (x[4] - x[3])/(x[4] + x[3])\n> (s2.ndvi <- st_apply(L7, c(\"x\", \"y\"), ndvi))\nstars object with 2 dimensions and 1 attribute\nattribute(s): > (s2.ndvi <- st_apply(L7, c(\"x\", \"y\"), ndvi))\nstars object with 2 dimensions and 1 attribute\nattribute(s):\nndvi\nMin. :-0.75342\n1st Qu.:-0.20301\nMedian :-0.06870\nMean\n:-0.06432\n3rd Qu.: 0.18667\nMax. : 0.58667\ndimension(s):\ns\ne\nu\nl\na\nv\nt\nn\ni\no\np\ns\ny\ns\nf\ne\nr\na\nt\nl\ne\nd\nt\ne\ns\nf\nf\no\no\nt\nm\no\nr\nf\nx\n1 349\n288776\n28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [x]\ny\n1 352 9120761 -28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [y] ( )\ns\ne\nu\nl\na\nv\nt\nn\ni\no\np\ns\ny\ns\nf\ne\nr\na\nt\nl\ne\nd\nt\ne\ns\nf\nf\no\no\nt\nm\no\nr\nf\nx\n1 349\n288776\n28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [x]\ny\n1 352 9120761 -28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [y] The same file can also be accessed using the proxy mechanism, which creates a link \nto the external entity, here a file: > L7p <- read_stars(fn, proxy=TRUE)\n> L7p\nstars_proxy object with 1 attribute in file:\n$L7_ETMs.tif\n[1] \"[...]/L7_ETMs.tif\"\ndimension(s): > L7p <- read_stars(fn, proxy=TRUE)\n> L7p\nstars_proxy object with 1 attribute in file:\n$L7_ETMs.tif\n[1] \"[...]/L7_ETMs.tif\" [1] \"[...]/L7_ETMs.tif\" dimension(s): ( )\ns\ne\nu\nl\na\nv\nt\nn\ni\no\np\ns\ny\ns\nf\ne\nr\na\nt\nl\ne\nd\nt\ne\ns\nf\nf\no\no\nt\nm\no\nr\nf\nx\n1 349\n288776\n28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [x]\ny\n1 352 9120761 -28.5 UTM Zone 25, Southern Hem... 3.2 \u0007Raster representations FALSE\nNULL [y]\nL\nL\nU\nN\nA\nN\nA\nN\nA\nN\nA\nN\n6\n1\nd\nn\na\nb s\ne\nu\nl\na\nv\nt\nn\ni\no\np\ns\ny\ns\nf\ne\nr\na\nt\nl\ne\nd\nt\ne\ns\nf\nf\no\no\nt\nm\no\nr\nf\nx\n1 349\n288776\n28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [x]\n1 352 9120761\n28 5 UTM Z\n25\nS\nh\nH\nFALSE\nNULL [ ] a\nt\nl\ne\nd\nt\ne\ns\nf\nf\no\no\nt\nm\no\nr\nf s\ne\nu\nl\na\nv\nt\nn\ni\no\np\ns\ny\ns\nf\ne\nr\na\nt\nl\ne\nd\nt\ne\ns\nf\nf\no\no\nt\nm\no\nr\nf\nx\n1 349\n288776\n28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [x] x\n1 349\n288776\n28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [x]\ny\n1 352 9120761 -28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [y]\nL\nL\nU\nN\nA\nN\nA\nN\nA\nN\nA\nN\n6\n1\nd\nn\na\nb 3 Progress in the R ecosystem for representing and handling spatial… 529 The same function can also be applied across the same two spatial dimensions \nof the array, but no calculation is carried out until the data are needed and the out-\nput resolution known, with the command needed to create the output stored in the \nobject: > (L7p.ndvi = st_apply(L7p, c(\"x\", \"y\"), ndvi))\nstars_proxy object with 1 attribute in file:\n$L7_ETMs.tif\n[1] \"[...]/L7_ETMs.tif\" > (L7p.ndvi = st_apply(L7p, c(\"x\", \"y\"), ndvi)) > (L7p.ndvi = st_apply(L7p, c(\"x\", \"y\"), ndvi)\nstars_proxy object with 1 attribute in file:\n$L7 ETMs tif p\npp y\np,\n,\ny\n,\nstars_proxy object with 1 attribute in file:\n$L7_ETMs.tif [1] \"[...]/L7_ETMs.tif\" dimension(s):\ns\ne\nu\nl\na\nv\nt\nn\ni\no\np\ns\ny\ns\nf\ne\nr\na\nt\nl\ne\nd\nt\ne\ns\nf\nf\no\no\nt\nm\no\nr\nf\nx\n1 349\n288776\n28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [x]\ny\n1 352 9120761 -28.5 UTM Zone 25, Southern Hem... 3.2 \u0007Raster representations FALSE\nNULL [y]\nL\nL\nU\nN\nA\nN\nA\nN\nA\nN\nA\nN\n6\n1\nd\nn\na\nb\ncall list:\n[[1]]\nst_apply(X = X, MARGIN = c(\"x\", \"y\"), FUN = ndvi) dimension(s): dimension(s):\ns\ne\nu\nl\na\nv\nt\nn\ni\no\np\ns\ny\ns\nf\ne\nr\na\nt\nl\ne\nd\nt\ne\ns\nf\nf\no\no\nt\nm\no\nr\nf\nx\n1 349\n288776\n28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [x]\ny\n1 352 9120761 -28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [y]\nL\nL\nU\nN\nA\nN\nA\nN\nA\nN\nA\nN\n6\n1\nd\nn\na\nb\ncall list:\n[[1]]\nst_apply(X = X, MARGIN = c(\"x\", \"y\"), FUN = ndvi) a\nt\nl\ne\nd\nt\ne\ns\nf\nf\no\no\nt\nm\no\nr\nf 1]]\n_apply(X = X, MARGIN = c(\"x\", \"y\"), FUN = ndvi) The array object can also be split, here on the band dimension, to yield a repre-\nsentation as six rasters in list form: > (x6 <- split(L7, \"band\"))\nstars object with 2 dimensions and 6 attributes\nattribute(s): stars object with 2 dimensions and 6 attributes\nattribute(s):\nX1\nX2\nX3\nX4\nMin. : 47.00\nMin. : 32.00\nMin. : 21.00\nMin. :\n9.00\n1st Qu.: 67.00\n1st Qu.: 55.00\n1st Qu.: 49.00\n1st Qu.: 52.00\nMedian : 78.00\nMedian : 66.00\nMedian : 63.00\nMedian : 63.00\nMean\n: 79.15\nMean\n: 67.57\nMean\n: 64.36\nMean\n: 59.24\n3rd Qu.: 89.00\n3rd Qu.: 79.00\n3rd Qu.: 77.00\n3rd Qu.: 75.00\nMax. :255.00\nMax. :255.00\nMax. :255.00\nMax. :255.00\nX5\nX6\nMin. :\n1.00\nMin. :\n1.00\n1st Qu.: 63.00\n1st Qu.: 32.00\nMedian : 89.00\nMedian : 60.00\nMean\n: 83.18\nMean\n: 59.98\n3rd Qu.:112.00\n3rd Qu.: 88.00\nMax. :255.00\nMax. :255.00\ndimension(s):\ns\ne\nu\nl\na\nv\nt\nn\ni\no\np\ns\ny\ns\nf\ne\nr\na\nt\nl\ne\nd\nt\ne\ns\nf\nf\no\no\nt\nm\no\nr\nf\nx\n1 349\n288776\n28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [x]\ny\n1 352 9120761 -28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [y] X1\nX2\nX3\nX4\nMin. : 47.00\nMin. : 32.00\nMin. : 21.00\nMin. 3.2 \u0007Raster representations :\n9.00\n1st Qu.: 67.00\n1st Qu.: 55.00\n1st Qu.: 49.00\n1st Qu.: 52.00\nMedian : 78.00\nMedian : 66.00\nMedian : 63.00\nMedian : 63.00\nMean\n: 79.15\nMean\n: 67.57\nMean\n: 64.36\nMean\n: 59.24\n3rd Qu.: 89.00\n3rd Qu.: 79.00\n3rd Qu.: 77.00\n3rd Qu.: 75.00\nMax. :255.00\nMax. :255.00\nMax. :255.00\nMax. :255.00\nX5\nX6\nMin. :\n1.00\nMin. :\n1.00\n1st Qu.: 63.00\n1st Qu.: 32.00\nMedian : 89.00\nMedian : 60.00\nMean\n: 83.18\nMean\n: 59.98\n3rd Qu.:112.00\n3rd Qu.: 88.00\nMax. :255.00\nMax. :255.00\ndimension(s):\ns\ne\nu\nl\na\nv\nt\nn\ni\no\np\ns\ny\ns\nf\ne\nr\na\nt\nl\ne\nd\nt\ne\ns\nf\nf\no\no\nt\nm\no\nr\nf dimension(s):\ns\ne\nu\nl\na\nv\nt\nn\ni\no\np\ns\ny\ns\nf\ne\nr\na\nt\nl\ne\nd\nt\ne\ns\nf\nf\no\no\nt\nm\no\nr\nf\nx\n1 349\n288776\n28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [x]\ny\n1 352 9120761 -28.5 UTM Zone 25, Southern Hem... FALSE\nNULL [y] These rasters may also be subjected to arithmetical operations, and as may be \nseen, explicit arithmetic on the six rasters has the same outcome as applying the \nsame calculation to the three-dimensional array: 1 3 530 R. S. Bivand > x6$mean <- (x6[[1]] + x6[[2]] + x6[[3]] + x6[[4]] + x6[[5]] + x6[[6]])/6\n> xm <- st_apply(L7, c(\"x\", \"y\"), mean)\n> all.equal(xm[[1]], x6$mean)\n[1] TRUE > x6$mean <- (x6[[1]] + x6[[2]] + x6[[3]] + x6[[4]] + x6[[5]] + x6[[6]])/6\n> xm <- st_apply(L7, c(\"x\", \"y\"), mean)\n> all.equal(xm[[1]], x6$mean)\n[1] TRUE The extension to a gridded temporal dimension or to irregular spatial and tem-\nporal entities is not particularly difficult, but it remains to document the possibili-\nties of the package more fully. 3.3 \u0007Visualization The classInt package (Bivand 2020a) for finding thematic mapping class inter-\nvals is used directly in plot methods in sf and stars, and in the tmap (Tennekes \n2018, 2020) and cartography (Giraud and Lambert 2016, 2017, 2020) packages. Lapa et al. (2001) (Leprosy surveillance in Olinda, Brazil, using spatial analysis \ntechniques) made available the underlying data set of Olinda census tracts (setor) \nin the Corrego Alegre 1970-72 / UTM zone 25S projection (EPSG:22525); we \nwill use the data set and the deprivation variable to point to visualization alterna-\ntives now available. > olinda <- st_read(\"olinda.gpkg\", quiet=TRUE) The style= argument gives the choice of method used for finding the number \nof classes specified, with \"fisher\" being a natural breaks method, and \"bclust\" \nbagged clustering from the e1071 package (Meyer et al. 2019): > library(classInt) > library(classInt)\n> cI_fisher <- classIntervals(olinda$\"DEPRIV\", n=7, style=\"fisher\")\n> set.seed(1)\n> cI_bclust <- classIntervals(olinda$\"DEPRIV\", n=7, style=\"bclust\")\nCommittee Member: 1(1) 2(1) 3(1) 4(1) 5(1) 6(1) 7(1) 8(1) 9(1) 10(1)\nComputing Hierarchical Clustering > cI_bclust <- classIntervals(olinda$\"DEPRIV\", n=7, style=\"bclust\")\nCommittee Member: 1(1) 2(1) 3(1) 4(1) 5(1) 6(1) 7(1) 8(1) 9(1) 10(1)\nComputing Hierarchical Clustering > cI_bclust <- classIntervals(olinda$\"DEPRIV\", n=7, style=\"bclust\")\nCommittee Member: 1(1) 2(1) 3(1) 4(1) 5(1) 6(1) 7(1) 8(1) 9(1) 10(1) Committee Member: 1(1) 2(1) 3(1) 4(1) 5(1) 6(1) 7(1) 8(1) 9(1) 10(1)\nComputing Hierarchical Clustering For a long time, the RColorBrewer package (Neuwirth 2014) colour palettes \nwere the obvious choice for thematic cartography, but more recently other packages, \nsuch as rcartocolor (Nowosad 2019), have become available, often as supersets of \nthe RColorBrewer palettes: > pal <- rcartocolor::carto_pal(7, \"SunsetDark\")\n> plot(cI_fisher, pal, xlab=\"DEPRIV\", ylab=\"\")\n> plot(cI_bclust, pal, xlab=\"DEPRIV\", ylab=\"\") > pal <- rcartocolor::carto_pal(7, \"SunsetDark\") > plot(cI_fisher, pal, xlab=\"DEPRIV\", ylab=\"\")\n> plot(cI_bclust, pal, xlab=\"DEPRIV\", ylab=\"\") Figure 3 shows plots of the two class interval schemes, with obvious differences \nbetween the two styles. Both styles are attempting to balance low within-class vari-\nance and high between-class variance. 1 3 1 3 Progress in the R ecosystem for representing and handling spatial… 531 0.0\n0.2\n0.4\n0.6\n0.8\n1.0\n0.0\n0.2\n0.4\n0.6\n0.8\n1.0\nfisher\n0.0\n0.2\n0.4\n0.6\n0.8\n1.0\n0.0\n0.2\n0.4\n0.6\n0.8\n1.0\nbclust\nFig. 3 Deprivation by census tract in Olinda, Brazil; empirical cumulative distribution function and class \nintervals for two class intervals: left panel: natural breaks; right panel: bagged clustering Fig. 3 Deprivation by census tract in Olinda, Brazil; empirical cumulative distribution function and class \nintervals for two class intervals: left panel: natural breaks; right panel: bagged clustering Figure 4 shows maps of the same variable with the same class intervals chosen \ninternally using classInt functionality, with the same palette. The syntax is a little \ndifferent, and here, the sf plot method default key has been turned off to permit \nside-by-side display. This method is really best at providing glimpses of the data, \nrather than at creating complete figures, in contrast to the richer functions in cartog-\nraphy; both use base graphics. Fig. > plot(olinda[,\"DEPRIV\"], nbreaks=7, breaks=\"fisher\", pal=pal, key.pos=NULL,\n+\nmain=\"\")\n> library(cartography)\n> choroLayer(olinda, var=\"DEPRIV\", method=\"fisher-jenks\", nclass=7, col=pal,\n+\nlegend.values.rnd=3) > olinda <- st_read(\"olinda.gpkg\", quiet=TRUE) 6 Thematic maps with ggplot2: left panel: default map of a continuous variable; right panel: grati-\ncule removed and palette modified Fig. 7 Thematic maps with ggplot2, natural breaks class intervals: left panel: natural breaks class inter-\nvals; right panel: bagged clustering class intervals Fig. 7 Thematic maps with ggplot2, natural breaks class intervals: left panel: natural breaks class inter-\nvals; right panel: bagged clustering class intervals We can also display the bagged clustering class intervals beside the natural \nbreaks map, again using ggplot2; Figure 7 shows the maps, but because a con-\ntinuous scale is still enforced, all that changes is the position of the breaks on \nthe key. > g3 <- g1 + theme_void() + scale_fill_gradientn(colours=pal,\n+\nbreaks=round(cI_bclust$brks, 3)) > g3 <- g1 + theme_void() + scale_fill_gradientn(colours=pal,\n+\nbreaks=round(cI_bclust$brks, 3)) > olinda <- st_read(\"olinda.gpkg\", quiet=TRUE) 4 Deprivation by census tract in Olinda, Brazil, natural breaks class intervals; left panel: sf plot \nmethod; right panel: cartography choropleth map Fig. 4 Deprivation by census tract in Olinda, Brazil, natural breaks class intervals; left panel: sf plot \nmethod; right panel: cartography choropleth map 3 532 R. S. Bivand Fig. 5 tmap output, natural breaks class intervals; left panel: object o1 without boundaries; right panel: \nobject o2 with boundaries Fig. 5 tmap output, natural breaks class intervals; left panel: object o1 without boundaries; right panel: \nobject o2 with boundaries Figure 5 shows how tmap functions use grid graphics to permit graphics objects to \nbe updated and then plotted using tmap_arrange(). Once again, the same class \nintervals are chosen internally using classInt, with the same palette. The left panel is \nupdated on the right to add thin boundaries between census tracts. tmap also offers \nsmall multiples of facets, for example thematic maps of the same variable observed at \nsuccessive time periods using the same class intervals. An extensive discussion of the \nuse of tmap is provided by Lovelace et al. (2019). > library(tmap)\n> o1 <- tm_shape(olinda) + tm_fill(\"DEPRIV\", style=\"fisher\", n=7, palette=pal)\n> o2 <- o1 + tm_borders(lwd=0.8) > library(tmap)\n> o1 <- tm_shape(olinda) + tm_fill(\"DEPRIV\", style=\"fisher\", n=7, palette=pal)\n> o2 <- o1 + tm_borders(lwd=0.8) > library(tmap) The ggplot2 package (Wickham et al. 2020) provides the geom_sf() facil-\nity for mapping sf objects: > library(ggplot2)\n> g1 <- ggplot(olinda) + geom_sf(aes(fill=DEPRIV))\n> g2 <- g1 + theme_void() + scale_fill_gradientn(colours=pal,\n+\nbreaks=round(cI_fisher$brks, 3)) This approach also builds on grid graphics. It is possible to set a theme that \ndrops the arguably unnecessary graticule, but there is a lot of intervention \nrequired to get a simple map. To get proper class intervals involves even more \nwork, because the package takes specific, not general, positions on how graphics \nare observed. ColorBrewer, for example, eschews continuous colour scales based \non cognitive research, but ggplot2 enforces them by default for continuous vari-\nables. Figure 6 shows the default choice of palette, updated to remove the unnec-\nessary graticule and to use the user-specified palette and class intervals: 1 3 1 3 3 Progress in the R ecosystem for representing and handling spatial… 533 Fig. 6 Thematic maps with ggplot2: left panel: default map of a continuous variable; right panel: grati-\ncule removed and palette modified Fig. 3.4 \u0007Reverse dependencies of the sp and sf packages R packages can possess forward or reverse dependencies. Forward or upstream \ndependencies are typically on R itself, a small number of packages whose func-\ntionalities are used in the package in question (by loading and attaching the pack-\nage (dependencies) or just loading the namespace of the package (imports)), and \npossibly external software libraries. Reverse or downstream dependencies are \npackages that themselves use the package in question by loading and attaching \nit, only loading its namespace or using it on demand (suggests). sp and sf were \nwritten carefully to minimize forward dependencies, with sp only depending on \nand importing packages included in every R distribution by default and sf adding \nCRAN contributed packages Rcpp and units required to build the package and 1 3 3 534 R. S. Bivand classInt for class intervals, DBI for interfacing spatial databases and magrittr \nfor piped operations, where none of these extra forward dependencies draws in \nmany other packages. classInt for class intervals, DBI for interfacing spatial databases and magrittr \nfor piped operations, where none of these extra forward dependencies draws in \nmany other packages. In sp, the compiled code (written in C) is self-contained and is made available to \nother packages, chiefly rgdal and rgeos, to link to their compiled code. rgdal links \nto sp and to the external libraries PROJ and GDAL. GDAL itself links to PROJ and \ncan link to GEOS and many other libraries needed for specific drivers. The external \nsoftware versions used may be reported using *_extSoftVersion, here using \nthe :: operator to avoid attaching the packages being queried: > rgdal::rgdal_extSoftVersion()\nGDAL GDAL_with_GEOS\nPROJ\nsp\n\"3.1.2\"\n\"TRUE\"\n\"7.1.0\"\n\"1.4-4\" The versions vary between platforms and by the installation method used; as \npackage maintainer, I often run with pre-release or latest versions of external soft-\nware to attempt to detect and mitigate changes before they impact users’ workflows. For rgdal, the versions of GDAL, PROJ and sp are reported, together with a test \nshowing whether GDAL was built linking to GEOS, something that affects the \nbehaviour of some drivers. The report for rgeos is simpler, only listing the versions \nof GEOS itself and sp. 3.4 \u0007Reverse dependencies of the sp and sf packages > rgeos::rgeos_extSoftVersion()\nGEOS\nsp\n\"3.8.1\" \"1.4-4\" In the case of sf, and because it brings together access to the GDAL and GEOS \nexternal libraries through Simple Features representation for vector objects, the \nexternal software versions supported are the union of those seen above, omitting \nlinkage to a separate package defining classes for objects. In addition, it reports \nwhich API is used for PROJ, either proj.h or not (the earlier proj_api.h). > sf_extSoftVersion()\nGEOS\nGDAL\nproj.4 GDAL_with_GEOS\nUSE_PROJ_H\n\"3.8.1\"\n\"3.1.2\"\n\"7.1.0\"\n\"true\"\n\"true\" Table 1 shows the structure of reverse dependency counts for sp and sf. Recursive \ndependencies traverse through the whole CRAN dependency tree; the first column \nof the table shows counts of “depends” and “imports” dependencies counted across \nthe whole tree. These split into 1285 only involving sp, 232 involving both pack-\nages and 65 only involving sf. If we additionally include “suggests” dependencies, \nboth packages may be used at least indirectly by all CRAN packages. The two right \ncolumns show the same counts, but only for packages’ first-order dependencies on \nsp, sf or both. We can note that of these for “depends” and “imports” dependencies, \n459 only involve sp, 63 involve both packages and 121 only involve sf. In the first 1 3 Progress in the R ecosystem for representing and handling spatial… Progress in the R ecosystem for representing and handling spatial…\nTable 1 Reverse dependency \ncounts for sp and sf, August \n2020, for recursive and non-\nrecursive reverse dependencies \ntaken as “Depends” and \n“Imports” only, and with \n“Suggests”\nRecursive\nRecursive \nw/suggests\nNot recursive\nNot recursive \nw/suggests\nSum sp\n1517\n16,619\n522\n629\nSum sf\n297\n16,619\n184\n277\nOnly sp\n1285\n0\n459\n513\nOnly sf\n65\n0\n121\n161\nBoth\n232\n16,619\n63\n116\n535 535 column, the number of packages only depending on sf is less than when we ignore \nrecursive dependencies in the third column, which is packages using sf that also use \nsp. The number of packages only using sf is encouraging, given that it first entered \nCRAN in October 2016. It is also encouraging that a fair number of these packages use both sp and sf, \nshowing existing packages are preserving legacy workflows, but also opening up for \nmore modern object representations. It takes time and effort to communicate the \ndesirability of migrating from sp representations to sf and probably stars. 3.4 \u0007Reverse dependencies of the sp and sf packages Although \nkeeping the R code running is feasible, including compiled code not using external \nsoftware, migration to sf is advisable. 4 \u0007Upstream software dependencies of the R‑spatial ecosystem When changes occur in upstream external software, R packages using these libraries \noften need to adapt, but package maintainers try very hard to shield users from any \nnegative consequences, so that legacy workflows continue to provide the same or \nat least similar results from the same data. The code shown in Bivand et al. (2008, \n(2013) is almost all run nightly on a platform with updated R packages and external \nsoftware. This does not necessarily trap all differences (figures are not compared), \nbut is helpful in detecting impacts of changes in packages or external software. It is \nalso very helpful that CRAN servers using the released and development versions of \nR, and with different versions of external software, also run nightly checks. Again, \nsometimes changes are only noticed by users, but quite often checks run by main-\ntainers and by CRAN alert us to impending challenges. Tracking the development \nmailing lists of the external software communities, all open source, can also show \nhow thinking is evolving. However, sometimes code tidying in external software \ncan have unexpected consequences, breaking not sf or sp with rgdal or rgeos, but a \npackage further downstream. Bivand (2014) discusses open-source geospatial soft-\nware stacks more generally, but here we will consider ongoing changes in PROJ and \nlinked changes in GDAL. We will use the example of the location of the Broad Street pump in Soho, London, \nrelated to the 1854 Cholera epidemic and Dr John Snow’s intervention Brody et al. 1 3 536 R. S. Bivand (2000), distributed with sf (from version 0.8–1). Although it was known that changes in \nupstream software would impact workflows, the extent of those impacts became clear \nusing a standard example following upgrading to PROJ 6 and GDAL 3 in 2019: (2000), distributed with sf (from version 0.8–1). Although it was known that changes in \nupstream software would impact workflows, the extent of those impacts became clear \nusing a standard example following upgrading to PROJ 6 and GDAL 3 in 2019: > bp_file <- system.file(\"gpkg/b_pump.gpkg\", package=\"sf\")\n> b_pump_sf <- st_read(bp_file, quiet=TRUE) > bp_file <- system.file(\"gpkg/b_pump.gpkg\", package=\"sf\")\n> b_pump_sf <- st_read(bp_file, quiet=TRUE) Before R packages upgraded the way coordinate reference systems were represented \nin early 2020, our Proj4 string representation suffered degradation. Taking the Proj4 \nstring defined in PROJ 5 for the British National Grid, we can see a +datum=OSGB36 \nkey–value pair. 4 \u0007Upstream software dependencies of the R‑spatial ecosystem But when processing this input with PROJ 6 and GDAL 3, this key is \nremoved. Checking, we can see that reading the input string appears to work, but the \noutput for the Proj4 string drops the +datum=OSGB36 key–value pair, introducing \ninstead the ellipse implied by that datum: > proj5 <- paste0(\"+proj=tmerc +lat_0=49 +lon_0=-2 +k=0.9996012717\",\n+\n\" +x_0=400000 +y_0=-100000 +datum=OSGB36 +units=m +no_defs\")\n> legacy <- st_crs(proj5)\n> proj6 <- legacy$proj4string\n> proj5_parts <- unlist(strsplit(proj5, \" \"))\n> proj6_parts <- unlist(strsplit(proj6, \" \"))\n> proj5_parts[!is.element(proj5_parts, proj6_parts)]\n[1] \"+datum=OSGB36\"\n> proj6_parts[!is.element(proj6_parts, proj5_parts)]\n[1] \"+ellps=airy\" > proj5 <- paste0(\"+proj=tmerc +lat_0=49 +lon_0=-2 +k=0.9996012717\n+\n\" +x_0=400000 +y_0=-100000 +datum=OSGB36 +units=m +no_defs\")\n> legacy <- st_crs(proj5)\n> proj6 <- legacy$proj4string\n> proj5_parts <- unlist(strsplit(proj5, \" \"))\n> proj6_parts <- unlist(strsplit(proj6, \" \"))\n> proj5_parts[!is.element(proj5_parts, proj6_parts)]\n[1] \"+datum=OSGB36\"\n> proj6_parts[!is.element(proj6_parts, proj5_parts)]\n[1] \"+ellps=airy\" We can emulate the problem seen following the release in May 2019 of GDAL 3.0.0 \nusing PROJ 6, by inserting the degraded Proj4 string into the Broad Street pump object. The coordinate reference system representation is now ignorant of the proper datum \nspecification: > b_pump_sf1 <- b_pump_sf > b_pump_sf1 <- b_pump_sf _p\np_\n_p\np_\n> st_crs(b_pump_sf1) <- st_crs(st_crs(b_pump_sf1)$proj4string) p\np\np\np\n> st_crs(b_pump_sf1) <- st_crs(st_crs(b_pump_sf1)$proj4string) > st_crs(b_pump_sf1) <- st_crs(st_crs(b_pump_sf1)$proj4string) Why does this matter? For visualization on a web map, for example using the \nmapview package, the projected geometries are transformed to the same WGS84 \nellipse and datum (EPSG:4326) that were used in PROJ 4 as a transformation hub. In leaflet, these are projected to Web Mercator (EPSG:3857). In mapview(), the \nsf::st_transform() function is used, so we will emulate this step before hand-\ning on the geometries for display. 4 \u0007Upstream software dependencies of the R‑spatial ecosystem The projected British National Grid point location is \nunchanged: 1 3 1 3 Progress in the R ecosystem for rep\n> all.equal(st_coordinate\n+\nst_coordinate\n[1] TRUE\nHowever, because the \nresentation of its coordin\nformed identically to WG\nFigure 8:\n> b_pump_sf_ll <- st_tran\n> b_pump_sf1_ll <- st_tra\n> st_distance(b_pump_sf_l\nUnits: [m]\n[,1]\n[1,] 125.0578 Progress in the R ecosystem for representing and handling spatial… 537 > all.equal(st_coordinates(st_geometry(b_pump_sf)),\n+\nst_coordinates(st_geometry(b_pump_sf1)))\n[1] TRUE However, because the one of the objects now has a degraded Proj4 string rep-\nresentation of its coordinate reference system, the output points, apparently trans-\nformed identically to WGS84, are now some distance apart, as is also shown in \nFigure 8: > b_pump_sf_ll <- st_transform(b_pump_sf, 4326)\n> b_pump_sf1_ll <- st_transform(b_pump_sf1, 4326)\n> st_distance(b_pump_sf_ll, b_pump_sf1_ll)\nUnits: [m]\n[,1]\n[1,] 125.0578 > b_pump_sf_ll <- st_transform(b_pump_sf, 4326) [,1]\n[1,] 125.0578 Once PROJ 6 and GDAL 3 had stabilized in the summer of 2019, we iden-\ntified the underlying threat as lying in the advertised degradation of GDAL’s \nexportToProj4() function. When reading raster and vector files, the coordinate ref-\nerence system representation using Proj4 strings would often be degraded, so that fur-\nther transformation within R (also using GDAL/PROJ functionality) would be at risk of \nmuch greater inaccuracy than with PROJ 5 and GDAL 2. Since then, sf, sp with rgdal Fig. 8 Displays made using mapview displays of the Broad Street pump, with the green point within 2 m \nof the pump location, and the red point in Ingestre Place because of the loss of the datum specification Fig. 8 Displays made using mapview displays of the Broad Street pump, with the green point within 2 m \nof the pump location, and the red point in Ingestre Place because of the loss of the datum specification Fig. 8 Displays made using mapview displays of the Broad Street pump, with the green point within 2 m \nof the pump location, and the red point in Ingestre Place because of the loss of the datum specification 1 3 3 538 R. S. 7 https://cran.r-project.org/web/packages/rgdal/vignettes/CRS_projections_transformations.html. 6 https://www.r-spatial.org/r/2020/03/17/wkt.html.\n7 8 State of https://github.com/rsbivand/sp of 18 August 2020 or later. 4 \u0007Upstream software dependencies of the R‑spatial ecosystem Bivand and raster have adopted the 2019 version of the “Well-Known Text” coordinate refer-\nence system representation WKT2-2019 (ISO 2019) instead of Proj4 strings to contain \ncoordinate reference system definitions.6 Accommodations have also been provided so \nthat the S3 class \"crs\" objects used in objects defined in sf, and the formal S4 class \n\"CRS\" objects used objects defined in sp and raster, can continue to attempt to support \nProj4 strings in addition, while other package maintainers and workflow users catch up.7 l\nFollowing an extended campaign of checking about 900 reverse dependencies \n(packages depending on sp, rgdal and others) and dozens of github issues, most of \nthe consequences of the switch to WKT2 among packages have now been addressed. Most recently (late August 2020), 115 packages have been offered rebuilt stored \nobjects that had included \"CRS\" objects without WKT2 definitions. i\nThis approach has ensured that spatial objects, whether created within R, read in from \nexternal data sources or read as stored objects, all have WKT2 string representations of \ntheir coordinate reference systems, and for backward compatibility can represent these in \naddition as Proj4 strings. Operations on objects should carry forward the new represen-\ntations, which should be written out to external data formats correctly. There is a minor \ndivergence between sf and sp (and thus rgdal): In sf, the axis order of the CRS is pre-\nserved as instantiated, but objects do not have their axes swapped to accord with authori-\nties unless sf::st_axis_order() is set TRUE. This can appear odd, because \nalthough the representation records a northings–eastings axis order, data are treated as \neastings–northings in plotting, variogram construction and so on: > st_crs(\"EPSG:4326\")\nCoordinate Reference System:\nUser input: EPSG:4326\nwkt:\nGEOGCRS[\"WGS 84\",\nDATUM[\"World Geodetic System 1984\",\nELLIPSOID[\"WGS 84\",6378137,298.257223563,\nLENGTHUNIT[\"metre\",1]]],\nPRIMEM[\"Greenwich\",0,\nANGLEUNIT[\"degree\",0.0174532925199433]],\nCS[ellipsoidal,2],\nAXIS[\"geodetic latitude (Lat)\",north,\nORDER[1],\nANGLEUNIT[\"degree\",0.0174532925199433]],\nAXIS[\"geodetic longitude (Lon)\",east,\nORDER[2],\nANGLEUNIT[\"degree\",0.0174532925199433]], ANGLEUNIT[\"degree\",0.0174532925199433]], ANGLEUNIT[\"degree\",0.0174532925199433]] g\nUSAGE[\nSCOPE[\"unknown\"],\nAREA[\"World\"],\nBBOX[-90,-180,90,180]],\nID[\"EPSG\",4326]] g\nUSAGE[\nSCOPE[\"unknown\"],\nAREA[\"World\"],\nBBOX[-90,-180,90,180]],\nID[\"EPSG\",4326]] USAGE[\nSCOPE[\"unknown\"],\nAREA[\"World\"],\nBBOX[-90,-180,90,180]],\nID[\"EPSG\",4326]] USAGE[\nSCOPE[\"unknown\"],\nAREA[\"World\"],\nBBOX[-90,-180,90,180]],\nID[\"EPSG\",4326]] ID[\"EPSG\",4326]] In sp/rgdal, attempts are made to ensure that axis order is in the form termed \nGIS, traditional or visualization that is always eastings–northings: 1 3 Progress in the R ecosystem for representing and handling spatial… > library(sp)\n> cat(wkt(CRS(\"EPSG:4326\")))\nGEOGCRS[\"WGS 84\",\nDATUM[\"World Geodetic System 1984\",\nELLIPSOID[\"WGS 84\",6378137,298.257223563,\nLENGTHUNIT[\"metre\",1]],\nID[\"EPSG\",6326]],\nPRIMEM[\"Greenwich\",0,\nANGLEUNIT[\"degree\",0.0174532925199433],\nID[\"EPSG\",8901]],\nCS[ellipsoidal,2],\nAXIS[\"longitude\",east,\nORDER[1],\nANGLEUNIT[\"degree\",0.0174532925199433,\nID[\"EPSG\",9122]]],\nAXIS[\"latitude\",north,\nORDER[2],\nANGLEUNIT[\"degree\",0.0174532925199433,\nID[\"EPSG\",9122]]],\nUSAGE[\nSCOPE[\"unknown\"],\nAREA[\"World\"],\nBBOX[-90,-180,90,180]]] > library(sp)\n> cat(wkt(CRS(\"EPSG:4326\")))\nGEOGCRS[\"WGS 84\",\nDATUM[\"World Geodetic System 1984\",\nELLIPSOID[\"WGS 84\",6378137,298.257223563,\nLENGTHUNIT[\"metre\",1]],\nID[\"EPSG\",6326]],\nPRIMEM[\"Greenwich\",0,\nANGLEUNIT[\"degree\",0.0174532925199433],\nID[\"EPSG\",8901]],\nCS[ellipsoidal,2],\nAXIS[\"longitude\",east,\nORDER[1],\nANGLEUNIT[\"degree\",0.0174532925199433,\nID[\"EPSG\",9122]]],\nAXIS[\"latitude\",north,\nORDER[2],\nANGLEUNIT[\"degree\",0.0174532925199433,\nID[\"EPSG\",9122]]],\nUSAGE[\nSCOPE[\"unknown\"],\nAREA[\"World\"],\nBBOX[-90,-180,90,180]]] Progress in the R ecosystem for representing and handling spatial… 539 9 nl is used to represent the verbatim new line character in the processed article. USAGE[ USAGE[\nSCOPE[\"unknown\"],\nAREA[\"World\"],\nBBOX[-90,-180,90,180]]] The probability of confusion increases when coercing from sf to sp and vice \nversa, with the representations most often remaining unchanged.8 > sf_from_sp <- st_crs(CRS(\"EPSG:4326\"))\n> o <- strsplit(sf_from_sp$wkt, nl)[[1]]\n> cat(paste(o[grep(\"CS|AXIS|ORDER\", o)], collapse=nl))\nCS[ellipsoidal,2],\nAXIS[\"longitude\",east,\nORDER[1],\nAXIS[\"latitude\",north,\nORDER[2], > sf_from_sp <- st_crs(CRS(\"EPSG:4326\"))\n> o <- strsplit(sf_from_sp$wkt, nl)[[1]]\n> cat(paste(o[grep(\"CS|AXIS|ORDER\", o)], collapse=nl))\nCS[ellipsoidal,2],\nAXIS[\"longitude\",east,\nORDER[1],\nAXIS[\"latitude\",north,\nORDER[2], > sf_from_sp <- st_crs(CRS(\"EPSG:4326\")) o <- strsplit(sf_from_sp$wkt, nl)[[1]] p\np\n> cat(paste(o[grep(\"CS|AXIS|ORDER\", o)], collapse=nl)) (p\n( [g\np(\n|\n|\nCS[ellipsoidal,2],\nAXIS[\"longitude\",east,\nORDER[1],\nAXIS[\"latitude\",north,\nORDER[2], > sp_from_sf <- as(st_crs(\"EPSG:4326\"), \"CRS\")\n> o <- strsplit(wkt(sp_from_sf), nl)[[1]]\n> cat(paste(o[grep(\"CS|AXIS|ORDER\", o)], collapse=nl))\nCS[ellipsoidal,2],\nAXIS[\"geodetic latitude (Lat)\",north,\nORDER[1],\nAXIS[\"geodetic longitude (Lon)\",east,\nORDER[2], > sp_from_sf <- as(st_crs(\"EPSG:4326\"), \"CRS\") > sp_from_sf <- as(st_crs(\"EPSG:4326\"), \"CRS\")\n> o <- strsplit(wkt(sp_from_sf), nl)[[1]]\n> cat(paste(o[grep(\"CS|AXIS|ORDER\", o)], collapse=nl)\nCS[ellipsoidal,2],\nAXIS[\"geodetic latitude (Lat)\",north,\nORDER[1],\nAXIS[\"geodetic longitude (Lon)\",east,\nORDER[2], > o <- strsplit(wkt(sp_from_sf), nl)[[1]] p\np\n> cat(paste(o[grep(\"CS|AXIS|ORDER\", o)], collapse=nl)) Both of these coercions are using the same underlying PROJ and GDAL versions, \nand the same PROJ metadata. Once work in progress is completed, coercions should \nrespect the setting of sf::st_axis_order(). 1 3 540 R. S. Bivand It may useful for users to know of other differences between sf and sp/rgdal. Transformation in sf uses code in GDAL, which in turn uses functions in PROJ; in \nsp/rgdal, PROJ is used directly for transformation. In order to demonstrate more of \nwhat is happening, let us coerce these sf objects to sp (they are both planar with an \nx–y axis order): > b_pump_sp <- as(b_pump_sf, \"Spatial\")\n> b_pump_sp1 <- as(b_pump_sf1, \"Spatial\") > b_pump_sp <- as(b_pump_sf, \"Spatial\")\n> b_pump_sp1 <- as(b_pump_sf1, \"Spatial\") We will also set up a temporary directory for use with the on-demand grid down-\nload functionality in PROJ 7; this must be done before rgdal is loaded: > td <- tempfile()\n> dir.create(td)\n> Sys.setenv(\"PROJ_USER_WRITABLE_DIRECTORY\"=td)\n> library(rgdal) > td <- tempfile()\n> dir.create(td)\n> Sys.setenv(\"PROJ_USER_WRITABLE_DIRECTORY\"=td)\n> library(rgdal) > td <- tempfile()\n> dir.create(td)\n> Sys.setenv(\"PROJ_USER_WRITABLE_DIRECTORY\"=td)\n> library(rgdal) > td <- tempfile() > td <- tempfile()\n> dir.create(td)\n( > dir.create(td) > library(rgdal) In sf, areas of interest need to be given by the users, while in transformation and \nprojection in rgdal, these are calculated from the object being projected or trans-\nformed. The provision of areas of interest is intended to reduce the number of candi-\ndate coordinate operations found by PROJ. USAGE[ > WKT <- wkt(b_pump_sp)\n> o <- list_coordOps(WKT, \"EPSG:4326\")\n> c(nrow(o), nrow(o[o$instantiable,]), sum(o$number_grids))\n[1] 8 7 1\n> aoi0 <- project(t(unclass(bbox(b_pump_sp))), WKT, inv=TRUE)\n> aoi <- c(t(aoi0 + c(-0.1, +0.1)))\n> o_aoi <- list_coordOps(WKT, \"EPSG:4326\", area_of_interest=aoi)\n> c(nrow(o_aoi), nrow(o_aoi[o_aoi$instantiable,]), sum(o_aoi$number_grids))\n[1] 5 4 1 rgdal::list_coordOps() accesses the PROJ metadata database to \nsearch through candidate coordinate operations, ranking them by accuracy, \nreturning a data frame of operations. When an area of interest is provided, candi-\ndates falling outside it are dropped. Coordinate operations that cannot be instanti-\nated because of missing grids are also listed. We can see here that when an area \nof interest is not given, 8 candidate operations are found when the WKT string \ncontains datum information. Of these, 7 may be instantiated, with 1 needing a \ngrid (here, the operation that cannot be instantiated). Three operations cease to be \ncandidates if we use an area of interest. In sp/rgdal, the coordinate operation last used is returned and can be retrieved \nusing rgdal::get_last_coordOp(); coordinate operations are repre-\nsented as pipelines (Knudsen and Evers 2017; Evers and Knudsen 2017), intro-\nduced in PROJ 5 and using the PROJ key–value pair notation:9 1 3 1 3 Progress in the R ecosystem for representing and handling spatial… 541 > b_pump_sp_ll <- spTransform(b_pump_sp, \"EPSG:4326\")\n> cat(strwrap(get_last_coordOp()), sep=nl)\n+proj=pipeline +step +inv +proj=tmerc +lat_0=49 +lon_0=-2 +k=0.999601\n+x_0=400000 +y_0=-100000 +ellps=airy +step +proj=push +v_3 +step +proj=cart\n+ellps=airy +step +proj=helmert +x=446.448 +y=-125.157 +z=542.06 +rx=0.15\n+ry=0.247 +rz=0.842 +s=-20.489 +convention=position_vector +step +inv\n+proj=cart +ellps=WGS84 +step +proj=pop +v_3 +step +proj=unitconvert\n+xy_in=rad +xy_out=deg > b_pump_sp_ll <- spTransform(b_pump_sp, \"EPSG:4326\")\n> cat(strwrap(get_last_coordOp()), sep=nl)\n+proj=pipeline +step +inv +proj=tmerc +lat_0=49 +lon_0=-2 +k=0.999601\n+x_0=400000 +y_0=-100000 +ellps=airy +step +proj=push +v_3 +step +proj=cart\n+ellps=airy +step +proj=helmert +x=446.448 +y=-125.157 +z=542.06 +rx=0.15\n+ry=0.247 +rz=0.842 +s=-20.489 +convention=position_vector +step +inv\n+proj=cart +ellps=WGS84 +step +proj=pop +v_3 +step +proj=unitconvert\n+xy_in=rad +xy_out=deg > b_pump_sp_ll <- spTransform(b_pump_sp, \"EPSG:4326\") > b_pump_sp_ll <- spTransform(b_pump_sp, \"EPSG:4326\") > b_pump_sp_ll <- spTransform(b_pump_sp, \"EP\n> cat(strwrap(get_last_coordOp()), sep=nl) p\np\np\np\np\np\np\n> cat(strwrap(get_last_coordOp()), sep=nl) +proj=pipeline +step +inv +proj=tmerc +lat_0=49 +lon_0=-2 +k=0.999601\n+x_0=400000 +y_0=-100000 +ellps=airy +step +proj=push +v_3 +step +proj=cart\n+ellps=airy +step +proj=helmert +x=446.448 +y=-125.157 +z=542.06 +rx=0.15\n+ry=0.247 +rz=0.842 +s=-20.489 +convention=position_vector +step +inv\n+proj=cart +ellps=WGS84 +step +proj=pop +v_3 +step +proj=unitconvert\n+xy_in=rad +xy_out=deg Here, we can see that an inverse projection from the specified Transverse Mer-\ncator projection is made to geographical coordinates, followed by a seven-param-\neter Helmert transformation to WGS84 ellipsoid and datum. 10 https://cdn.proj.org. > enable_proj_CDN()\n[1] \"Using: /tmp/Rtmp5keMtw/file1c4592b3b37c2\"\n> list.files(td)\ncharacter(0) > o <- list_coordOps(WKT, \"EPSG:4326\", area_of_interest=aoi)\n> c(nrow(o), nrow(o[o$instantiable,]), sum(o$number_grids))\n[1] 5 5 1 > o <- list_coordOps(WKT, \"EPSG:4326\", area_of_interest=aoi)\n> c(nrow(o), nrow(o[o$instantiable,]), sum(o$number_grids))\n[1] 5 5 1 > o <- list_coordOps(WKT, \"EPSG:4326\", area_of_interest=aoi)\n> c(nrow(o), nrow(o[o$instantiable,]), sum(o$number_grids))\n[1] 5 5 1 > b_pump_sp_llg <- spTransform(b_pump_sp, \"EPSG:4326\") > cat(strwrap(get_last_coordOp()), sep=nl) +proj=pipeline +step +inv +proj=tmerc +lat_0=49 +lon_0=-2 +k=0.999601 +x_0=400000 +y_0=-100000 +ellps=airy +step +proj=hgridshift +grids=uk_os_OSTN15_NTv2_OSGBtoETRS.tif +step +proj=unitconvert +xy_in=rad\n+xy_out=deg Now the downloaded grid is cached in the database in the designated CDN direc-\ntory and may be used for other transformations using the same operation. > list.files(td)\n[1] \"cache.db\"\n> file.size(file.path(td, list.files(td)[1]))\n[1] 319488\n> disable_proj_CDN() > list.files(td)\n[1] \"cache.db\"\n> file.size(file.path(td, list.files(td)[1]))\n[1] 319488\n> disable_proj_CDN() Once again, the distance between the point transformed from the sf object as read \nfrom file and the point with the degraded coordinate reference system emulating the \neffect of the change in behaviour of GDAL’s exportToProj4() in GDAL 6 and \nlater is about 125 m. Using the CDN shifts the output point by 1.7 m. For confirma-\ntion, the output transformed coordinates for the sp and sf objects using the Helmert \ntransformation are the same. > c(spDists(b_pump_sp1_ll, b_pump_sp_ll),\n+\nspDists(b_pump_sp_llg, b_pump_sp_ll))*1000\n[1] 125.057683\n1.751474\n> all.equal(unname(coordinates(b_pump_sp_ll)),\n+\nunname(st_coordinates(st_geometry(b_pump_sf_ll))))\n[1] TRUE > c(spDists(b_pump_sp1_ll, b_pump_sp_ll),\n+\nspDists(b_pump_sp_llg, b_pump_sp_ll))*1000\n[1] 125.057683\n1.751474\n> all.equal(unname(coordinates(b_pump_sp_ll)),\n+\nunname(st_coordinates(st_geometry(b_pump_sf_ll))))\n[1] TRUE > c(spDists(b_pump_sp1_ll, b_pump_sp_ll),\n+\nspDists(b_pump_sp_llg, b_pump_sp_ll))*1000\n[1] 125.057683\n1.751474\n> all.equal(unname(coordinates(b_pump_sp_ll)),\n+\nunname(st_coordinates(st_geometry(b_pump_sf_ll))))\n[1] TRUE Although it appears that most of the consequences of the change in representa-\ntion of coordinate reference systems from Proj4 to WKT2 strings have now been \naddressed, we still see signs on the mailing list and on Twitter that users, natu-\nrally directing their attention to their analytical or visualization work, may still \nbe confused. The extent of the spatial cluster of R packages is so great that it will \nundoubtedly take time before the dust settles. However, we trust that the operation \nof upgrading representations is now largely complete. Multiple warnings issued in \nsp workflows, now noisily drawing attention to possible degradations in workflows, \nwill by default be muted when sp 1.5 and rgdal 1.6 are released. USAGE[ The parameters are \ncontained in the best instantiable coordinate operation retrieved from the PROJ \ndatabase. > o <- list_coordOps(wkt(b_pump_sp1), \"EPSG:4326\", area_of_interest=aoi)\n> cat(nrow(o), o$ballpark, nl)\n1 TRUE > b_pump_sp1_ll <- spTransform(b_pump_sp1, \"EPSG:4326\") > cat(strwrap(get_last_coordOp()), sep=nl) Going on to the case of the degraded representation, only 1 operation is found, \nwith only ballpark accuracy. With our emulation of the dropping of +datum= sup-\nport in GDAL’s exportToProj4(), we see that the coordinate operation pipe-\nline only contains the inverse projection step, accounting for the observed shift of \nthe Broad Street pump to Ingestre Place. Finally, sp/rgdal may use the provision of on-demand downloading of transfor-\nmation grids to provide more accuracy (CDN, from PROJ 7).10 Before finding and \nchoosing to use a coordinate operation using an on-demand downloaded grid, the \ndesignated directory is empty: > enable_proj_CDN()\n[1] \"Using: /tmp/Rtmp5keMtw/file1c4592b3b37c2\"\n> list.files(td)\ncharacter(0) Using the CDN, all the candidate operations are instantiable, and the pipeline \nnow shows a horizontal grid transformation rather than a Helmert transformation. 1 3 R. S. Bivand 542 5 \u0007Outlook As has already been mentioned, either most sp-based workflows continue to func-\ntion using sf objects or changes have been made in packages like spdep and its mod-\nelling counterpart spatialreg to permit scripts and packages using these packages \nto continue to function. Some workflows require more attention than others, but 1 3 Progress in the R ecosystem for representing and handling spatial… 543 the transition to sf from sp, rgdal and rgeos should be unproblematic. Shifting to \nnew visualization packages like tmap, cartography and mapview should also be \nrelatively easy, and use of sf and the new visualization packages should certainly \nbecome standard for new research and teaching. It may take a little longer for stars \nto find its place, but work here is continuing, and will stabilize before long. Similar \nremarks apply to transition from raster to terra. The key challenges for handling spatial data using R concern the upstream \nsoftware libraries in the open-source geospatial software stack, not just PROJ as \ndescribed above or changes in validity requirements in GEOS and other libraries, \nbut also the opportunities opened up by access to cloud-based earth observation data \nstreams. Because incoming data may take forms as yet not provided for, even where \nGDAL drivers become available, changes in object representations may become \nnecessary. In particular, this relates to spatio-temporal data, where trajectory data \nare especially demanding. These data representation challenges are actually oppor-\ntunities for users and developers to continue cooperating to contribute to making the \nR ecosystem for handling and analysing spatial and spatio-temporal data even more \ncapable and performant. Finally, please note that a CODECHECK certificate for this \npaper is available at https://doi.org/10.5281/zenodo.4003848. CODECHECK is an \nopen-science initiative to facilitate sharing of computer programs and results pre-\nsented in scientific publications (see https://codecheck.org.uk/) Funding Open Access funding provided by Norwegian School of Economics. Funding Open Access funding provided by Norwegian School of Economics. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, \nwhich permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as \nyou give appropriate credit to the original author(s) and the source, provide a link to the Creative Com-\nmons licence, and indicate if changes were made. The images or other third party material in this article \nare included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the \nmaterial. 5 \u0007Outlook If material is not included in the article’s Creative Commons licence and your intended use is \nnot permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission \ndirectly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licen\nses/by/4.0/. nding Open Access funding provided by Norwegian School of Economics. References https\nCRAN.R-project.org/package=rgeos, R package version 0.5-3 Bivand R, Pebesma E, Gomez-Rubio V (2008) Applied spatial data analysis with R. Springer, New Y Bivand R, Pebesma E, Gomez-Rubio V (2008) Applied spatial data analysis with R. Springer, New Yor\nBivand R, Pebesma E, Gomez-Rubio V (2013) Applied spatial data analysis with R, 2nd edn. 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Comput Geosci \n19:627–655 Rowlingson B, Diggle P (2017) splancs: spatial and space-time point pattern analysis. https://CRAN.R-\nproject.org/package=splancs, R package version 2.01-40 Sawicka K, Heuvelink GB, Walvoort DJ (2018) Spatial uncertainty propagation analysis with the spup R \npackage. R J 10(2):180–199. https://doi.org/10.32614/RJ-2018-047 Sawicka K, Heuvelink GB, Walvoort DJ (2018) Spatial uncertainty propagation analysis with the spup R \npackage. R J 10(2):180–199. https://doi.org/10.32614/RJ-2018-047\nTennekes M (2018) tmap: thematic maps in R. J Stat Softw 84(6):1–39 Tennekes M (2018) tmap: thematic maps in R. J Stat Softw 84(6):1–39 Tennekes M (2020) tmap: thematic maps. https://CRAN.R-project.org/package=tmap, R package versi\n3.1 Venables WN, Ripley BD (2002) Modern applied statistics with S, 4th edn. Springer, New York Warmerdam F (2008) The geospatial data abstraction library. In: Hall GB, Leahy M (eds) Open \napproaches in spatial data handling. Springer, Berlin, pp 87–104 Wickham H (2014) Tidy data. J Stat Softw 59(10):1–23. https://doi.org/10.18637/jss.v059.i10 Wickham H, Chang W, Henry L, Pedersen TL, Takahashi K, Wilke C, Woo K, Yutani H (2020) ggplot2: \ncreate elegant data visualisations using the grammar of graphics. https://CRAN.R-project.org/packa\nge=ggplot2, R package version 3.3.2 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published \nmaps and institutional affiliations. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published \nmaps and institutional affiliations. 1 3 1 3 1 3"
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Infrared Spectroscopy of Bilberry Extract Water-in-Oil Emulsions: Sensing the Water-Oil Interface
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Biosensors
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Infrared Spectroscopy of Bilberry Extract Water-in-Oil
Emulsions: Sensing the Water-Oil Interface 2
Erlangen Graduate School in Advanced Optical Technologies, University of Erlangen-Nuremberg,
Erlangen 91052, Germany 3
School of Engineering, University of Aberdeen, Aberdeen AB24 3UE, UK
4
Section I: Food Process Engineering, Institute of Process Engineering in Life Sciences,
Karlsruhe Institute of Technology, Karlsruhe 76131, Germany; heike.schuchmann@kit.edu
5
Present affiliation: BASF SE, Ludwigshafen 67056, Germany; kerstin.frank@basf.com
*
Correspondence: jkiefer@uni-bremen.de; Tel.: +49-421-218-64777 3
School of Engineering, University of Aberdeen, Aberdeen AB24 3UE, UK
4
Section I: Food Process Engineering, Institute of Process Engineering in Life Sciences,
Karlsruhe Institute of Technology, Karlsruhe 76131, Germany; heike.schuchmann@kit.edu
5
Present affiliation: BASF SE, Ludwigshafen 67056, Germany; kerstin.frank@basf.com
*
Correspondence: jkiefer@uni-bremen.de; Tel.: +49-421-218-64777 3
School of Engineering, University of Aberdeen, Aberdeen AB24 3UE, UK
4
Section I: Food Process Engineering, Institute of Process Engineering in Life Sciences,
Karlsruhe Institute of Technology, Karlsruhe 76131, Germany; heike.schuchmann@kit.edu
5
Present affiliation: BASF SE, Ludwigshafen 67056, Germany; kerstin.frank@basf.com
*
Correspondence: jkiefer@uni-bremen.de; Tel.: +49-421-218-64777 Academic Editor: Torsten Frosch Received: 16 February 2016; Accepted: 8 April 2016; Published: 14 April 2016 Received: 16 February 2016; Accepted: 8 April 2016; Published: 14 April 2016 Abstract: Water-in-oil (w/o) emulsions are of great interest in many areas of the life sciences,
including food technology, bioprocess engineering, and pharmaceuticals. Such emulsions are complex
multi-component systems and the molecular mechanisms which lead to a stable emulsion are yet
to be fully understood. In this work, attenuated total reflection (ATR) infrared (IR) spectroscopy is
applied to a series of w/o emulsions of an aqueous anthocyanin-rich bilberry extract dispersed in a
medium chain triglyceride (MCT) oil phase. The content of the emulsifier polyglycerin-polyricinoleat
(PGPR) has been varied systematically in order to investigate whether or not its concentration has an
impact on the molecular stabilization mechanisms. The molecular stabilization is accessed by a careful
analysis of the IR spectrum, where changes in the vibrational frequencies and signal strengths indicate
alterations of the molecular environment at the water/oil interface. The results suggest that adding
emulsifier in excess of 1% by weight does not lead to an enhanced stabilization of the emulsion. Keywords: MCT oil; hydrogen bonding; interfacial layer; vibrational spectroscopy; functional
food; anthocyanin biosensors biosensors biosensors biosensors 1. Introduction p
p
p
A few years ago, our group extended the ATR-FTIR approach to the analysis of water in the
interfacial layer of water-in-oil (w/o) emulsions [10]. Making use of the difference in absorption
between the oil and the water phase in a selected range of the spectrum allowed the nature of the
interfacial molecular interactions to be analyzed. The OH vibrational stretching modes were utilized
as sensors for the molecular environment. It was found that the emulsifier weakens the hydrogen
bonding network in the interfacial water layer leading to a stabilized water droplet and a decreased
tendency of water droplets to coalesce. In the present work, we apply the above ATR-FTIR method to study the effect of varying the
emulsifier content on the molecular stabilization mechanism. The samples under investigation are of
interest in the area of food technology and food chemistry: an emulsion where an aqueous anthocyanin
solution in terms of a bilberry extract is dispersed in a medium chain triglyceride (MCT) oil stabilized
with polyglycerine-polyricinoleat (PGPR) as emulsifier. Anthocyanin compounds are among the most
important hydrophilic plant pigments [11,12]. They are considered as food additives owing to their
beneficial anti-oxidant, anti-carcinogenic as well as immune modulating effects [12–15]. Unfortunately,
anthocyanins exhibit relatively low chemical stability. Environmental stresses such as pH-values above
4, thermal stress, and the presence of oxygen or certain enzymes accelerate their degradation [16,17]. This poses a challenge in the processing of anthocyanin-containing foods. One possibility to overcome
this problem and to protect the bioactive ingredients against environmental stresses is the formulation
of multiple emulsions, e.g., a water-in-oil-in-water (w/o/w) system. In such a multiphase system,
the sensitive molecules are incorporated into aqueous droplets with a stable chemical composition
and pH value for an optimal anthocyanin stability [18,19]. The water droplets are then dispersed
in a continuous oil phase. In a further step, this w/o emulsion is dispersed in another aqueous
phase. It is needless to say that the formulation of a stable w/o/w emulsion requires both experience
and a good understanding of the interfacial phenomena. Despite the effort in the production, such
multiple emulsions can offer benefits beyond the mere protection of bioactive ingredients. 1. Introduction Emulsions are multiphase systems comprising of two or more liquids that are immiscible with
each other. Typically, the dispersed phase in the form of small droplets is evenly distributed in the
continuous phase. To stabilize the emulsion and to avoid macroscopic phase separation, emulsifiers in
terms of surface-active substances (surfactants) are admixed. These surfactants are normally soluble,
predominantly in one phase, but they exhibit functional groups that can interact with the other phase
at the interfacial boundary. On the one hand, the emulsifier represents an additive, which ensures
emulsion stability. On the other hand, its concentration should be limited to a minimum in order to
save costs and to avoid undesirable effects. For example, in the area of food technology, the emulsifier
may alter the taste when its concentration reaches a certain threshold. Hence, in order to optimize
emulsion systems there is a need to understand the mechanisms at the interface between the dispersed
and the continuous phase. This is the key to develop strategies for selecting the best emulsifier for a
given system and minimize the required concentration. An important step towards understanding the molecular mechanisms is the development of
suitable analytical techniques offering high sensitivity for the interactions at the interface. Vibrational Biosensors 2016, 6, 13; doi:10.3390/bios6020013 www.mdpi.com/journal/biosensors www.mdpi.com/journal/biosensors 2 of 11 Biosensors 2016, 6, 13 spectroscopic methods have proven their capability for studying intra- and intermolecular interactions
in pure substances and mixtures [1–3]. They have also been successfully applied to emulsions. Jorgensen et al. [4] used transmission-mode FTIR spectroscopy to study structural changes of proteins
in w/o emulsions. The same method was employed by Zhou et al. [5] and Valero et al. [6] to study
water structures in AOT/alkane/water micro-emulsions of different composition. An attempt to
distinguish between interfacial and bulk water IR signals in AOT reverse micelles was made by
Sechler et al. [7]. They carried out a detailed analysis of the spectra recorded in emulsions with
a systematically varied ratio between interfacial and bulk water by varying the droplet diameters. Kemsley et al. [8] used attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR)
to quantitatively analyze the fat content of cream samples. Nickolov et al. [9] applied the same
technique to investigate water structures in a nanoparticle synthesis which was realized in emulsified
droplets acting as micro-reactors. They focused on structural changes inside the droplets during the
precipitation process. 2.2. ATR Spectroscopy The measurements were carried out on a Bruker Vertex v70 FTIR spectrometer equipped with
a diamond ATR accessory. The number of reflections at the surface is 1, and the reflection angle
is 45˝. Spectra were recorded taking 16 scans with a nominal resolution of 1 cm´1. All spectra were
corrected for water vapor and carbon dioxide interference. In order to check the reproducibility of the
measurement, three spectra of each sample were recorded and subtracted from each other pairwise. This subtraction resulted in statistical noise across the entire spectrum. From this, we concluded that
statistical errors in the data can be considered to be negligible. To carry out a measurement, a droplet of the emulsion was placed on the diamond crystal and
covered with a cap. The basics of studying w/o emulsions with ATR-IR spectroscopy were described
in detail in [10] and Figure 1 shows the situation near the crystal surface. The surface of the ATR
crystal is predominantly wetted by the oil phase as no significant contribution from bulk water was
observed in the emulsion spectra. On the left hand side of the drawing in Figure 1, the schematic
intensity distribution of the evanescent field is plotted as a function of distance from the surface. It
represents a single-exponential decay in the absence of absorbing molecules. When a wavelength
is considered at which significant absorption in the oil phase takes place, the penetration depth is
reduced starting at the surface. On the other hand, when the wavelength of interest is absorbed in the
water phase only, the initial intensity decay at the surface remains the same as in the figure, but at the
w/o interface the decay constant changes abruptly and the decay is enhanced inside the droplet. As a
simplified picture, this can be considered as the product of two exponential functions, one taking into
account the medium without absorption and one taking into account the absorption effects via the
Beer-Lambert law. In our previous work [10] it was shown that the resulting thickness of the interfacial water layer
that is probed is only a few hundred nanometers. This means that the signals are dominated by the
interfacial water rather than from bulk water. Moreover, the probed thickness in terms of its 1/e value
is independent of the distance of a droplet from the surface. 2.1. Sample Preparation Anthocyanin-enriched w/o emulsions were prepared as follows. Aqueous phase: For the
preparation of the aqueous anthocyanin solutions, 0.05% (w/w) anthocyanin-rich bilberry extract
(Kaden Biochemical GmbH, Hamburg, Germany) was dissolved in distilled water under constant
stirring for 30 min via a magnetic stirrer at room temperature. Insoluble solid extract particles were
separated by filtration (Sartorius Stedim biotech Germany, Filter Discs, Grade 388). Oil phase: The
emulsifier-containing oil-phase was prepared by dissolving 1, 5, or 10 wt % polyglycerin-polyricinoleat
(PGPR 4150, Palsgaard®, Juelsminde, Denmark) in MCT-oil (Medium Chain Triglyceride, Schuman und
Sohn GmbH, Germany). The samples were stirred for 1 h via a propeller mixer at 40 ˝C. Emulsion: The
w/o emulsions were then prepared via a high pressure homogenizer (MF 110 EH-30, Microfluidics®,
Westwood, MA, USA) operated at 1000 bar pressure difference, where the water phase (30 wt %) was
emulsified in the surrounding oil phase (70 wt %). The diameter of the water droplets in the emulsion
was about 2 µm (Sauter mean diameter). 1. Introduction For example,
during the digestive process, the multiple emulsion structure of several cladding layers composed of a
triglyceride phase and several emulsifiers and further stabilizing ingredients can enable a triggered
release of the encapsulated molecules at different locations in the gastrointestinal tract. Previous work
has shown that oil droplets can sustain stomach conditions [20,21]. This makes them a perfect shell
for anthocyanin protection in the gastric passage. It was also shown that the protective effect of the
capsule structure and a possible targeted release of the encapsulated matter at the effective location
strongly depends on the emulsion microstructure [22]. The present study focuses on the initial w/o emulsion system, as the interface between the
aqueous anthocyanin solution and the oil is crucial for the behavior from a functional food point of
view. The aim of this work is to investigate the effect of the emulsifier concentration on the molecular
stabilization mechanism. For this purpose, w/o emulsions are studied and the emulsifier concentration
in the oil phase is varied between 1% and 10% by weight, which represents the practically relevant 3 of 11 Biosensors 2016, 6, 13 range. The samples are analyzed using ATR-FTIR spectroscopy, for which we have shown previously
that the main signals originate from only a thin water layer (below 0.5 µm) at the w/o interface [10]. In addition, we present a vibrational spectroscopic analysis of the individual components of the
emulsion system. 3.1.1. Bilberry Extract Solution
3.1.1. Bilberry Extract Solution
3.1.1. Bilberry Extract Solution The IR spectrum of the aqueous solution of the bilberry extract is shown in Figure 2. Due to the
very small content of bilberry extract, the spectrum is virtually identical to the spectrum of pure
water. It is dominated by broad bands, where the most prominent features are the OH bending
mode at 1636 cm−1 and the broad OH stretching band ranging from 2800 to 3700 cm−1. Our focus will
be on the stretching band, the shape of which is an indicator of the hydrogen-bonding environment. In other words, we utilize water as a molecular sensor for studying the effects of the more
complicated oil and emulsifier molecules. The IR spectrum of the aqueous solution of the bilberry extract is shown in Figure 2. Due to
the very small content of bilberry extract, the spectrum is virtually identical to the spectrum of pure
water. It is dominated by broad bands, where the most prominent features are the OH bending mode
at 1636 cm´1 and the broad OH stretching band ranging from 2800 to 3700 cm´1. Our focus will be on
the stretching band, the shape of which is an indicator of the hydrogen-bonding environment. In other
words, we utilize water as a molecular sensor for studying the effects of the more complicated oil and
emulsifier molecules. The IR spectrum of the aqueous solution of the bilberry extract is shown in Figure 2. Due to the
very small content of bilberry extract, the spectrum is virtually identical to the spectrum of pure
water. It is dominated by broad bands, where the most prominent features are the OH bending
mode at 1636 cm−1 and the broad OH stretching band ranging from 2800 to 3700 cm−1. Our focus will
be on the stretching band, the shape of which is an indicator of the hydrogen-bonding environment. In other words, we utilize water as a molecular sensor for studying the effects of the more
complicated oil and emulsifier molecules. Figure 2. IR spectrum of aqueous bilberry extract solution. The small peak around 2300 cm−1 is an
experimental artifact of the diamond crystal involved in the ATR measurement. Fo a detailed a aly i
the OH ba d
e o ded i
the bilbe y e t a t a d the e
ul io
a
Figure 2. IR spectrum of aqueous bilberry extract solution. 3. Results and Discussion
h
ll
3. Results and Discussion
3. Results and Discussion This section will present the results and their interpretation. We start with the vibrational
analysis of the individual components of the emulsions and then proceed to the emulsion samples. This section will present the results and their interpretation. We start with the vibrational analysis
of the individual components of the emulsions and then proceed to the emulsion samples. This section will present the results and their interpretation. We start with the vibrational
analysis of the individual components of the emulsions and then proceed to the emulsion samples. This section will present the results and their interpretation. We start with the vibrational
analysis of the individual components of the emulsions and then proceed to the emulsion samples. This section will present the results and their interpretation. We start with the vibrational analysis
of the individual components of the emulsions and then proceed to the emulsion samples. This section will present the results and their interpretation. We start with the vibrational
analysis of the individual components of the emulsions and then proceed to the emulsion samples. 3.1. Vibrational Analysis of the Individual Components
3.1. Vibrational Analysis of the Individual Components
3.1. Vibrational Analysis of the Individual Components 3.1. Vibrational Analysis of the Individual Components
3.1. Vibrational Analysis of the Individual Components
3.1. Vibrational Analysis of the Individual Components 2.2. ATR Spectroscopy The intensity distribution of the evanescent field is
illustrated as a function of distance from the surface. 2.2. ATR Spectroscopy Therefore, heterogeneities in the emulsion
(e.g., in terms of droplet concentration) will not affect the measurement. However, it must be kept in
mind that this is only true for the spectral regions where water and not the oil or the emulsifier absorb. Consequently, we focus on the OH stretching region in our analysis. 4 of 11
4
f 11 Biosensors 2016, 6, 13 osensors 2016, 6, 13
4 of
osensors 2016, 6, 13
4 of
Figure 1. Schematic of the measurement principle. The infrared (IR) radiation is propagating in the
attenuated total reflection (ATR) crystal. It undergoes total internal reflection at the surface, where
the crystal is in contact with the emulsion sample. The intensity distribution of the evanescent field is
illustrated as a function of distance from the surface. Figure 1. Schematic of the measurement principle. The infrared (IR) radiation is propagating in the
attenuated total reflection (ATR) crystal. It undergoes total internal reflection at the surface, where
the crystal is in contact with the emulsion sample. The intensity distribution of the evanescent field is
illustrated as a function of distance from the surface. osensors 2016, 6, 13
4 o
Figure 1. Schematic of the measurement principle. The infrared (IR) radiation is propagating in the
attenuated total reflection (ATR) crystal. It undergoes total internal reflection at the surface, where
the crystal is in contact with the emulsion sample. The intensity distribution of the evanescent field is
illustrated as a function of distance from the surface. Figure 1. Schematic of the measurement principle. The infrared (IR) radiation is propagating in the
attenuated total reflection (ATR) crystal. It undergoes total internal reflection at the surface, where
the crystal is in contact with the emulsion sample. The intensity distribution of the evanescent field is
illustrated as a function of distance from the surface. Figure 1. Schematic of the measurement principle. The infrared (IR) radiation is propagating in the
attenuated total reflection (ATR) crystal. It undergoes total internal reflection at the surface, where
the crystal is in contact with the emulsion sample. The intensity distribution of the evanescent field is
illustrated as a function of distance from the surface. Figure 1. Schematic of the measurement principle. The infrared (IR) radiation is propagating in the
attenuated total reflection (ATR) crystal. It undergoes total internal reflection at the surface, where
the crystal is in contact with the emulsion sample. 3.1.1. Bilberry Extract Solution
3.1.1. Bilberry Extract Solution
3.1.1. Bilberry Extract Solution The small peak around 2300 cm−1 is an
experimental artifact of the diamond crystal involved in the ATR measurement. F
d t il d
l
i
th
OH b
d
d d i
th
bilb
t
t
d th
l i
Figure 2. IR spectrum of aqueous bilberry extract solution. The small peak around 2300 cm´1 is an
experimental artifact of the diamond crystal involved in the ATR measurement. Figure 2. IR spectrum of aqueous bilberry extract solution. The small peak around 2300 cm−1 is an
experimental artifact of the diamond crystal involved in the ATR measurement. Figure 2. IR spectrum of aqueous bilberry extract solution. The small peak around 2300 cm−1 is an
experimental artifact of the diamond crystal involved in the ATR measurement. Figure 2. IR spectrum of aqueous bilberry extract solution. The small peak around 2300 cm´1 is an
experimental artifact of the diamond crystal involved in the ATR measurement. 5 of 11 Biosensors 2016, 6, 13 Biosensors 2016, 6, 13 For a detailed analysis, the OH bands recorded in the bilberry extract and the emulsions are
de-convolved. The de-convolved spectrum will be presented in Section 3.2, but the results for the
aqueous bilberry extract are discussed in the following. The OH stretching band was fitted by a sum
of six individual Gaussian profiles using a least squares fit algorithm. The fitting parameters were
the line position, the line width, and the line intensity. The number of profiles was fixed to allow
an interpretation in line with Schmidt and Miki [23] and our previous work [10,24,25]. The results
of the fitting procedure are summarized in Table 1. The center wavenumbers slightly differ from
our earlier emulsion study [10], but it must be noted that the present spectra were recorded on a
different instrument. The lines at 3625 and 2950 cm´1 together exhibit only about 5% of the total OH
band intensity and are therefore not considered in the following discussion. The low wavenumber
component at 3117 cm´1 can be assigned to tetra-coordinated, fully hydrogen-bonded water. The
higher wavenumber components indicate water, which is not fully coordinated and hence exhibits
stronger covalent bonds vibrating at higher frequency [3]. The 3230 cm´1 mode can be assigned to
symmetrically hydrogen-bonded water, the 3375 cm´1 mode to asymmetrically hydrogen-bonded
water, and the 3522 cm´1 mode to weakly hydrogen-bonded water. Table 1. Fitting parameters of the aqueous bilberry extract solution spectrum. 3.1.1. Bilberry Extract Solution
3.1.1. Bilberry Extract Solution
3.1.1. Bilberry Extract Solution Center frequency, peak
width, and the percentage intensity with respect to the overall band by peak area are given for the six
individual Gaussian profiles. Table 1. Fitting parameters of the aqueous bilberry extract solution spectrum. Center frequency, peak
width, and the percentage intensity with respect to the overall band by peak area are given for the six
individual Gaussian profiles. Profile
Center Wavenumber in cm´1
Width in cm´1
Intensity in %
peak 1
3625
94
2.24
peak 2
3522
155
12.48
peak 3
3375
188
39.27
peak 4
3230
172
29.07
peak 5
3117
200
14.14
peak 6
2950
200
2.80
1 2 Emulsifier 3.1.2. Emulsifier 3.1.2. Emulsifier The chemical structure of polyglycerin-polyricinoleat (PGPR) is depicted in Figure 3a. It
emphasizes the backbone of polyglycerin and the substituents, R, represent H atoms, or a ricinoleic
acid moiety or a polyricinoleic acid moiety. The structure of ricinoleic acid is shown in Figure 3b. It is an unsaturated omega-9 fatty acid and it is the only naturally occurring fatty acid exhibiting a
hydroxyl group. This hydroxyl group allows the formation of polyricinoleic acid macromolecules via
esterification. Figure 3c shows the 3D sketch of diricinoleic acid as an example. The emulsifier PGPR
is strongly lipophilic and virtually not soluble in water at all. However, as it possesses several ether
groups and a few OH groups with permanent dipoles it can interact with the water phase and stabilize
the droplets by weakening the water-water hydrogen bonds and hence lowering the propensity to
coalesce with other droplets. Figure 4 displays the IR spectrum of PGPR. The most predominant feature is the C=O stretching
mode at 1732 cm´1. Further distinct peaks can be found in the CH stretching region of the spectrum
around 3000 cm´1. Strong peaks are located 2854, 2924, and 2954 (shoulder) cm´1, as well as a
weaker peak at 3011 cm´1. They can be assigned to symmetric and anti-symmetric stretching of
CH2 and CH3 groups. The OH stretching appears as a broad band between 3100 and 3600 cm´1. At the low wavenumber wing, the OH stretching band overlaps with the CH stretching peaks, and
hence care must be taken in the interpretation of this part of the spectrum. In the fingerprint region,
a multitude of mainly CC stretching and CH bending modes is present resulting in a very dense
structure of overlapping lines. A detailed analysis of this part of the spectrum would require the aid of
computational methods and is beyond the scope of the present study. 6 of 11 Biosensors 2016, 6, 13 Figure 3. Chemical structure of the emulsifier polyglycerin-polyricinoleat (PGPR). (a) General
structure where R can be a hydrogen atom or a ricinoleic acid or a polyricinoleic acid substituent;
(b) Structure of ricinoleic acid; (c) 3D model of the structure of diricinoleic acid. Figure 3. Chemical structure of the emulsifier polyglycerin-polyricinoleat (PGPR). (a) General structure
where R can be a hydrogen atom or a ricinoleic acid or a polyricinoleic acid substituent; (b) Structure of
ricinoleic acid; (c) 3D model of the structure of diricinoleic acid. 3.1.2. Emulsifier Biosensors 2016, 6, 13
6 of 11
Figure 3. Chemical structure of the emulsifier polyglycerin-polyricinoleat (PGPR). (a) General
structure where R can be a hydrogen atom or a ricinoleic acid or a polyricinoleic acid substituent; Biosensors 2016, 6, 13
6 of 11 Figure 3. Chemical structure of the emulsifier polyglycerin-polyricinoleat (PGPR). (a) General
structure where R can be a hydrogen atom or a ricinoleic acid or a polyricinoleic acid substituent;
(b) Structure of ricinoleic acid; (c) 3D model of the structure of diricinoleic acid. Figure 3. Chemical structure of the emulsifier polyglycerin-polyricinoleat (PGPR). (a) General structure
where R can be a hydrogen atom or a ricinoleic acid or a polyricinoleic acid substituent; (b) Structure of
ricinoleic acid; (c) 3D model of the structure of diricinoleic acid. Figure 3. Chemical structure of the emulsifier polyglycerin-polyricinoleat (PGPR). (a) General
structure where R can be a hydrogen atom or a ricinoleic acid or a polyricinoleic acid substituent; Figure 3. Chemical structure of the emulsifier polyglycerin-polyricinoleat (PGPR). (a) General
structure where R can be a hydrogen atom or a ricinoleic acid or a polyricinoleic acid substituent;
(b) Structure of ricinoleic acid; (c) 3D model of the structure of diricinoleic acid. Figure 3. Chemical structure of the emulsifier polyglycerin-polyricinoleat (PGPR). (a) General structure
where R can be a hydrogen atom or a ricinoleic acid or a polyricinoleic acid substituent; (b) Structure of
ricinoleic acid; (c) 3D model of the structure of diricinoleic acid. Figure 3. Chemical structure of the emulsifier polyglycerin-polyricinoleat (PGPR). (a) General
structure where R can be a hydrogen atom or a ricinoleic acid or a polyricinoleic acid substituent; Figure 4. IR spectrum of the PGPR emulsifier. The small peak around 2300 cm−1 is an experimental
artifact of the diamond crystal involved in the ATR measurement. 3.1.3. MCT Oil and Oil Phase
The oil used is a medium chain triglyceride i e a triglyceride typically with aliphatic C6 to C12
( )
( )
Figure 4. IR spectrum of the PGPR emulsifier. The small peak around 2300 cm−1 is an experimental
artifact of the diamond crystal involved in the ATR measurement. Figure 4. IR spectrum of the PGPR emulsifier. The small peak around 2300 cm´1 is an experimental
artifact of the diamond crystal involved in the ATR measurement. rum of the PGPR emulsifier. The small peak around 2300 cm−1
mond crystal involved in the ATR measurement. 3.1.3. 3.1.2. Emulsifier MCT Oil and Oil Phase
The oil used is a medium chain triglyceride i e a triglyceride typically with aliphatic C6 to C12
Figure 4. IR spectrum of the PGPR emulsifier. The small peak around 2300 cm−1 is an experimental
artifact of the diamond crystal involved in the ATR measurement. Figure 4. IR spectrum of the PGPR emulsifier. The small peak around 2300 cm´1 is an experimental
artifact of the diamond crystal involved in the ATR measurement. fatty acid chains. Figure 5 sh
3.1.3. MCT Oil and Oil Phase
The oil used is a mediu
3.1.3. MCT Oil and Oil Phase The small peak around 2300 cm´1
is an experimental artifact of the diamond crystal involved in the ATR measurement. compared to PGPR. Again, in the fingerprint region a multitude of mainly CC stretching and CH
bending modes is present resulting in a very dense structure of overlapping lines. Figure 5 IR spectrum of the medium chain triglyceride (MCT) oil The small peak around 2300 cm−1 Figure 5. IR spectrum of the medium chain triglyceride (MCT) oil. The small peak around 2300 cm−1
is an experimental artifact of the diamond crystal involved in the ATR measurement. Figure 5. IR spectrum of the medium chain triglyceride (MCT) oil. The small peak around 2300 cm´1
is an experimental artifact of the diamond crystal involved in the ATR measurement. i
I
f h
d
h
l
d
l
h
ll
k
d ,
p
y
y
g
shows the IR spectra of the mixtures that were used as continuous phase in the emulsions. They
contained 1%, 5%, or 10%wt PGPR. The spectra are very similar as a consequence. The pure oil
spectrum is shown as a reference. Distinct changes with the varied emulsifier concentration can be
seen at a closer look only. For this purpose, a zoom in of the oil C=O peak around 1744 cm−1 is shown
in Figure 6 as well. It becomes clear that the peak absorbance is reduced systematically with
increasing emulsifier content. Moreover, aside from the change in the peak strength, the zoomed
spectrum indicates a small shift as the oil peak at 1744 cm−1 decreases and, at the same time, the
PGPR peak at 1732 cm−1 increases. This results in the appearance of an isosbestic point at 1735 cm−1,
see inset in Figure 6. As aforesaid, the PGPR and oil spectra are very similar due to the structural similarity. Figure 6
shows the IR spectra of the mixtures that were used as continuous phase in the emulsions. They
contained 1%, 5%, or 10%wt PGPR. The spectra are very similar as a consequence. The pure oil
spectrum is shown as a reference. Distinct changes with the varied emulsifier concentration can be
seen at a closer look only. For this purpose, a zoom in of the oil C=O peak around 1744 cm´1 is
shown in Figure 6 as well. It becomes clear that the peak absorbance is reduced systematically with
increasing emulsifier content. fatty acid chains. Figure 5 sh
3.1.3. MCT Oil and Oil Phase
The oil used is a mediu
3.1.3. MCT Oil and Oil Phase Moreover, aside from the change in the peak strength, the zoomed
spectrum indicates a small shift as the oil peak at 1744 cm´1 decreases and, at the same time, the PGPR
peak at 1732 cm´1 increases. This results in the appearance of an isosbestic point at 1735 cm´1, see
inset in Figure 6. is an experimental artifact of the diamond crystal involved in the ATR measurement. As aforesaid, the PGPR and oil spectra are very similar due to the structural similarity. Figure 6
shows the IR spectra of the mixtures that were used as continuous phase in the emulsions. They
contained 1%, 5%, or 10%wt PGPR. The spectra are very similar as a consequence. The pure oil
spectrum is shown as a reference. Distinct changes with the varied emulsifier concentration can be
seen at a closer look only. For this purpose, a zoom in of the oil C=O peak around 1744 cm−1 is shown
in Figure 6 as well. It becomes clear that the peak absorbance is reduced systematically with
increasing emulsifier content. Moreover, aside from the change in the peak strength, the zoomed
spectrum indicates a small shift as the oil peak at 1744 cm−1 decreases and, at the same time, the
PGPR peak at 1732 cm−1 increases. This results in the appearance of an isosbestic point at 1735 cm−1,
see inset in Figure 6. Figure 6. IR spectra of the oil phases containing different amounts of emulsifier. The percentages in
the legend are wt % PGPR. The zoomed in region shows the C=O stretching peak. The small peak
around 2300 cm−1 is an experimental artifact of the diamond crystal involved in the ATR
measurement. The zoomed in region in the circle shows the isosbestic point at 1735 cm−1. Figure 6. IR spectra of the oil phases containing different amounts of emulsifier. The percentages in
the legend are wt % PGPR. The zoomed in region shows the C=O stretching peak. The small peak
around 2300 cm−1 is an experimental artifact of the diamond crystal involved in the ATR
measurement. The zoomed in region in the circle shows the isosbestic point at 1735 cm−1. Figure 6. IR spectra of the oil phases containing different amounts of emulsifier. The percentages in the
legend are wt % PGPR. The zoomed in region shows the C=O stretching peak. fatty acid chains. Figure 5 sh
3.1.3. MCT Oil and Oil Phase
The oil used is a mediu
3.1.3. MCT Oil and Oil Phase Further distinct peaks can be found in the CH stretching region at 2854, 2923,
and 2956 (shoulder) cm−1 as well as a weaker peak at 3007 cm−1 Their positions are almost identical C=O stretching mode, but it is located at 1744 cm´1. In other words, it is blue-shifted by 12 cm´1 with
respect to the band at 1732 cm´1 in PGPR. This indicates that the C=O bond in the oils is stronger than
in the emulsifier. Further distinct peaks can be found in the CH stretching region at 2854, 2923, and
2956 (shoulder) cm´1, as well as a weaker peak at 3007 cm´1. Their positions are almost identical
compared to PGPR. Again, in the fingerprint region a multitude of mainly CC stretching and CH
bending modes is present resulting in a very dense structure of overlapping lines. to the emulsifier spectrum discussed in preceding section. The most predominant feature is again
the C=O stretching mode, but it is located at 1744 cm−1. In other words, it is blue-shifted by 12 cm−1
with respect to the band at 1732 cm−1 in PGPR. This indicates that the C=O bond in the oils is stronger
than in the emulsifier. Further distinct peaks can be found in the CH stretching region at 2854, 2923,
and 2956 (shoulder) cm−1, as well as a weaker peak at 3007 cm−1. Their positions are almost identical
compared to PGPR. Again, in the fingerprint region a multitude of mainly CC stretching and CH
bending modes is present resulting in a very dense structure of overlapping lines. Biosensors 2016, 6, 13
7 of 11
to the emulsifier spectrum discussed in preceding section. The most predominant feature is again
the C=O stretching mode, but it is located at 1744 cm−1. In other words, it is blue-shifted by 12 cm−1
with respect to the band at 1732 cm−1 in PGPR. This indicates that the C=O bond in the oils is stronger
than in the emulsifier. Further distinct peaks can be found in the CH stretching region at 2854, 2923,
and 2956 (shoulder) cm−1 as well as a weaker peak at 3007 cm−1 Their positions are almost identical Figure 5. IR spectrum of the medium chain triglyceride (MCT) oil. The small peak around 2300 cm−1
is an experimental artifact of the diamond crystal involved in the ATR measurement. Figure 5. IR spectrum of the medium chain triglyceride (MCT) oil. fatty acid chains. Figure 5 sh
3.1.3. MCT Oil and Oil Phase
The oil used is a mediu
3.1.3. MCT Oil and Oil Phase g y
,
,
g y
yp
y
p
fatty acid chains. Figure 5 shows the corresponding IR spectrum. The overall appearance is similar
The oil used is a medium chain triglyceride, i.e., a triglyceride typically with aliphatic C6 to C12
fatty acid chains. Figure 5 shows the corresponding IR spectrum. The overall appearance is similar to
the emulsifier spectrum discussed in preceding section. The most predominant feature is again the 7 of 11
11 Biosensors 2016, 6, 13 Biosensors 2016, 6, 13 C=O stretching mode, but it is located at 1744 cm´1. In other words, it is blue-shifted by 12 cm´1 with
respect to the band at 1732 cm´1 in PGPR. This indicates that the C=O bond in the oils is stronger than
in the emulsifier. Further distinct peaks can be found in the CH stretching region at 2854, 2923, and
2956 (shoulder) cm´1, as well as a weaker peak at 3007 cm´1. Their positions are almost identical
compared to PGPR. Again, in the fingerprint region a multitude of mainly CC stretching and CH
bending modes is present resulting in a very dense structure of overlapping lines. to the emulsifier spectrum discussed in preceding section. The most predominant feature is again
the C=O stretching mode, but it is located at 1744 cm−1. In other words, it is blue-shifted by 12 cm−1
with respect to the band at 1732 cm−1 in PGPR. This indicates that the C=O bond in the oils is stronger
than in the emulsifier. Further distinct peaks can be found in the CH stretching region at 2854, 2923,
and 2956 (shoulder) cm−1, as well as a weaker peak at 3007 cm−1. Their positions are almost identical
compared to PGPR. Again, in the fingerprint region a multitude of mainly CC stretching and CH
bending modes is present resulting in a very dense structure of overlapping lines. Biosensors 2016, 6, 13
7 of 11
to the emulsifier spectrum discussed in preceding section. The most predominant feature is again
the C=O stretching mode, but it is located at 1744 cm−1. In other words, it is blue-shifted by 12 cm−1
with respect to the band at 1732 cm−1 in PGPR. This indicates that the C=O bond in the oils is stronger
than in the emulsifier. 3.2. Spectroscopy of Emulsions
3.2. Spectroscopy of Emulsions The de-convolved OH bands of the aqueous bilberry extract solution and the three emulsions
with varied emulsifier content are displayed in Figure 7. As described in Section 3.1.1, six individual
Gaussian profiles have been used to fit the band. In each panel of Figure 7, the thick red line shows
the experimental data, the thin brown line represents the sum of the Gaussian profiles, which are
displayed in various colors individually. In order to get an overview the Tables 2–4 compare the center
frequencies, the peak widths, and the percentage contribution of the individual Gaussian profiles. The de-convolved OH bands of the aqueous bilberry extract solution and the three emulsions
with varied emulsifier content are displayed in Figure 7. As described in Section 3.1.1, six individual
Gaussian profiles have been used to fit the band. In each panel of Figure 7, the thick red line shows
the experimental data, the thin brown line represents the sum of the Gaussian profiles, which are
displayed in various colors individually. In order to get an overview the Tables 2–4 compare the
center frequencies, the peak widths, and the percentage contribution of the individual Gaussian profiles. Figure 7. IR spectra of the emulsions in the OH stretching region and the fitted Gaussian profiles. (a) Aqueous bilberry extract solution; (b) Emulsion with 1% PGPR; (c) Emulsion with 5% PGPR;
(d) Emulsion with 10% PGPR. Figure 7. IR spectra of the emulsions in the OH stretching region and the fitted Gaussian profiles. (a) Aqueous bilberry extract solution; (b) Emulsion with 1% PGPR; (c) Emulsion with 5% PGPR;
(d) Emulsion with 10% PGPR. Figure 7. IR spectra of the emulsions in the OH stretching region and the fitted Gaussian profiles. (a) Aqueous bilberry extract solution; (b) Emulsion with 1% PGPR; (c) Emulsion with 5% PGPR;
(d) Emulsion with 10% PGPR. Figure 7. IR spectra of the emulsions in the OH stretching region and the fitted Gaussian profiles. (a) Aqueous bilberry extract solution; (b) Emulsion with 1% PGPR; (c) Emulsion with 5% PGPR;
(d) Emulsion with 10% PGPR. Figure 7. IR spectra of the emulsions in the OH stretching region and the fitted Gaussian profiles. (a) Aqueous bilberry extract solution; (b) Emulsion with 1% PGPR; (c) Emulsion with 5% PGPR;
(d) Emulsion with 10% PGPR. Figure 7. IR spectra of the emulsions in the OH stretching region and the fitted Gaussian profiles. fatty acid chains. Figure 5 sh
3.1.3. MCT Oil and Oil Phase
The oil used is a mediu
3.1.3. MCT Oil and Oil Phase The small peak around
2300 cm´1 is an experimental artifact of the diamond crystal involved in the ATR measurement. The
zoomed in region in the circle shows the isosbestic point at 1735 cm´1. a of the oil phases containing different amounts of emulsifier. % PGPR. The zoomed in region shows the C=O stretching pe
i
i
t l
tif
t
f th
di
d
t l i around 2300 cm is an experimental artifact of the diamond crystal involved in the ATR
measurement. The zoomed in region in the circle shows the isosbestic point at 1735 cm−1. Figure 6. IR spectra of the oil phases containing different amounts of emulsifier. The percentages in
the legend are wt % PGPR. The zoomed in region shows the C=O stretching peak. The small peak
around 2300 cm−1 is an experimental artifact of the diamond crystal involved in the ATR
measurement. The zoomed in region in the circle shows the isosbestic point at 1735 cm−1. Figure 6. IR spectra of the oil phases containing different amounts of emulsifier. The percentages in the
legend are wt % PGPR. The zoomed in region shows the C=O stretching peak. The small peak around
2300 cm´1 is an experimental artifact of the diamond crystal involved in the ATR measurement. The
zoomed in region in the circle shows the isosbestic point at 1735 cm´1. 8 of 11
8 of 11 Biosensors 2016, 6, 13
Bi
2016 6 13 3.2. Spectroscopy of Emulsions
3.2. Spectroscopy of Emulsions (a) Aqueous bilberry extract solution; (b) Emulsion with 1% PGPR; (c) Emulsion with 5% PGPR;
(d) Emulsion with 10% PGPR. Table 2. Center frequencies in cm−1 of the six individual Gaussian profiles obtained from fitting the
infrared OH bands of the aqueous solution and the three emulsions. Table 2. Center frequencies in cm´1 of the six individual Gaussian profiles obtained from fitting the
infrared OH bands of the aqueous solution and the three emulsions. Table 2. Center frequencies in cm−1 of the six individual Gaussian profiles obtained from fitting the
infrared OH bands of the aqueous solution and the three emulsions. Table 2. Center frequencies in cm´1 of the six individual Gaussian profiles obtained from fitting the
infrared OH bands of the aqueous solution and the three emulsions. Profile
Aqueous
Solution
Emulsion with
1% PGPR
Emulsion with
5% PGPR
Emulsion with
10% PGPR
peak 1
3625
3625
3625
3624
peak 2
3522
3544
3544
3542
peak 3
3375
3416
3416
3414
peak 4
3230
3291
3290
3290
peak 5
3117
3198
3198
3198
peak 6
2950
3025
3025
3025
Profile
Aqueous
Solution
Emulsion with 1%
PGPR
Emulsion with 5%
PGPR
Emulsion with
10% PGPR
peak 1
3625
3625
3625
3624
peak 2
3522
3544
3544
3542
peak 3
3375
3416
3416
3414
peak 4
3230
3291
3290
3290
peak 5
3117
3198
3198
3198
peak 6
2950
3025
3025
3025 9 of 11 Biosensors 2016, 6, 13 Table 3. Peak widths in cm´1 of the six individual Gaussian profiles obtained from fitting the infrared
OH bands of the aqueous solution and the three emulsions. Profile
Aqueous
Solution
Emulsion with 1%
PGPR
Emulsion with 5%
PGPR
Emulsion with
10% PGPR
peak 1
94
94
94
94
peak 2
155
155
155
155
peak 3
188
188
188
188
peak 4
172
172
172
172
peak 5
200
195
195
195
peak 6
200
160
160
160 Table 4. Percentage intensity with respect to the overall band by peak area of the six individual Gaussian
profiles obtained from fitting the infrared OH bands of the aqueous solution and the three emulsions. 3.2. Spectroscopy of Emulsions
3.2. Spectroscopy of Emulsions Profile
Aqueous
Solution
Emulsion with 1%
PGPR
Emulsion with 5%
PGPR
Emulsion with
10% PGPR
peak 1
2.24
2.68
2.64
2.61
peak 2
12.48
13.23
12.61
12.76
peak 3
39.27
41.34
42.61
41.90
peak 4
29.07
18.06
17.80
17.41
peak 5
14.14
20.14
19.85
20.00
peak 6
2.80
4.55
4.49
5.32 In the emulsions, the OH band and its contributions represented by the Gaussian profiles change
significantly, which is in agreement with our previous study [10]. Aside from the peak at 3625 cm´1,
which exhibits very low intensity in all cases, the peaks are blue-shifted in the emulsions with respect
to the aqueous solution, see Table 2. This means that the corresponding covalent OH bonds are
strengthened, which in turn indicates that the entire hydrogen-bonding network in the probed water
layer at the interface is weakened by the interaction with emulsifier molecules. The peak widths
in Table 3 reveal no or negligible differences between the aqueous solution and the emulsions. The
same is true in terms of the percentage contribution to the overall band for most of the peaks, see
Table 4. Only Peaks 4 and 5, corresponding to the profiles centered at 3230 and 3117 cm´1 respectively
in the aqueous solution show a significant alteration. The peak at 3230 cm´1, which was assigned
to symmetrically hydrogen-bonded water, is reduced in the emulsion by approximately one third
with respect the aqueous system. On the other hand, the 3117 cm´1 peak, which was assigned to
tetra-coordinated water, gains about 50% at the same time. This indicates that the fraction of fully
hydrogen-bonded water molecules increases in the emulsion. However, it must be kept in mind that
the frequency of the peak is blue-shifted with respect to the aqueous solution case. Hence, the data
indicate that the network of these fully hydrogen-bonded molecules is significantly weaker than in
bulk water. An interesting finding arises from the data in the Tables 2–4 as the changes of the spectroscopic
parameters with the variation of the emulsifier concentration can be considered negligible. This
indicates that the molecular stabilization at the w/o interface is rather independent of the emulsifier
concentration. In other words, adding emulsifier in excess of 1% seems to not lead to an improved
emulsion stability. 3.2. Spectroscopy of Emulsions
3.2. Spectroscopy of Emulsions From an engineering and an economic point of few, this finding is rather important
as it indicates that a low amount of PGPR is sufficient to stabilize the w/o emulsion. References 1. Kiefer, J.; Noack, K.; Bartelmess, J.; Walter, C.; Dörnenburg, H.; Leipertz, A. Vibrational structure of the
polyunsaturated fatty acids eicosapentaenoic acid and arachidonic acid studied by infrared spectroscopy. J. Mol. Struct. 2010, 965, 121–124. [CrossRef] 1. Kiefer, J.; Noack, K.; Bartelmess, J.; Walter, C.; Dörnenburg, H.; Leipertz, A. Vibrational structure of the
polyunsaturated fatty acids eicosapentaenoic acid and arachidonic acid studied by infrared spectroscopy. J. Mol. Struct. 2010, 965, 121–124. [CrossRef] 2. Noack, K.; Leipertz, A.; Kiefer, J. Molecular interactions and macroscopic effects in binary mixtures of an
imidazolium ionic liquid with water, methanol, and ethanol. J. Mol. Struct. 2012, 1018, 45–53. [CrossRef]
3. Joseph, J.; Jemmis, E.D. Red-, blue-, or no-shift in hydrogen bonds: A unified explanation. J. Am. Chem. Soc. 2007, 129, 4620–4632. [CrossRef] [PubMed] 2. Noack, K.; Leipertz, A.; Kiefer, J. Molecular interactions and macroscopic effects in binary mixtures of an
imidazolium ionic liquid with water, methanol, and ethanol. J. Mol. Struct. 2012, 1018, 45–53. [CrossRef] 2. Noack, K.; Leipertz, A.; Kiefer, J. Molecular interactions and macroscopic effects in binary mixtures of an
imidazolium ionic liquid with water, methanol, and ethanol. J. Mol. Struct. 2012, 1018, 45–53. [CrossRef]
3. Joseph, J.; Jemmis, E.D. Red-, blue-, or no-shift in hydrogen bonds: A unified explanation. J. Am. Chem. Soc. 2007 129 4620 4632 [C
R f] [P bM d] imidazolium ionic liquid with water, methanol, and ethanol. J. Mol. Struct. 2012, 1018, 45 53. [CrossRef]
3. Joseph, J.; Jemmis, E.D. Red-, blue-, or no-shift in hydrogen bonds: A unified explanation. J. Am. Chem. Soc. 2007, 129, 4620–4632. [CrossRef] [PubMed] . Jorgensen, L.; Van de Weert, M.; Vermehren, C.; Bjerregaard, S.; Frokjaer, S. Probing structural change
proteins incorprated into water-in-oil emulsions. J. Pharm. Sci. 2004, 93, 1847–1859. [CrossRef] [PubMe . Zhou, G.W.; Li, G.Z.; Chen, W.J. Fourier-transform infrared investigation on water states and
conformations of Aerosol-OT in reverse microemulsions. Langmuir 2002, 18, 4566–4571. [CrossRef] conformations of Aerosol OT in reverse microemulsions. Langmuir 2002, 18, 4566 4571. [CrossRef]
6. Valero, M.;
Sanchez, F.;
Gomez-Herrera, C.;
Lopez-Cornejo, P. Study of water solubilized in
AOT/n-decane/water microemulsions. Chem. Phys. 2008, 345, 65–72. [CrossRef] 7. Sechler, T.D.; DelSole, E.M.; Deak, J.C. Measuring properties of interfacial and bulk water regions in a reverse
micelle with IR spectroscopy: A volumetric analysis of the inhomogeneously broadened OH band. J. Colloid
Interface Sci. 2010, 346, 391–397. [CrossRef] [PubMed] 8. Kemsley, E.K.; Appleton, G.P.; Wilson, R.H. Abbreviations The following abbreviations are used in this manuscript: The following abbreviations are used in this manuscript: PGPR:
PolyGlycerine-PolyRicinoleat
MCT:
Medium Chain Triglyceride
IR:
InfraRed
FTIR:
Fourier-Transform InfraRed
ATR:
Attenuated Total Reflection PGPR:
PolyGlycerine-PolyRicinoleat
MCT:
Medium Chain Triglyceride
IR:
InfraRed
FTIR:
Fourier-Transform InfraRed
ATR:
Attenuated Total Reflection 4. Conclusions In the present work, the vibrational spectra of an aqueous bilberry extract solution, a
polyglycerine-polyricinoleat emulsifier and a medium chain triglyceride oil were studied by IR
spectroscopy. Based on the vibrational analysis, the IR spectra of a series of water-in-oil emulsions 10 of 11 Biosensors 2016, 6, 13 comprising of the above components were studied. The emulsifier concentration was varied
systematically. The OH stretching vibration band was utilized as a sensor for the molecular interactions
at the water-oil interface leading to a stabilization of the emulsion. A detailed analysis of the OH band
revealed that changing the emulsifier content has negligible effects on the stabilization of the emulsion. This is at least true for the concentration range studied, i.e., 1%–10%wt. However, in all emulsion
spectra, the individual contributions to the OH stretching band are significantly blue-shifted. In other
words, the hydrogen-bonding network in the water phase is weakened as a result of the presence of
the emulsifier. Overall, we can conclude that ATR-IR spectroscopy is a useful tool for studying w/o emulsions. In particular, the OH stretching region of water in the spectrum represents a highly sensitive means of
sensing changes in the molecular environment. Acknowledgments: This research project was supported by the FEI (Forschungskreis der Ernährungsindustrie e.V.,
Bonn), the AiF and the Ministry of Economics and Technology, AiF-Project No. 15612 N. Furthermore, we would
like to thank Kaden Biochemical GmbH, Germany, for supplying us with bilberry extract, Palsgaard®Denmark
for supplying us with PGPR 4150. Author Contributions: Johannes Kiefer and Heike P. Schuchmann conceived and designed the experiments;
Kerstin Frank and Florian M. Zehentbauer performed the experiments; Johannes Kiefer, Florian M. Zehentbauer,
and Kerstin Frank analyzed the data; all authors contributed to the interpretation of the data; all authors
contributed to the preparation of the manuscript with Johannes Kiefer putting together the first draft. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References Quantitative analysis of emulsions using attenuated total
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during the synthesis of oxalate precursor nanoparticles. Colloids Surf. A 2004, 232, 93–99. [CrossRef] 9. Nickolov, Z.S.; Paruchuri, V.; Shah, D.O.; Miller, J.D. FTIR-ATR studies of water structure in reverse micelles
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water-in-oil emulsion. Appl. Spectrosc. 2011, 65, 1024–1028. [CrossRef] [PubMed] 11. Newsome, A.G.; Culver, C.A.; van Breemen, R.B. Nature’s Palette: The Search for Natural Blue Colorants. J. Agric. Food Chem. 2014, 62, 6498–6511. [CrossRef] [PubMed] 12. Tsuda, T. Anthocyanins as Functional Food Factors—Chemistry, Nutrition and Health Promotion. Food Sci. Technol. Res. 2012, 18, 315–324. [CrossRef] 13. Prior, R.L.; Cao, G.H.; Martin, A.; Sofic, E.; McEwen, J.; O'Brien, C.; Lischner, N.; Ehlenfeldt, M.; Kalt, W.;
Krewer, G.; et al. Antioxidant capacity as influenced by total phenolic and anthocyanin content, maturity,
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in commercial black currant juices by simple high-performance liquid chromatography. Investigation of
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parameters and food additives on the color and stability of isolated anthocyanins in food. PhD Dissertation,
Christian-Albrecht-Universität zu Kiel, Kiel, Germany, 2005. 18. Frank, K.; Köhler, K.; Schuchmann, H.P. Formulation of labile hydrophilic ingredients in multiple emulsions:
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bioactives. J. Dispers. Sci. Technol. 2011, 32, 1–6. [CrossRef] 19. References Baum, M.; Schantz, M.; Leick, S.; Berg, S.; Betz, M.; Frank, K.; Rehage, H.; Schwarz, K.; Kulozik, U.;
Schuchmann, H.P.; et al. Is the antioxidative effectiveness of a bilberry extract influenced by encapsulation? J. Sci. Food Agric. 2014, 94, 2301–2307. [CrossRef] [PubMed] 20. Ax, K. Emulsionen und Liposomen als Trägersysteme für Carotinoide. PhD Dissertation, Uni
Karlsruhe, Karlsruhe, Germany, 2004. 21. Ribeiro, H.S.; Schuchmann, H.P.; Engel, R.; Briviba, K.; Walz, E. Encapsulation of Caroteniods and Vitamins. In Encapsulation Technologies for Food Active Ingredients and Food Processing; Zuidam, N.J., Nedovic, V.A., Eds.;
Springer: Heidelberg, Germany, 2009. 22. Palzer, S. Food structures for nutrition, health and wellness. Trends Food Sci. Technol. 2009, 20, 194–200. [CrossRef] 23. Schmidt, D.A.; Miki, K. Structural correlations in liquid water: A new interpretation of IR spectroscopy. J. Phys. Chem. A 2007, 111, 10119–10122. [CrossRef] [PubMed] 24. Wallace, V.M.; Dhumal, N.R.; Zehentbauer, F.M.; Kim, H.J.; Kiefer, J. Revisiting the Aqueous Solutions
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article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
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Introduzione
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Mauro Lombardi, University of Florence, Italy, mauro.lombardi@unifi.it, 0000-0002-3234-7039
FUP Best Practice in Scholarly Publishing (DOI 10.36253/fup_best_practice)
Mauro Lombardi, Introduzione, pp. 9-13, © 2021 Author(s), CC BY 4.0 International, DOI 10.36253/978-88-
5518-310-9.02, in Mauro Lombardi, Transizione ecologica e universo fisico-cibernetico. Soggetti, strategie,
lavoro, © 2021 Author(s), content CC BY 4.0 International, metadata CC0 1.0 Universal, published by
Firenze University Press (www.fupress.com), ISSN 2704-5919 (online), ISBN 978-88-5518-310-9 (PDF),
DOI 10.36253/978-88-5518-310-9 Introduzione Stiamo vivendo in un’epoca molto particolare della storia dell’Umanità, che
ha creato un potenziale tecnico-scientifico senza pari, grazie al quale è possibi-
le elaborare la rappresentazione digitale dei processi dalla nano-scala alla scala
globale. Il Pianeta è iperconnesso da dispositivi che elaborano flussi informati-
vi generati da entità individuali e collettive, da esseri viventi e non. Individui e
collettività possono esprimere emozioni e argomentazioni in una sorta di ‘Ba-
bele organizzata’ e senza limiti, che nella vulgata è il web. I confini tra settori
produttivi e attività economico-sociali sono diventati permeabili e confusi, nel
senso che per ogni attività e output (materiali e immateriali) è sempre più spes-
so necessario esplorare molteplici domini conoscitivi, quindi essere inseriti in
strutture interattive, tendenzialmente multi-scala. In uno scenario di questo tipo
l’innovazione tecnico-scientifica mostra un’accelerazione, che autorevoli studiosi
stimano esponenziale, quindi capace di innescare mutamenti generalizzati nei
modelli mentali, nei comportamenti strategici ed operativi, nelle competenze,
nei sistemi di credenze e di valori su cui si fondano le società. A tutto questo bi-
sogna poi aggiungere che flussi globali di informazioni e persone cambiano le
relazioni tra le culture e gli assetti geo-politici con esiti imprevedibili. gt
g
p
p
L’insieme dei fenomeni e dei processi sinteticamente indicati delineano, per-
tanto, un nuovo scenario, contraddistinto da alcune peculiarità: mutamenti ge-
neralizzati e profondi, crisi di interi assetti tecno-economici, emergere di nuovi
oligopoli globali (chiamati moligopoli al capitolo 3, par. 3), spinte irresistibili a
cambiare la visioni individuali e collettive. TRANSIZIONE ECOLOGICA E UNIVERSO FISICO-CIBERNETICO Su queste basi non deve sorprendere che Centri di Ricerca internazionali e la
maggioranza degli studiosi di molte discipline mettano al centro della riflessione
due aspetti cruciali: 1) i mutamenti in atto nei sotto-sistemi, che compongono il
sistema globale interconnesso; 2) le molte difficoltà con cui si devono misurare
i processi decisionali a qualsiasi livello e in ogni ambito. i p
q
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Ciò che sta accadendo, inoltre, pone l’intera Umanità di fronte a sfide epocali:
1) l’evento pandemico mette in discussione le strutture sanitarie praticamente
in quasi tutti i Paesi del mondo. 2) È concreto il rischio che la contrazione eco-
nomico-produttiva, conseguente al lockdown dei primi mesi del 2020 e a quello
in atto all’inizio del 2021, si trasformi in un periodo recessivo del quale non è
possibile stimare durata e intensità. Introduzione Da ciò consegue il diffondersi di incertezze
e ansietà, individuale e collettiva, per i timori di perdere il posto di lavoro e le
aspettative di una diminuzione del proprio reddito. 3) La dinamica tecno-eco-
nomica è sempre più spinta verso l’aumento della potenza computazionale e l’e-
laborazione di sistemi di software (Intelligenza Artificiale e Machine Learning,
algoritmi di editing genomico, modelli di simulazione ingegneristica e sociale). I risultati sono non di rado imprevedibili sul piano delle attività socio-economi-
che, tecnico-scientifiche e sanitarie. 4) Si impone la necessità di cambiare stili
di vita, modelli di produzione e consumo, meccanismi e modalità di relazioni
sociali ed economiche, circuiti di feedback che aumentano incertezza e ansietà,
mentre i processi economico-produttivi divengono sempre meno comprensibili,
alla luce dei numerosi fattori causali che interagiscono nel determinare dinami-
che fuori controllo. 5) La numerosità di fattori e processi che interagiscono sia
a livello di nano-scala che sul piano globale incrementa il grado di complessità
dell’evoluzione socio-economica, a cui si aggiunge – come si illustrerà nel capi-
tolo 1 – la stretta compenetrazione tra le innumerevoli componenti sistemiche
del ‘Sistema-Terra’. Dalle interconnessioni globali e dalla complessità evolutiva scaturiscono sfi-
de epocali per l’Umanità e l’intero Pianeta: effetti dell’evento pandemico ancora
fuori controllo; sfruttamento eccessivo delle risorse naturali non riproducibili,
tra cui le cosiddette ‘terre rare’, essenziali per l’evoluzione della potenza compu-
tazionale e dell’Intelligenza Artificiale (d’ora in poi IA); variazioni climatiche
indotte da una serie di comportamenti alla lunga distruttivi (crescita di grandi
insediamenti urbani, deforestazione, colture intensive), inquinamento marino
(plastiche, sostanze chimiche, luce artificiale urbana che altera i ritmi circadia-
ni di specie ittiche, National Geographic 2020); alterazione dell’Oceano e della
criosfera a causa dei cambiamenti climatici e delle emissioni di CO2 in continuo
aumento (NOAA 2021). Le implicazioni sull’evoluzione del Pianeta Terra, da un lato, e dall’altro le
trasformazioni radicali e velocemente diffuse a livello globale sono sempre più
al centro delle analisi dei maggiori centri di ricerca internazionali. È infatti quasi
unanime la consapevolezza che l’evento pandemico sia una prova ulteriore che
l’intera Umanità e il Pianeta Terra siano vicini ad un tipping point, ovvero che
stiano vivendo una critical transition, caratterizzata dalla presenza di almeno
quattro crisi congiunte: climatica, energetica, pandemica, economico-produttiva 10 INTRODUZIONE (vedi Cap. 3). Esse sono il risultato dell’evoluzione verso un mondo iperconnesso
(Ubiquitous computing, Ubiquitous connectivity) e caratterizzato dalla creazione
di strutture interattive globali. Introduzione Tutto ciò ha determinato processi di feedback
positivi, con un incremento della complessità sistemica a livello locale e globa-
le e il diffondersi di livelli crescenti di incertezza e di potenziale instabilità. In
tale quadro i processi decisionali di imprese e individui – oltre che di organiz-
zazioni e apparati istituzionali – devono misurarsi con sfide epocali e quindi
occorre delineare nuovi strumenti di analisi e metodologie operative, al fine di
orientarsi in uno scenario di evoluzione complessa e multidimensionale, acce-
lerata dall’incremento della potenza computazionale disponibile per chiunque. In uno scenario globale investito da fenomeni e processi di tale portata è lo-
gico che manchino coordinate generali e criteri applicativi per nuove regole di
azione. È d’altra parte evidente come le sfide e i rischi siano così rilevanti da dover
indurre a ripensare sistemi di pensiero consolidati e schemi operativi, allo scopo
di agire in modo da evitare esiti distruttivi. L’alternativa all’inerzia e al deleterio
arroccamento nel modo di pensare standard, non più appropriato, è effettuare
uno sforzo serio e sistematico di riflessione, che parta dalla ricerca di strumenti
e metodi che aiutino a comprendere i mutamenti in atto. Ciò può avvenire se,
grazie ad un open mindset, si cerca di elaborare e adottare schemi teorici, sulla ba-
se dei quali analizzare e descrivere in modo pregnante intuizioni razionali circa
nuove tendenze tecno-economiche. Lo scopo deve essere quello di intravedere,
data l’impossibilità di prevederli, trend morfologici e strutturali emergenti nelle
interconnessioni che si sviluppano nel Sistema-Terra, al fine di progettare inter-
venti correttivi ed evitare eventi catastrofici o gravemente distruttivi. ti
gt
Alla luce delle considerazioni iniziali, tre sono i punti fondamentali da assu-
mere per orientare l’analisi e la riflessione: 1) bisogna assegnare centralità alla
conoscenza dei sistemi complessi, in merito ai quali è doveroso creare e diffon-
dere cultura tecnico-scientifica, manageriale ed etico-filosofica, dal momento
che la posta in gioco appare sempre più il futuro dell’Umanità e degli esseri vi-
venti, insieme a quello della Terra. 2) La prospettiva sistemica richiede un pro-
fondo cambiamento dell’orizzonte temporale del quadro teorico e dei processi
decisionali: è cruciale, infatti, la capacità di guardare al medio-lungo periodo,
essenziale per prendere decisioni per disegni operativi nel breve coerenti con le
visioni di lungo termine. 3) La natura sistemica globale e l’orizzonte temporale
prolungato portano con sé il problema di come coordinare micro- e macro-com-
portamenti (individuali e collettivi). Introduzione Ciò significa che occorrono lo sviluppo e
la diffusione di una mentalità strategica, che sia in grado di realizzare processi
e tendenze a varia scala in direzioni non deleterie. tfi Si tratta naturalmente di questioni di elevata difficoltà sia sul piano teorico
che pratico, con implicazioni multidimensionali: tecnico-scientifiche, economi-
che, sociali, politiche, istituzionali, geo-politiche. La piena consapevolezza della
posta in gioco può essere un ottimo punto di partenza per cercare di elaborare
strumenti e meccanismi atti a sintonizzare micro e macro-decisioni con traiet-
torie evolutive meno dannose per tutti. Si tratta poi di pensare a dispositivi di
varia natura, che consentano di verificare puntualmente se la progettazione in- 11 TRANSIZIONE ECOLOGICA E UNIVERSO FISICO-CIBERNETICO novativa assecondi o meno direttrici verso una dinamica sostenibile sul piano
economico e ambientale. L’ambito in cui si muove la nostra analisi è prevalentemente economico, più
precisamente quello delle attività umane finalizzate alla soddisfazione di bisogni,
che nell’orizzonte odierno sembrano assumere sempre più natura immateriale,
anche se alcune parti del mondo devono ancora fare i conti con la preponderanza
di necessità materiali largamente insoddisfatte. Questo libro tenta di risponde-
re alle esigenze indicate seguendo un preciso itinerario analitico e progettuale,
proponendo al contempo strumenti teorici ed operativi. t p
p
p
p
Il lavoro è pertanto così articolato. Il Capitolo 1 introduce il concetto di
universo fisico-cibernetico e le sfide per l’umanità che da esso scaturiscono. Il
Capitolo 2 si concentra sull’analisi di alcune peculiarità dell’era odierna: com-
plessità, incertezza e ansietà, conseguenti alla Grande Accelerazione che segna
il passaggio dall’Olocene all’Antropocene, era in cui la ‘forza tellurica’ delle at-
tività umane sembra più potente delle forze geofisiche ed astronomiche. Queste
ultime per secoli hanno influenzato l’evoluzione sulla Terra, mentre oggi gli ef-
fetti dei comportamenti umani producono effetti globali apparentemente privi di
controllo. Come agire per riprenderne il controllo? Bisogna imparare molto dalla
Natura e dalla sua evoluzione, dalla quale trarre una serie di lezioni essenziali:
incessante capacità di apprendimento, adattatività, continua sperimentazione.t p
ppt
p
Nel Capitolo 3 la trattazione approfondisce quella che molte discipline de-
finiscono ‘transizione critica’ nell’evoluzione del Sistema-Terra e della storia
umana, in quanto emergono segnali di uno scenario caratterizzato da una crisi
sistemica globale. Viene argomentato come tale crisi si manifesti in un mondo
divenuto ‘universo fisico-digitale’. Introduzione Una fase di transizione critica richiede il ri-
pensamento complessivo della società in cui viviamo, per cui centri di ricerca
e società di consulenza internazionali tematizzano modelli di riorganizzazione
generale di imprese e attività, con l’introduzione di varie definizioni sintetiche:
«The Great Reset» (World Economic Forum); «dal capitalismo degli sharehol-
ders al capitalismo degli stakeholders» (Boston Consulting Group); «long term
capitalism» (McKinsey Global Institute). t L’apparato teorico e la concettualizzazione operativa consentono nel Capito-
lo 4 di avviare un itinerario strategico-cognitivo, che inizia con l’analisi delle sfide
globali dell’era odierna, tra cui i cambiamenti dei processi economico-produttivi
e i mutamenti tendenziali nel mondo del lavoro, delle competenze, delle strategie
appropriate per creare occupazione, misurandosi in primis la questione cruciale
dei prossimi decenni: il rapporto tra Intelligenza umana Intelligenza Artificia-
le (par. 6). Per fronteggiare le sfide globali nel Capitolo 5 vengono proposti uno
schema generale (Frame) e una metodologia appropriata, la cui formulazione
sintetica è l’adaptive strategic learning. ftt Nel Capitolo 6 viene effettuato un approfondimento delle traiettorie tecno-
economiche, rispetto alle quali viene esplicitato in modo specifico per ciascuna
di esse uno schema generale di intervento strategico, basato su precisi elemen-
ti: Identificazione delle missions, individuazione degli obiettivi e degli attori
coinvolti, indicatori ai fini della valutazione ex-ante ed ex-post. Il ruolo degli at- 12 INTRODUZIONE tori individuali e collettivi viene approfondito nel Capitolo 7, insieme agli stru-
menti tecnici ed operativi considerati idonei per valutare le politiche dirette a
innescare e favorire processi innovativi all’altezza delle sfide globali, preceden-
temente indicate. Sempre in questo capitolo si mette alla prova sul piano empi-
rico lo schema teorico introdotto nei capitoli precedenti e arricchito da concetti
desunti da Ackoff (1972) e Gharajedaghi (2011) con l’interpretazione di come
alcune regioni italiane, considerate molto dinamiche dalla letteratura sull’ana-
lisi dei sistemi locali, hanno reagito alla dinamica tecno-economica pre-Covid.ir g
p
Il Capitolo 8, infine, propone un frame di design thinking, funzionale al ripen-
samento degli indicatori adatti per progettare e valutare le politiche dell’inno-
vazione, in modo che gli interventi strategici siano coerenti con gli elementi del
frame tecnico-scientifico esposto nel corso di tutto il libro e le cui linee essen-
ziali sono richiamate nelle Conclusioni. NOAA, US Department of Commerce. 2021. How much oxygen comes from the ocean?
At least of Earth’s oxygen comes from the Ackoff, R.L., e F.E. Emery. 1972. On Purposeful Systems. Chicago: Aldine-Atherton.
Gharajedaghi, J. 2011. Systems Thinking: Managing Chaos and Complexity. Amsterdam:
Elsevier.
National Geographic. 2020. Marine pollution, explained. <https://www.
nationalgeographic.com/environment/oceans/critical-issues-marine-pollution/>
(2021-03-10).
NOAA, US Department of Commerce. 2021. How much oxygen comes from the ocean?
At least of Earth’s oxygen comes from the Bibliografia Ackoff, R.L., e F.E. Emery. 1972. On Purposeful Systems. Chicago: Aldine-Atherton. Gharajedaghi, J. 2011. Systems Thinking: Managing Chaos and Complexity. Amsterdam:
Elsevier. National Geographic. 2020. Marine pollution, explained. <https://www. nationalgeographic.com/environment/oceans/critical-issues-marine-pollution/>
(2021-03-10). NOAA, US Department of Commerce. 2021. How much oxygen comes from the ocean? At least of Earth’s oxygen comes from the Ackoff, R.L., e F.E. Emery. 1972. On Purposeful Systems. Chicago: Aldine-Atherton. Gharajedaghi, J. 2011. Systems Thinking: Managing Chaos and Complexity. Amsterdam:
Elsevier. 13 13
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Production of schizophyllan from distiller’s dried grains with solubles by diverse strains of Schizophyllum commune
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© 2013 Sutivisedsak et al.; licensee Springer. 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. Production of schizophyllan from distiller’s dried
grains with solubles by diverse strains of
Schizophyllum commune Nongnuch Sutivisedsak, Timothy D Leathers* and Neil PJ Price * Correspondence: Tim.Leathers@ars.usda.gov
Renewable Product Technology Research Unit, National Center for
Agricultural Utilization Research, Agricultural Research Service, U.S.
Department of Agriculture, 1815 North University Street, Peoria, IL 61604,
USA Open Access Open Access Abstract Eleven diverse strains of Schizophyllan commune were examined for production of the biopolymer schizophyllan
from agricultural biomass. Strains were grown in malt extract (ME) basal medium containing 1% (w/v) distiller’s
dried grains with solubles (DDGS), an abundant coproduct of fuel ethanol production by the dry grind process. Ten
of 11 strains tested produced more than 2 g schizophyllan/L. Two strains, ATCC 20165 and CBS 266.60, produced
more than 10 g schizophyllan/L. Schizophyllan from these strains was similar to commercial product in terms of
solution viscosity, molecular weight, and surface tension properties, suggesting that they would be equivalent in
biomaterial applications. Keywords: Distiller’s dried grains with solubles; DDGS; Schizophyllum commune; Schizophyllan schizophyllan for biomaterials applications. Schizo-
phyllan could fit into the integrated biorefinery con-
cept as a value-added bioproduct from biomass. One
readily available type of agricultural biomass is dis-
tiller’s dried grains with solubles (DDGS), an abun-
dant coproduct of fuel ethanol production from corn
by the dry-grind process (Rosentrater et al. 2012). We recently demonstrated the efficient utilization of
DDGS for schizophyllan production by S. commune
commercial production strain ATCC 38548 (Sutivisedsak
et al. 2013). Sutivisedsak et al. SpringerPlus 2013, 2:476
http://www.springerplus.com/content/2/1/476 Sutivisedsak et al. SpringerPlus 2013, 2:476
http://www.springerplus.com/content/2/1/476 a SpringerOpen Journal a SpringerOpen Journal Background g
Schizophyllan is a homoglucan with a β-1,3-linked
backbone and single β-1,6-linked glucose side chains
at every third residue (Figure 1), produced by the fun-
gus Schizophyllum commune (Rau 1999; Rau 2002). Schizophyllan is commercially produced for use in anti-
cancer therapies and as a bioactive cosmetics ingredient
(Rau 2002). However, schizophyllan also has unique
physical properties of high viscosity, film formation, and
thermal stability that suggest bulk biomaterials applica-
tions. Schizophyllan can form oxygen-impermeable films
for food preservation (Schulz et al. 1992), and it has
been tested for use in enhanced petroleum recovery
(Rau et al. 1992b). Despite the fact that S. commune is an ubiquitous
mushroom found world-wide, commercial production
and most research studies have involved a single strain,
ATCC 38548 (Prokop et al. 1992; Rau et al. 1992a;
Sanroman and Nunez 1993; Shu and Hsu 2011). Few
additional strains have been studied for schizophyllan
production, such as NRCM and CGMCC 5.113 (Kumari
et al. 2008; Li et al. 2011). In the current study we
compare 11 diverse strains of S. commune for their
ability to produce schizophyllan in medium contai-
ning DDGS. Although
glucose
is
used
in
conventional
pro-
duction of schizophyllan, S. commune can utilize a
number of sugars and agricultural residues for poly-
saccharide production (Steiner et al. 1987; Leathers
et al. 2006; Gao and Zhou 2008; Kumari et al. 2008;
Shu and Hsu 2011). Inexpensive agricultural bio-
mass residues could be appropriate for production of Sutivisedsak et al. SpringerPlus 2013, 2:476 Sutivisedsak et al. SpringerPlus 2013, 2:476
http://www.springerplus.com/content/2/1/476 Page 2 of 6 O
OH
OH
HO
HO
O
OH
O
HO
O
O
OH
O
HO
HO
O
O
OH
O
HO
HO
n
Schizophyllan repeat unit
Figure 1 Chemical structure of the schizophyllan repeat unit. Schizophyllan repeat unit Figure 1 Chemical structure of the schizophyllan repeat unit. Figure 1 Chemical structure of the schizophyllan repeat unit. Results and discussion schizophyllan from agricultural biomass appears to be a
common trait among strains of S. commune. Two of
these strains, ATCC 20165 and CBS 266.60, produced
more than 10 g schizophyllan/L (Table 2). These yields
are comparable to those obtained from commercial
production strain ATCC 38548 (Sutivisedsak et al. 2013). Schizophyllan preparations from strains ATCC
20165 and CBS 266.60 were further examined for so-
lution viscosity, molecular weight, and surface tension
properties. Production of schizophyllan by diverse strains of
Schizophyllum commune A set of 11 S. commune strains was chosen for this study,
representing diverse isolation sources and sites (Table 1). Only non-clinical isolates were used because the goal
was to identify strains with potential for industrial pro-
duction. Ten of these strains produced more than 2 g
schizophyllan/L in ME basal medium containing 1%
(w/v) DDGS (Table 2). Thus, the capacity to produce Table 1 Strains of Schizophyllum commune used in this study
Strain
Substrate of isolation
Depositors
Country
Designation
Synonym
ATCC 20165
Kyowa Ferm. Ind. Co., Ltd. 6 F2
ATCC 26262
CA Raper
699
ATCC 26890
Y Koltin
700
ATCC 28095
Loblolly pine log
ER Toole
R-8
ATCC 44200
J.G.H. Wessels, Jun 1981
USA
4-39
CBS 341.81
CBS 199.27
Conserved stem, Hevea
A. van Luijk, Koloniaal Inst., May 1927
MUCL 1008
CBS 249.69
In sawmill, Intsia bijuga
M.R. Monsalud, Univ. of the Philippines, Mar 1969
Philippines
Daedalea elegans, FPRI10
CBS 266.60
Mahogany wood
IMI, Mar 1960
UK
FPRL 9, IMI 061312
CBS 579.83
Decaying jute cutting
H. Esterbauer, Oct 1983
CBS 109645
Litter
López, Aug 2001
Colombia
NRRL A-23867
P.A. Lemke
ATCC 38548
Elm twig
M.G. Paice
Delmar Table 1 Strains of Schizophyllum commune used in this study Page 3 of 6 Page 3 of 6 Sutivisedsak et al. SpringerPlus 2013, 2:476
http://www.springerplus.com/content/2/1/476 Table 2 Total biomass and production of schizophyllan
(dry weights) by strains of Schizophyllum communea
Strain
Total biomass (g/l)
Schizophyllan (g/l)
ATCC 20165
6.4 ± 0.5
10.1 ± 0.7
ATCC 26262
8.7 ± 0.1
8.3 ± 0.5
ATCC 26890
12.4 ± 0.4
5.9 ± 0.5
ATCC 28095
9.4 ± 0.5
2.2 ± 0.2
ATCC 44200
13.2 ± 0.2
0.6 ± 0.1
CBS 199.27
10.7 ± 0.5
2.4 ± 0.1
CBS 249.69
11.5 ± 0.4
3.4 ± 0.1
CBS 266.60
4.7 ± 0.8
12.7 ± 0.1
CBS 579.83
12.3 ± 0.1
2.1 ± 0.2
CBS 109645
13.2 ± 0.4
2.1 ± 0.1
NRRL A-23867
10 ± 0.1
2.3 ± 0.2
a Cultures grown for 8 days on 1% (w/v) distiller’s dried grains with solubles
(DDGS) in malt extract basal medium. Table 2 Total biomass and production of schizophyllan
(dry weights) by strains of Schizophyllum communea of 4.6 × 106 and 5.2 × 106, respectively. The values
are consistent with literature values of 6–12 × 106
(Rau 1999). The interfacial tension of 0.3% (w/v) aqueous solutions
of schizophyllan from S. Conclusions a Cultures grown for 8 days on 1% (w/v) distiller’s dried grains with solubles
(DDGS) in malt extract basal medium. Ten of 11 diverse strains of S. commune produced
schizophyllan from agricultural biomass. Two of these
strains, ATCC 20165 and CBS 266.60, produced yields
comparable to those from a commercial production
strain. The physical properties of schizophyllan solutions
from these strains also were comparable to those of
commercial schizophyllan, suggesting that they would be
equivalent in biomaterial applications. Properties of schizophyllan solutions from S. commune
strains ATCC 20165 and CBS 266.60 Schizophyllan from S. commune strains ATCC 20165
and CBS 266.60 was structurally analyzed by Hete-
ronuclear Single Quantum Coherence-NMR (Figure 2). The spectra were essentially identical to those of both a
commercial schizophyllan standard and schizophyllan
from commercial strain ATCC 38548. Spectra showed
two anomeric sugar signals at 4.55 ppm and 4.20 ppm,
due to the β-1,3-linked glucose and β-1,6-linked glucose,
respectively. These signals correlated to overlapping
13C signals at 103.4 ppm, consistent with β-linked
glucosyl residues. Other carbohydrate signals were
apparent in the 2.7 – 4.0 ppm region for 1H, and 55 –
85 ppm for
13C nuclei. Characteristic methylene –
CH2 signals were apparent at 3.40 and 3.60 ppm,
coupled to a single 13C signal at 61 ppm. These were
assigned to the C-6 position of the backbone glucose
residues carrying a 1,6-glucose branch. These data
are consistent with the isolated polysaccharides being
schizophyllan. Production of schizophyllan by diverse strains of
Schizophyllum commune commune strains ATCC 20165
and CBS 266.60 were 52 ± 1.9 and 55 ± 2.8 dy/cm,
respectively (standard deviations shown). By compa-
rison, schizophyllan from commercial strain ATCC
38548 showed similar values of 58 ± 3.5 dy/cm, while
pure water exhibits an interfacial tension of 72 dy/cm
(Dunlap et al. 2011). Thus, schizophyllan solutions
from strains ATCC 20165 and CBS 266.60 showed
relatively low surface activity, similar to commercial
preparations (Rau 1999). Strains and culture conditions Strains and culture conditions
Schizophyllum commune strains used in this study were
obtained from the American Type Culture Collection,
Manassas, VA (ATCC strains), the ARS Culture Col-
lection at the National Center for Agricultural Uti-
lization Research, Peoria IL (NRRL strain) and the
Centraalbureau voor Schimmelcultures, Utrecht, The
Netherlands (CBS strains). Malt extract (ME) basal
medium contained 2% (w/v) malt extract and 0.1%
(w/v)
peptone. Strains
were
grown
on
ME
solid
medium containing 2% (w/v) glucose and 2.5% (w/v)
agar at 28°C for 7–10 days. A 7 mm × 7 mm square
of
mycelium
was
used
to
inoculate
250
mL
of
ME basal medium containing 2% (w/v) glucose in a
500 ml fluted Erlenmeyer flask with three 10 mm
glass beads. This preinoculum culture was incubated
at 240 rpm for 4–5 days at 30°C. Experimental cul-
tures containing 1% (w/v) of DDGS in 150 mL of ME
basal medium (without glucose) in 500 mL flasks
were inoculated with 1.5 mL of preinoculum (1% v/v)
and incubated at 240 rpm for 8 days at 30°C. DDGS
was obtained from the National Corn-to-Ethanol Re-
search Center, Edswardville, IL. All experiments were
carried out in triplicate and standard deviations are
shown. The solution viscosity properties of 0.3% (w/v) aque-
ous solutions of schizophyllan from strains ATCC 20165
and CBS 266.60 were compared with those of com-
mercial strain ATCC 38548 (Figure 3). These strains
exhibited nearly identical viscosity and shear thinning
properties, characteristic of the pseudoplastic flow be-
havior of schizophyllan solutions (Rau 1999). HPSEC was used to characterize the molecular weight
of schizophyllan. Schizophyllan produced by S. com-
mune strains ATCC 20165 and CBS 266.60 exhibited
heterodisperse peaks with maximum molecular weights Sutivisedsak et al. SpringerPlus 2013, 2:476
http://www.springerplus.com/content/2/1/476 Sutivisedsak et al. SpringerPlus 2013, 2:476
http://www.springerplus.com/content/2/1/476 Sutivisedsak et al. SpringerPlus 2013, 2:476
http://www.springerplus.com/content/2/1/476 Page 4 of 6 Page 4 of 6 Figure 2 HSQC 2D-NMR spectra of schizophyllans from strains CBS 266.6 (top) and ATCC 20165 (lower). Figure 2 HSQC 2D-NMR spectra of schizophyllans from strains CBS 266.6 (top) and ATCC 20165 (lower). Figure 2 HSQC 2D-NMR spectra of schizophyllans from strains CBS 266.6 (top) and ATCC 20165 (lower). air-dried overnight to reduce the amount of ethanol, and
then lyophilized. Isolation of schizophyllan Whole culture suspensions were homogenized for 20 sec
(Power Gen 700, Fisher Scientific) and centrifuged at
6,166 × g for 1 h at 4°C. The supernatants were collected
and the insoluble pellets (containing mycelia and re-
sidual DDGS) were resuspended in 100 mL of deionized
water, homogenized, and centrifuged as before. The pel-
lets were dried under vacuum for 48 h at 60°C. The su-
pernatants were combined and one volume of 95%
ethanol was added. After 1 h at 4°C, precipitates were
collected by centrifugation at 6,166 × g for 1 h at 4°C, Authors’ contributions NS, TL, and NP participated in the conception and design of the study,
interpreted experimental results, and helped draft the manuscript. NS
produced and characterized schizophyllan from diverse strains of
Schizophyllum commune. NP conducted the NMR studies of schizophyllan
from selected strains and controls. All authors read and approved the
final manuscript. NMR analysis Solution NMR spectra were recorded on a Bruker
AMX 500 spectrometer at normal probe temperature
with standard instrument settings. Deuterated dime-
thylsulfoxide (d6-DMSO) was used as the solvent. All
chemical shifts were referenced to tetramethylsilane
at 0 ppm. Sutivisedsak et al. SpringerPlus 2013, 2:476
http://www.springerplus.com/content/2/1/476 Page 5 of 6 Figure 3 Solution viscosity properties of 0.3% (w/v) aqueous solutions of schizophyllan produced by Schizophyllum commune on DDGS
in ME basal medium. Figure 3 Solution viscosity properties of 0.3% (w/v) aqueous solutions of schizophyllan produced by Schizophyllum commune on DDGS
in ME basal medium. Competing interests The authors declare that they have no competing interests. Abbreviations DDGS: Distiller’s dried grains with solubles; d6-DMSO: Deuterated
dimethylsulfoxide; HPSEC: High performance size exclusion chromatography;
ME: Malt extract basal medium; NMR: Nuclear magnetic resonance;
USDA: United States Department of Agriculture. Schizophyllan molecular weights were determined by
size exclusion chromatography as previously described,
using a Shodex SB-806 M high performance size exclu-
sion chromatography (HPSEC) column (Showa Denko,
Tokyo, Japan) and eluted with 0.05 M sodium nitrate at
a flow rate of 0.5 ml/min (Leathers et al. 2010). The
column was calibrated with a set of eight pullulan mo-
lecular weight standards ranging from 5.8 × 103 Da to
1.66 × 106 Da (Showa Denko, Tokyo, Japan). Separations
were monitored using a Shodex OR-1 optical rotation
detector (Showa Denko). Solution viscosity properties Expert technical assistance was provided by Erika Hertenstein, Melinda S. Nunnally, Suzanne Unser, and Andrew J. Thomas. The authors sincerely thank
Christopher Dunlap for surface tension measurements. This project was
supported by Agriculture and Food Research Initiative Competitive Grant no. 2010-65504-20377 from the USDA National Institute of Food and Agriculture. Mention of any trade names or commercial products in this publication is
solely for the purpose of providing specific information and does not imply
recommendation or endorsement by the US Department of Agriculture. USDA is an equal opportunity provider and employer. Solution viscosity was measured using a TA Instruments
(New Castle, DE) ARES LS-1 controlled strain rheome-
ter with a 25 mm titanium parallel plate. All tests were
performed at 25°C using a peltier plate. Steady rate
sweeps were used to determine the viscosity of samples
from 0.01-100 s-1. The Cross model was used to deter-
mine the zero shear viscosity, which was 460 Pa.s. Received: 30 May 2013 Accepted: 19 September 2013
Published: 22 September 2013 References Surface activity was determined using the pendant drop
method (Dunlap et al. 2011). Samples were analyzed
using the FTA 4000 surface tension instrument (First
Ten Angstorms Inc., USA). Measurements were made
using 22 gauge blunt needles with 7 μL drops. The re-
ported values are the average of triplicate cultures. Dunlap CA, Schisler DA, Price NP, Vaughn SF (2011) Cyclic lipopeptide profile of
three Bacillus subtilis strains; antagonists of Fusarium head blight. J Microbiol
49:603–609 Dunlap CA, Schisler DA, Price NP, Vaughn SF (2011) Cyclic lipopeptide profile of
three Bacillus subtilis strains; antagonists of Fusarium head blight. J Microbiol
49:603–609 Gao L, Zhou S (2008) Optimization of extraction technology of schizophyllan
produced by solid fermentation. Shipin Gongye Keji 29:214–216
Kumari M, Survase SA, Singhal RS (2008) Production of schizophyllan using
Schizophyllum commune NRCM. Biores Technol 99:1036–1043 Page 6 of 6 Page 6 of 6 doi:10.1186/2193-1801-2-476
Cite this article as: Sutivisedsak et al.: Production of schizophyllan from
distiller’s dried grains with solubles by diverse strains of Schizophyllum
commune. SpringerPlus 2013 2:476. Sutivisedsak et al. SpringerPlus 2013, 2:476
http://www.springerplus.com/content/2/1/476 Sutivisedsak et al. SpringerPlus 2013, 2:476
http://www.springerplus.com/content/2/1/476 Leathers TD, Nunnally MS, Price NP (2006) Co-production of schizophyllan and
arabinoxylan from corn fiber. Biotechnol Lett 28:623–626 Leathers TD, Nunnally MS, Cote GL (2010) Optimization of process conditions for
enzymatic modification of alternan using dextranase from Chaetomium
erraticum. Carbohydr Pol 81:732–736 Li W, Zhou P, Yu L (2011) Statistical optimization of the medium composition by
response surface methodology to enhance schizophyllan production by
Schizophyllum commune. Zeitschrift Naturforschung C J Biosci 66:173–181 Prokop A, Rapp P, Wagner F (1992) Production of extracellular β-1,3/β-1,6-glucan
by mono- and dikaryons of Schizophyllum commune. Exp Mycol 16:197–206 Rau U (1999) Production of schizophyllan. In: Bucke C (ed) Methods in
biotechnology, vol 10, carbohydrate biotechnology protocols. Humana Press,
Totowa, pp 43–55 Rau U (2002) Schizophyllan. In: Vandamme EJ, De Baets S, Steinbuchel A (ed)
Biopolymers, vol 6, polysaccharides II: polysaccharides from eukaryotes. Wiley-VCH, Weinheim, pp 61–91 Rau U, Gura E, Olszewski E, Wagner F (1992a) Enhanced glucan formation of
filamentous fungi by effective mixing, oxygen limitation and fed-batch
processing. J Ind Microbiol 9:19–26 Rau U, Haarstrick A, Wagner F (1992b) Suitability of schizophyllan solutions for
polymerflooding of petroleum reservoirs with high temperature and stability. Chemie Ingenieur Technik 64:576–577 Rosentrater KA, Ileleji K, Johnston DB (2012) Manufacturing of fuel ethanol and
distillers grains - current and evolving processes. In: Liu K, Rosentrater KA (ed)
Distillers Grains: Production, Properties, and Utilization. CRC Press, Boca
Raton, pp 73–102 Rosentrater KA, Ileleji K, Johnston DB (2012) Manufacturing of fuel ethanol and
distillers grains - current and evolving processes. In: Liu K, Rosentrater KA (ed)
Distillers Grains: Production, Properties, and Utilization. CRC Press, Boca
Raton, pp 73–102 Sanroman MA, Nunez MJ (1993) Production of extracellular polysaccharides. Anales de la Asociacion Quimica Argentina 81:15–23 Sanroman MA, Nunez MJ (1993) Production of extracellular polysaccharides. Anales de la Asociacion Quimica Argentina 81:15–23 g
Schulz D, Rau U, Wagner F (1992) Characteristics of films prepared from native
and modified branched β-1,3-D-glucans. Carbohydr Pol 18:295–299
Shu C, Hsu H (2011) Production of schizophyllan glucan by Schizophyllum
commune ATCC 38548 from detoxificated hydrolysate of rice hull. J Taiwan
Inst Chem Eng 42:387–393 Schulz D, Rau U, Wagner F (1992) Characteristics of films prepared from native
and modified branched β-1,3-D-glucans. Carbohydr Pol 18:295–299 Schulz D, Rau U, Wagner F (1992) Characteristics of films prepared from native
and modified branched β-1,3-D-glucans. Sutivisedsak et al. SpringerPlus 2013, 2:476
http://www.springerplus.com/content/2/1/476 Carbohydr Pol 18:295–299
Shu C, Hsu H (2011) Production of schizophyllan glucan by Schizophyllum
commune ATCC 38548 from detoxificated hydrolysate of rice hull. J Taiwan
Inst Chem Eng 42:387–393 Shu C, Hsu H (2011) Production of schizophyllan glucan by Schizophyllum
commune ATCC 38548 from detoxificated hydrolysate of rice hull. J Taiwan
Inst Chem Eng 42:387–393 Steiner W, Lafferty RM, Gomes I, Esterbauer H (1987) Studies on a wild strain of
Schizophyllum commune: cellulase and xylanase production and formation of
the extracellular polysaccharide schizophyllan. Biotechnol Bioeng 30:169–178 Steiner W, Lafferty RM, Gomes I, Esterbauer H (1987) Studies on a wild strain of
Schizophyllum commune: cellulase and xylanase production and formation of
the extracellular polysaccharide schizophyllan. Biotechnol Bioeng 30:169–178 Steiner W, Lafferty RM, Gomes I, Esterbauer H (1987) Studies on a wild strain of
Schizophyllum commune: cellulase and xylanase production and formation of
the extracellular polysaccharide schizophyllan. Biotechnol Bioeng 30:169–178 Steiner W, Lafferty RM, Gomes I, Esterbauer H (1987) Studies on a wild strain of
Schizophyllum commune: cellulase and xylanase production and formation of
the extracellular polysaccharide schizophyllan. Biotechnol Bioeng 30:169–178
Sutivisedsak N, Leathers TD, Nunnally MS, Price NPJ, Biresaw G (2013) Utilization
of agricultural biomass in the production of the biopolymer schizophyllan. J Ind Microbiol Biotechnol 40:105–112 Sutivisedsak N, Leathers TD, Nunnally MS, Price NPJ, Biresaw G (2013) Utilization
of agricultural biomass in the production of the biopolymer schizophyllan. J Ind Microbiol Biotechnol 40:105–112 doi:10.1186/2193-1801-2-476
Cite this article as: Sutivisedsak et al.: Production of schizophyllan from
distiller’s dried grains with solubles by diverse strains of Schizophyllum
commune. SpringerPlus 2013 2:476. doi:10.1186/2193-1801-2-476
Cite this article as: Sutivisedsak et al.: Production of schizophyllan from
distiller’s dried grains with solubles by diverse strains of Schizophyllum
commune. SpringerPlus 2013 2:476. Submit your manuscript to a
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English
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A chiral phosphazane reagent strategy for the determination of enantiomeric excess of amines
|
Chemical science
| 2,022
|
cc-by
| 11,243
|
aYusuf Hamied Department of Chemistry, Cambridge University, Lenseld Road,
Cambridge, CB2 1EW, UK. E-mail: dsw1000@cam.ac.uk
bThe Faraday Institution Quad One, Harwell Science and Innovation Campus, Didcot,
UK
† Electronic supplementary information (ESI) available: synthetic procedures and
analytical data, NMR and X-ray data, DFT calculations. CCDC 2105705–2105719
and 2157891–2157896. For ESI and crystallographic data in CIF or other
electronic format see https://doi.org/10.1039/d2sc01692c A chiral phosphazane reagent strategy for the
determination of enantiomeric excess of amines† Cite this: Chem. Sci., 2022, 13, 5398 Andrew J. Peel,*a Alexandros Terzopoulos,a Rajesh B. Jethwa,
a
Dipanjana Choudhury,a Hao-Che Niu,a Andrew D. Bond,
a Jonathan Slaughter
ab
and Dominic S. Wright
*a All publication charges for this article
have been paid for by the Royal Society
of Chemistry Received 23rd March 2022
Accepted 12th April 2022
DOI: 10.1039/d2sc01692c
rsc.li/chemical-science Methods for measuring enantiomeric excess (ee) of organic molecules by NMR spectroscopy provide rapid
analysis using a standard technique that is readily available. Commonly this is accomplished by chiral
derivatisation of the detector molecule (producing a chiral derivatisation agent, CDA), which is reacted
with the mixture of enantiomers under investigation. However, these CDAs have almost exclusively been
based on carbon frameworks, which are generally costly and/or difficult to prepare. In this work,
a
methodology
based
on
the
readily
prepared
inorganic
cyclodiphosph(III)azane
CDA
ClP(m-
NtBu)2POBorn (Born ¼ endo-(1S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl) is shown to be highly effective
in the measurement of ee’s of chiral amines, involving in situ reaction of the chiral amines (R*NH2) with
the P–Cl bond of the CDA followed by quaternization of the phosphorus framework with methyl iodide. This results in sharp 31P NMR signals with distinct chemical shift differences between the diastereomers
that are formed, which can be used to obtain the ee directly by integration. Spectroscopic, X-ray
structural and DFT studies suggest that the NMR chemical shift differences between diastereomers is
steric in origin, with the sharpness of these signals resulting from conformational locking of the bornyl
group relative to the P2N2 ring induced by the presence of the P(V)-bonded amino group (R*NH). This
study showcases cheap inorganic phosphazane CDAs as simple alternatives to organic variants for the
rapid determination of ee. Received 23rd March 2022
Accepted 12th April 2022
DOI: 10.1039/d2sc01692c
rsc.li/chemical-science © 2022 The Author(s). Published by the Royal Society of Chemistry Introduction The reaction of [ClPNtBu]2
16 with the chiral, naturally
occurring alcohol ()-menthol has been reported previously,23c
however, the bicyclic alcohol endo-()-borneol was selected as
a chiral auxiliary in the current study on the basis that it should
exhibit less conformational freedom than menthol (and is
nonetheless inexpensive). Furthermore, it has been reported
that the formation of borneol derivatives, where O–H is replaced
by O–X (X s H) reduces the number of accessible conformers
that can arise due to rotation about the O–X bond, and this was
anticipated to be of benet in developing a CDA.29 The proto-
type CDA was prepared by reacting [ClPNtBu]2 with endo-
()-borneol in a 1 : 1 ratio in THF in the presence of Et3N
(Scheme 3). Aer trituration with Et2O and extraction into
toluene, the crude product was recrystallised from n-pentane to
give
ClP(m-NtBu)2POBorn
(Born
¼
bornyl,
endo-(1S)-1,7,7-
trimethylbicyclo[2.2.1]heptan-2-yl) (1) in 50% yield (from two
fractions). 1H NMR spectroscopy in C6D6 showed signals
belonging to bornyl and tBu groups in a 1 : 2 ratio (see ESI
Fig. S1†). Meanwhile, 31P{1H} NMR spectroscopy revealed two
doublets, at d 190.5 ppm and 140.0 ppm, which could be
attributed to the –Cl and –OR substituted P atoms of a P2N2
ring, respectively. The observed inequivalence of the 1H NMR
signals of the tBu groups within the P2N2 ring unit can be
attributed to the effect of spatial orientation of the chiral axil-
lary, though evidently the inuence is small, with a separation
of d 0.02 ppm between these signals. Furthermore, this sepa-
ration appears to be solvent dependent as only one singlet is Molecules based on phosphorus–nitrogen skeletons are of
interest in this regard because of the similarity between the C–C
and P–N bond dissociation energies (346 kJ mol1 vs. Introduction Scheme 1
The reaction of an achiral pyridyl aluminate with a chiral
alcohol, producing diastereoisomeric dimers. The result is the asym-
metrisation of the reporter R2-groups of the pyridyl rings (marked in
magenta, green, brown and purple), which can be detected by 1H NMR
spectroscopy. R1, R2 ¼ alkyl. Scheme 2
The concept of forming diastereomeric cyclodiphosph(III)
azanes, where R* is a chiral auxiliary and R0*H is a chiral analyte. 2, in which one of the P atoms is bonded to a chiral auxiliary
(R*) while the other has a reactive P–Cl bond. The highly reac-
tive nature of this bond means that reaction with an acidic H-
atom of a chiral analyte (R0*H) leads to a pair of diastereo-
mers. An important feature of cyclodiphosphazanes of this type
is the general thermodynamic preference for the cis isomer in
the solution and solid states,28 so that the chiral groups R* and
R0* should be in close proximity. This, it was thought, should
induce the maximum effect on the chemical shis of the R and
S isomers of R0*. Scheme 1
The reaction of an achiral pyridyl aluminate with a chiral
alcohol, producing diastereoisomeric dimers. The result is the asym-
metrisation of the reporter R2-groups of the pyridyl rings (marked in
magenta, green, brown and purple), which can be detected by 1H NMR
spectroscopy. R1, R2 ¼ alkyl. Scheme 1
The reaction of an achiral pyridyl aluminate with a chiral
alcohol, producing diastereoisomeric dimers. The result is the asym-
metrisation of the reporter R2-groups of the pyridyl rings (marked in
magenta, green, brown and purple), which can be detected by 1H NMR
spectroscopy. R1, R2 ¼ alkyl. Scheme 1
The reaction of an achiral pyridyl aluminate with a chiral
alcohol, producing diastereoisomeric dimers. The result is the asym-
metrisation of the reporter R2-groups of the pyridyl rings (marked in
magenta, green, brown and purple), which can be detected by 1H NMR
spectroscopy. R1, R2 ¼ alkyl. discrimination relies on the in situ formation of diastereomeric
dimers, rather than monomeric diastereomers (Scheme 1).12
This, together with the substantial ionic character of organo-
metallic CDAs of this type, leads to hydrolytic sensitivity and
oxygen intolerance, which are major drawbacks to bench-top
applications. Introduction convenient determination of enantiomeric excess (ee) using
NMR spectroscopy.5 Current methods involve the use of
enantiomerically-pure compounds as chiral auxiliaries.6 A
number are based on non-covalent interactions, including
chiral
solvating
agents
(CSAs),7
chiral
lanthanide
shi
reagents,8 ion-pairing agents5c and liquid crystals.9 However,
chiral derivatisation agents (CDAs)10 that form a covalent bond
with the chiral analyte are perhaps the most commonly used
species for determining ee's. Their popularity stems from the
robustness of the covalent link between the auxiliary and ana-
lyte, greater freedom in solvent selection and their added
potential for use in assignment of absolute conguration.11 The creation of chiral molecules plays a major role in numerous
chemical processes, from natural product synthesis to the
design of new materials. Crucial to the search for efficient
methods of producing enantiopure substances is the ability to
detect and measure chiral composition.1 Many methods are
established for the determination of the chiral purity of
a sample, including optical rotation and circular dichroism,2
gas chromatography (GC) or liquid chromatography (HPLC)3
with a chiral stationary phase. Methods based on NMR spec-
troscopy have been studied extensively and remain at the fore-
front of this area due to the simplicity and availability of this
technique and the potential for fast turnaround.4 The reaction of an organic CDA with a chiral analyte typically
creates a new C–X bond that has a relatively high bond disso-
ciation energy and low-to-moderate bond polarity, e.g. C–N or
C–O. The kinetic stability of these bonds is essential to prevent
interchange of chiral components during analysis, and this has
led to the dominance of organic chemistry in chiral derivatisa-
tion. So far, however, the potential of readily prepared inorganic
CDAs has rarely been explored. Success in this endeavour
hinges on selection of inorganic frameworks that can compete
with the stability of classical carbon-based systems. A recent
example is pyridyl aluminate reagents which can be used for the
chiral discrimination of alcohols, but their mechanism of In the last few decades, considerable effort has gone into
developing new methodologies and reagents for the rapid and © 2022 The Author(s). Published by the Royal Society of Chemistry 5398 | Chem. Sci., 2022, 13, 5398–5412 5398 Edge Article Edge Article Chemical Science Scheme 2
The concept of forming diastereomeric cyclodiphosph(III)
azanes, where R* is a chiral auxiliary and R0*H is a chiral analyte. Results and discussion Scheme 3
Synthesis of 1. Introduction 290 kJ mol1,
respectively) and their low polarity (DcP–N ¼ 0.85).13 A particularly
well-developed subset of P–N compounds are cyclodiphosph(III)
azanes [XP(NR)]2 (X ¼ halogen, -OR, -N(H)R, -NR2, -R; R ¼ organic
group).14 Many different examples are accessible from the inex-
pensive precursor [ClPNtBu]2, which has excellent stability and is
easy to functionalise.15,16 Cyclodiphosphazane molecules have
been investigated extensively as building blocks to inorganic
macrocycles,17–20
as
ligands
in
supramolecular
coordination
complexes21 and in MOFs.22 Furthermore, they have found appli-
cations in homogeneous catalysis,23 anti-tumour metallodrug
development24 and anion recognition/transport.25,26 A role for P–N
compounds in the chiral realm is emerging with a recent report on
chiral amino-phosphonium salts which exhibit highly enantiose-
lective recognition of chiral molecules, including carboxylic acids,
amines
and
alcohols,
through
non-covalent
interactions.27
However, in this case the ready detection of chirality and the
measurement of ee's was not possible. Inspired by this, and our
earlier work on aluminates, we decided to explore the potential of
cyclodiphosphazanes to build covalent chiral reagents that can be
used to probe enantiomeric excess and absolute conguration
using NMR spectroscopy. Edge Article Fig. 1
The molecular structure of 1 (ellipsoids at 30% probability) with
H atoms omitted for clarity. One representative molecule from the
asymmetric unit is depicted. The other molecule has essentially
identical conformation, aside from rotation of one of the tBu groups. Selected bond lengths (˚A) and angles (): P1–N1 1.682(3), P1–N2
1.695(3), P2–N1 1.732(3), P2–N2 1.729(3), P1–Cl1 2.1619(3), P2–O1
1.612(2),
P1–N1–P2
97.93(13),
P1–N2–P2
97.52(13),
N1–P1–N2
82.77(13), N1–P2–N2 80.36(12), N1–P1–Cl1 104.05(10), N2–P1–Cl1
104.14(10), N1–P2–O1 106.83(12), N2–P2–O1 106.80(12). Reactions of 1 with the amines (R)-1-phenylethylamine and
(S)-1-phenylethylamine (both of which are benchmark chiral
amine reagents in the literature) were undertaken to explore its
potential as a CDA. In situ-prepared 1 was reacted in a 1 : 1 ratio
with (R)- or (S)-1-phenylethylamine in the presence of excess
Et3N (Scheme 4). Recrystallisation of the solid residues from
toluene gave crystals of (R)-PhCH(Me)NHP(m-NtBu)2POBorn (4-
R) or (S)-PhCH(Me)NHP(m-NtBu)2POBorn (4-S). The solid-state structures of 4-R and 4-S are signicantly
different. In 4-R there are three independent molecules in the
asymmetric unit (see ESI Fig. S55 and S56†). Two of these (one
of which is shown in Fig. 2a) are similar, but distinct from the
conformation of the third (Fig. 2b). The differences arise largely
due to pivoting around the P–O and C–O bonds at the bornyl
group (see ESI†). None of these arrangements allow for a good
‘t’ of the groups on the chiral amine within the pocket offered
by the endo face of the bornyl ring and their conformations are
probably dictated by packing effects, suggesting relatively small
energy differences between them. Fig. 1
The molecular structure of 1 (ellipsoids at 30% probability) with
H atoms omitted for clarity. One representative molecule from the
asymmetric unit is depicted. The other molecule has essentially
identical conformation, aside from rotation of one of the tBu groups. Selected bond lengths (˚A) and angles (): P1–N1 1.682(3), P1–N2
1.695(3), P2–N1 1.732(3), P2–N2 1.729(3), P1–Cl1 2.1619(3), P2–O1
1.612(2),
P1–N1–P2
97.93(13),
P1–N2–P2
97.52(13),
N1–P1–N2
82.77(13), N1–P2–N2 80.36(12), N1–P1–Cl1 104.05(10), N2–P1–Cl1
104.14(10), N1–P2–O1 106.83(12), N2–P2–O1 106.80(12). In contrast to 4-R, the solid-state structure of 4-S reveals
a single unique molecule (in space group P212121) and the
structural effect of inverting the stereochemistry at the a-carbon Fig. 2
Two of the independent molecules in the asymmetric unit of 4-
R, showing different conformations at the bornyl group (see ESI
Fig. S55† for ellipsoid representations and selected metric parameters). Establishing the methodology Based on the mechanism of chiral discrimination we had
observed previously using pyridyl aluminate reagents, involving
the formation of diastereomeric dimers (Scheme 1), we
reasoned that the more rigid P2N2 ring units of cyclo-
diphosph(III)azanes could form the basis of a new type of inor-
ganic CDA. Our prototype CDA was of type A, shown in Scheme Scheme 3
Synthesis of 1. Scheme 3
Synthesis of 1. Chem. Sci., 2022, 13, 5398–5412 | 5399 Chem. Sci., 2022, 13, 5398–5412 | 5399 © 2022 The Author(s). Published by the Royal Society of Chemistry Chemical Science Edge Article Edge Article Selected bond lengths (˚A) and angles
(): P1–N1 1.724(2), P1–N2 1.7373(19), P2–N1 1.6967(19), P2–N2
1.7136(19), P1–N 3 1.671(2), P2–O1 1.6492(16); P1–N1–P2 99.04(9),
N1–P2–N2 81.48(9), P2–N2–P1 97.88(9), N2–P1–N1 80.03(9), N3–
P1–N1 106.45(10), N3–P1–N2 106.71(10), O1–P2–N1 103.69(9) O1–
P2–N2 104.35(9). Edge Article
Chemical Science Chemical Science Edge Article Edge Article Fig. 3
Molecular structure of 4-S (ellipsoids at 30% probability) with H
atoms on C omitted for clarity. Selected bond lengths (˚A) and angles
(): P1–N1 1.724(2), P1–N2 1.7373(19), P2–N1 1.6967(19), P2–N2
1.7136(19), P1–N 3 1.671(2), P2–O1 1.6492(16); P1–N1–P2 99.04(9),
N1–P2–N2 81.48(9), P2–N2–P1 97.88(9), N2–P1–N1 80.03(9), N3–
P1–N1 106.45(10), N3–P1–N2 106.71(10), O1–P2–N1 103.69(9) O1–
P2–N2 104.35(9). Fig. 3
Molecular structure of 4-S (ellipsoids at 30% probability) with H
atoms on C omitted for clarity. Selected bond lengths (˚A) and angles
(): P1–N1 1.724(2), P1–N2 1.7373(19), P2–N1 1.6967(19), P2–N2
1.7136(19), P1–N 3 1.671(2), P2–O1 1.6492(16); P1–N1–P2 99.04(9),
N1–P2–N2 81.48(9), P2–N2–P1 97.88(9), N2–P1–N1 80.03(9), N3–
P1–N1 106.45(10), N3–P1–N2 106.71(10), O1–P2–N1 103.69(9) O1–
P2–N2 104.35(9). Fig. 4
(a) Expansions of the 1H NMR spectra of 4-R and 4-S in CDCl3;
(b) numbering scheme for bornyl ring hydrogens (blue ¼ exo, green ¼
endo) and (c) an expansion of the 31P{1H} NMR spectra of 4-R and 4-S. of the amine is unexpectedly large. While in 4-R the phenyl
group projects away from the bornyl group, in 4-S it faces it,
tting inside the pocket on the endo face (Fig. 3). Some steric
repulsion between the Ph- and tBu-groups is evident (also in 4-
R), which involves ‘pushing down’ on the tBu group [the sums of
the bond angles at N1 and N2 in 4-S are 359 and 347,
respectively]. The structural features seen in 4-R and 4-S would be expected
to translate into measurable differences in the NMR spectra of
these diastereomers if their solid-state structures were retained
in solution. However, at a glance this does not appear to be the
case. A comparison of the 1H NMR spectra of 4-R and 4-S in
CDCl3 reveals few differences, with the notable exceptions being
the methyl resonances of the bornyl group and of the two
hydrogens of the bornyl framework in the region d 2.3–2.1 ppm
(see Fig. 4a and b). Regarding the chiral amine substituents, the
resonance associated with the CH group, centred around
d 4.9 ppm, is broadened signicantly in both compounds, while
the N–H resonances, centred around d 3.3 ppm, remain as sharp
multiplets. © 2022 The Author(s). Published by the Royal Society of Chemistry Edge Article observed in more polar CDCl3. X-ray diffraction revealed that 1
crystallises in the Sohncke space group P21 (consistent with its
enantiopurity), with two molecules in the asymmetric unit
(Fig. 1). These molecules have essentially identical confor-
mations, except for rotation of one tBu group. The P2N2 rings
in 1 remain intact, as also observed in the only other solid-
state structure of an alkoxy(chloro)phosphazane, Hyp2N2P2(-
Cl)OCH(CF3)2 (Hyp ¼ (Me3Si)3Si).30 The P2N2 ring can be
described as kite-shaped, which is a result of the different P–N
bond lengths and angles at phosphorus associated with the
OBorn (P–Nav ¼ 1.689 ˚A; N–P–Nav ¼ 80.3) and Cl groups (P–
Nav ¼ 1.732 ˚A; N–P–Nav ¼ 82.9) that arise due to differences in
electronegativity. These groups are arranged cis to one another
in 1, as is seen in most other cyclodiphosphazanes in the solid
state.14 NMR spectroscopy on batches of the crude product showed
the only signicant impurities to be starting material, the
disubstituted product [tBuNPOBorn]2 (2), and phosphinic acid-
type hydrolysis product H(O)P(m-tBuN)2POBorn (3) (see ESI for
full characterisation and Fig. S53 and S54† for molecular
structures).31,32 Signicantly, for preparative purposes, puri-
cation of crude 1 was not necessary. Scheme 4
Synthesis of 4-R and 4-S. Fig. 2
Two of the independent molecules in the asymmetric unit of 4-
R, showing different conformations at the bornyl group (see ESI
Fig. S55† for ellipsoid representations and selected metric parameters). Fig. 2
Two of the independent molecules in the asymmetric unit of 4-
R, showing different conformations at the bornyl group (see ESI
Fig. S55† for ellipsoid representations and selected metric parameters). Scheme 4
Synthesis of 4-R and 4-S. © 2022 The Author(s). Published by the Royal Society of Chemistry 5400 | Chem. Sci., 2022, 13, 5398–5412 5400 | Chem. Sci., 2022, 13, 5398–5412 Fig. 3
Molecular structure of 4-S (ellipsoids at 30% probability) with H
atoms on C omitted for clarity. Selected bond lengths (˚A) and angles
(): P1–N1 1.724(2), P1–N2 1.7373(19), P2–N1 1.6967(19), P2–N2
1.7136(19), P1–N 3 1.671(2), P2–O1 1.6492(16); P1–N1–P2 99.04(9),
N1–P2–N2 81.48(9), P2–N2–P1 97.88(9), N2–P1–N1 80.03(9), N3–
P1–N1 106.45(10), N3–P1–N2 106.71(10), O1–P2–N1 103.69(9) O1–
P2–N2 104.35(9). Edge Article Fig. 3
Molecular structure of 4-S (ellipsoids at 30% probability) with H
atoms on C omitted for clarity. Edge Article Meanwhile, 31P{1H} NMR spectroscopy shows very
broad peaks at d 142 and 99 ppm in CDCl3 (Fig. 4c) that suggest
that dynamic processes are occurring, such as exo–endo inter-
conversion of the substituents (resulting from rotation of the
P–O or P–N bonds; the resultant conformers denoted Oexo/Oendo
and Nexo/Nendo, respectively) and/or cis–trans isomerism.23
Cooling of 4-R in CD2Cl2 (down to 233 K) only resulted in
sharpening of the 31P NMR resonances, and the fact that the
chemical shis remained in the region expected for the cis
isomer (ESI Fig. S34b†) suggest that endo-exo interconversion is
more likely to be responsible for the signal broadening at room
temperature.33 Fig. 4
(a) Expansions of the 1H NMR spectra of 4-R and 4-S in CDCl3;
(b) numbering scheme for bornyl ring hydrogens (blue ¼ exo, green ¼
endo) and (c) an expansion of the 31P{1H} NMR spectra of 4-R and 4-S. predicted.28b Hence, whilst we believe Oexo–Nendo–Oexo–Nexo
interconversion to be the dominant uxional process, cis–trans
isomerism cannot be ruled out. In contrast, calculations based
on the experimentally observed cis Oexo–Nendo isomer show that
rotation of the chiral Ph(Me)CH group in 4-S about the C–N
bond, switching the C–H bond from facing the P2N2 ring unit
(as in the observed solid-state structure) to away from the P2N2
ring unit, is thermodynamically unfavourable (with an activa-
tion energy of around 45.6 kJ mol1) (see ESI Table S7 and
Fig. S81†). DFT calculations on 4-S in chloroform indicate that its
various conformers possess energies in very close proximity (see
ESI Table S6 and Fig. S74–S80†); the three most stable are
shown in Fig. 5. Though NMR spectroscopy hints at the pref-
erence for cis isomers in solution, and the calculations reveal
a small energy difference between the Oexo–Nendo and Oexo–Nexo
forms of these, energetically accessible trans isomers are also While these results clearly show measurable differences in
the 1H NMR spectra of the diastereomers formed from the © 2022 The Author(s). Published by the Royal Society of Chemistry Chem. Sci., 2022, 13, 5398–5412 | 5401 Chemical Science Edge Article Edge Article Fig. 5
Optimised structures for the three most stable conformers of
4-S and their relative energies compared to the cis Oexo–Nendo
conformer (experimentally observed in the solid-state structure). Calculations are at the B3LYP/TZVP level and included an applied
polarised continuum model (PCM) using the dielectric constant for
chloroform to model the solution structure. Edge Article Analogous isomers of 4-R
are depicted in ESI Fig. S82 and S83.† Fig. 5
Optimised structures for the three most stable conformers of Fig. 5
Optimised structures for the three most stable conformers of
4-S and their relative energies compared to the cis Oexo–Nendo
conformer (experimentally observed in the solid-state structure). Calculations are at the B3LYP/TZVP level and included an applied
polarised continuum model (PCM) using the dielectric constant for
chloroform to model the solution structure. Analogous isomers of 4-R
are depicted in ESI Fig. S82 and S83.† Fig. 5
Optimised structures for the three most stable conformers of
4-S and their relative energies compared to the cis Oexo–Nendo
conformer (experimentally observed in the solid-state structure). Calculations are at the B3LYP/TZVP level and included an applied
polarised continuum model (PCM) using the dielectric constant for
chloroform to model the solution structure. Analogous isomers of 4-R
are depicted in ESI Fig. S82 and S83.† reactions of 1, the small chemical shiseparations encountered
in a mixture would lead to overlap of signals and make it
impossible to measure enatiomeric excess. Similarly, the oper-
ation of uxional processes at ambient temperature eliminates
31P NMR spectroscopy as a viable spectroscopic alternative in
these P(III) dimers. Attempts to obtain sharp 31P NMR signals by
formation of an internally coordinated lithiate (with Li envis-
aged to bridge the N and O atoms) were unsuccessful. However
previous reports on the selective methylation of phosphorus
atoms in cyclodiphosphazanes using methyl iodide hinted at
another option.21b,c Quaternisation has been observed to
provide advantages for this type of P/N compound such as
improved solubility in polar solvents and even air/water
stability. Therefore we reasoned that quaternisation might
also suppress the uxional processes observed above and
provide a further means of increasing the difference in the
chemical shis between the P atoms in diasteromeric pairs. 4-R
and 4-S were therefore combined with excess methyl iodide in n-
hexane which led to the formation of the salts [{(R)-PhCH(Me)
NH}P(Me)(m-NtBu)2POBorn]I
(5-R)
and
[{(S)-PhCH(Me)NH}
P(Me)(m-NtBu)2POBorn]I (5-S) (Scheme 5, Fig. 6). Fig. 6
(a) The molecular structure of 5-R (ellipsoids at 30% probability)
and with H atoms on C omitted for clarity. Edge Article Selected bond lengths (˚A)
and angles (): P1–N1 1.640(3), N1–P2 1.739(3), P2–N2 1.741(3), N2–P1
1.655(3), P1–N3 1.602(3), P2–O1 1.629(2), P1–C27 1.782(4); P1–N1–P2
97.11(16), N1–P2–N2 80.34(14), P2–N2–P1 96.48(15), N1–P1–N2
85.90(15), N1–P1–N3 116.25(17), N2–P1–N3 116.89(15), N1–P2–O1
103.91(14), N2–P2–O1 100.75(13), N1–P1–C27 116.07(17), N2–P1–
C27 116.09(17), N3–P1–C27 105.41(18). (b) The molecular structure of
5-S (ellipsoids at 30% probability) and with H atoms on C omitted for
clarity. Selected bond lengths (˚A) and angles (): P1–N1 1.646(3), N1–P2
1.755(2), P2–N2 1.731(3), N2–P1 1.640(3), P1–N3 1.600(3), P2–O1
1.627(2), P1–C27 1.781(3); P1–N2–P2 96.37(13), N1–P2–N2 80.06(12),
P2–N2–P1 97.54(14), N2–P1–N2 86.03(13), N1–P1–N3 115.37(14),
N2–P1–N3 117.40(14), N1–P2–O1 103.46(12), N2–P2–O1 100.54(11),
N1–P1–C27
117.55(16),
N2–P1–C27
114.83(16),
N3–P1–C27
105.45(15). Following recrystallisation from THF, X-ray diffraction
reveals that the amino/alkoxide substituents of 5-R and 5-S
remain in a cis relationship with respect to each other and
conrms that selective reaction of the more electron-rich
amine-substituted P atom has occurred in both cases, leaving
the O-substituted P and N atom of the chiral amino groups
untouched. The orientation of the substituents of the amino
groups in 5-R is identical to one of the molecules in 4-R
(specically the one depicted in Fig. 2b), and the conformation of 5-S is identical to 4-S. Not unexpectedly, the exocyclic P–N
bond lengths associated with the P(V) centre in 5-R (1.602(3) ˚A)
and 5-S (1.600(3) ˚A) are all shortened with respect to the cor-
responding P(III) centre in 4-R (1.667(5) ˚A [average]) and 4-S
(1.671(2) ˚A). While the asymmetry in these cycles does not allow
for a direct comparison, it is evident that a shortening of the
endocyclic PV–N bonds also occurs to within the range 1.640–
1.655 ˚A (compared with 1.716–1.747 ˚A for 4-R and 4-S). Osten-
sibly, due to bond shortening there is an expansion of the
Nchiral–P–Nring angles around the newly generated PV centre
(range 103.7–108.1 for 4-R/4-S; 115.3–117.4 for 5-R/5-S). Nonetheless, the effect on the exocyclic N/O distance between
the amino-N and alkoxide-O atoms is marginal (being ca. 4 ˚A in
every case). Both 5-R and 5-S show near linear interactions
between the N–H hydrogen atoms and the nearby iodide ions in
their lattices, at distances comparable to those reported in the of 5-S is identical to 4-S. © 2022 The Author(s). Published by the Royal Society of Chemistry Edge Article Not unexpectedly, the exocyclic P–N
bond lengths associated with the P(V) centre in 5-R (1.602(3) ˚A)
and 5-S (1.600(3) ˚A) are all shortened with respect to the cor-
responding P(III) centre in 4-R (1.667(5) ˚A [average]) and 4-S
(1.671(2) ˚A). While the asymmetry in these cycles does not allow
for a direct comparison, it is evident that a shortening of the
endocyclic PV–N bonds also occurs to within the range 1.640–
1.655 ˚A (compared with 1.716–1.747 ˚A for 4-R and 4-S). Osten-
sibly, due to bond shortening there is an expansion of the
Nchiral–P–Nring angles around the newly generated PV centre
(range 103.7–108.1 for 4-R/4-S; 115.3–117.4 for 5-R/5-S). Nonetheless, the effect on the exocyclic N/O distance between
the amino-N and alkoxide-O atoms is marginal (being ca. 4 ˚A in
every case). Both 5-R and 5-S show near linear interactions
between the N–H hydrogen atoms and the nearby iodide ions in
their lattices, at distances comparable to those reported in the Scheme 5
Selective reaction of 4-R and 4-S with methyl iodide. Scheme 5
Selective reaction of 4-R and 4-S with methyl iodide. © 2022 The Author(s). Published by the Royal Society of Chemistry 5402 | Chem. Sci., 2022, 13, 5398–5412 Edge Article Chemical Science literature for N–H/I contacts (2.65 ˚A for 5-R, 2.68 ˚A for 5-S
based on calculated positions; literature value 2.69 ˚A).34 The
most important conclusion from this structural analysis is that
despite clear changes in bonding within the P2N2 ring, inter-
actions between the exocyclic substituents are not signicantly
affected by methylation. NMR spectroscopy reveals much more pronounced differ-
ences between parent molecules 4-R/4-S and iodide salts 5-R/5-S. In CDCl3, the most noticeable change is the shiof the N–H
resonance, from ca. d 3.2 ppm to d 8.4 ppm, which is associated
with incorporation of the P(V) centre (Fig. 7a).24a The increased
electron withdrawing character of this P atom is also evident
from deshielding of the Ph- and Me-group hydrogens. Mean-
while, the CH resonances of the Ph(Me)CH groups, formerly
broad singlets centred at d 4.94 ppm in 4-R/4-S, transform into
clearly dened multiplets, at d 4.65 and 4.68 ppm for 5-R and 5-S,
respectively. This was the rst indication that the dynamic
processes evident in solutions of 4-R and 4-S are suppressed by
methylation in 5-R and 5-S. Edge Article This was reasoned to arise from
enhanced N(p) / P–N(s*) donation upon oxidation of the PN(III)
centre in 4-R/4-S to PN(V) increasing the barrier to P–N bond
rotation. Possibly as result of this, the separation of the inequi-
valent
1H NMR resonances of the
tBu groups is amplied
approximately twofold moving from 4-R/4-S to 5-R/5-S, as the
phenyl ring is more rigidly located with respect to each tBu group. The bornyl ring hydrogens still appear at slightly different posi-
tions in the new diastereomers but the differences between 5-R
and 5-S are no larger than between 4-R and 4-S (Fig. 7b). On the
other hand, the bornyl Me-peaks show a more distinctive pattern,
whereby Me* (Fig. 7c) appears more deshielded in 5-S (d 0.96
ppm) than in 5-R (d 0.92 ppm), consistent with Me* being posi-
tioned further from the shielding Ph-ring. 31P{1H} NMR spectroscopy provides more concrete evidence
of the change in the dynamics by showing the disappearance of
the broad resonances for 4-R/4-S (ca. d 140 and 100 ppm), which
are replaced by sharp doublets, at d 122.7 and 38.8 ppm for 5-R,
and d 123.0 and 38.8 ppm for 5-S (Fig. 8a). The signicance of
this is that the d 0.3 ppm separation in chemical shiof the O-
bound P atom is sufficient to avoid overlap (see Fig. 8b), in turn
allowing for accurate integration of the 31P NMR peaks. This
provided the necessary proof of principle for the use of cyclo-
diphosphazanes as CDAs since integration can be used directly
to determine the ee. This is described in more detail later in this
section. In the meantime, it is worth noting that the physical
characteristics of diastereomers 5-R and 5-S are substantially
different. For example, the decomposition temperature of 5-S is
some 20 C higher than 5-R. More importantly, from the reac-
tion of the roughly 50 : 50 mixture of (S)- and (R)-1-phenyleth-
ylamine with 1 followed by in situ quaternisation with methyl
iodide, 5-S is selectively crystallised, as veried by X-ray crys-
tallography and analysis of the bulk crystalline material by 31P
NMR spectroscopy (Fig. 8c and d). The ratio of 5-S and 5-R was
found to be 93 : 7. Edge Article Condi-
tions: (A) Et3N (2 eq.), THF, 4 h at RT; (B) methyl iodide (3 eq.), THF, 3 h
at RT. The reaction was stopped after stages (A) and (B) by removal of
the volatiles. Below are shown expansions of the 31P NMR spectra for
scalemic mixtures of 1-phenylethylamine. The determined %ee's are
given on the right of the spectra, and their predicted ee's based on the
ratios of the enantiomers added are shown in parentheses. The ee's
predicted for the pure enantiomers are those stated by the supplier
(Acros Organics). Edge Article Fig. 9
Method for the in situ derivatisation and analysis of a non-
enantiopure amine (in this case 1-phenylethylamine) using 1. Condi-
tions: (A) Et3N (2 eq.), THF, 4 h at RT; (B) methyl iodide (3 eq.), THF, 3 h
at RT. The reaction was stopped after stages (A) and (B) by removal of
the volatiles. Below are shown expansions of the 31P NMR spectra for
scalemic mixtures of 1-phenylethylamine. The determined %ee's are
given on the right of the spectra, and their predicted ee's based on the
ratios of the enantiomers added are shown in parentheses. The ee's
predicted for the pure enantiomers are those stated by the supplier
(Acros Organics). Fig. 8
Expanded regions of (a) 31P{1H} NMR spectra of 5-R and 5-S; (b)
31P NMR spectra of 5-R and 5-S; (c) an aliquot from a reaction mixture
containing roughly equal portions of (R)- and (S)-1-phenylethylamine
and (d) the product of this reaction following recrystallization. compositions, with the main error in the measurement of the ee
being due to the sensitivity of 31P NMR as well as the presence of
trace amounts of either isomer in the commercially-supplied
samples of the R- and S-isomers. Having established the ability to distinguish 5-R and 5-S in
their isolated form using 31P NMR spectroscopy, we developed
an in situ methodology that can be used to measure enantio-
meric excess in mixtures of commercial (R)/(S)-1-phenylethyl-
amine. Predetermined mixtures of these amines on the 50 mmol
scale (yielding sufficient product for NMR spectroscopy) were
treated in situ with 1 and then methyl iodide (Fig. 9, top). In all
cases, some hydrolysis of 1 was unavoidable (producing 3),
however, residual 1 in the reaction mixture indicated that
sufficient CDA was available to consume all of the amine added. The results of 31P NMR spectroscopy (Fig. Edge Article 9, bottom) show that
the in situ method works well across a range of chiral Edge Article The tendency of these iodide salts to crys-
tallise presents a promising avenue for further exploration,
since the presence of a heavy atom allows reliable assignment of
absolute
structure—essential
in
assessing
a
reaction's
stereoselectivity. Fig. 7
Expansions of the 1H NMR spectra of 4-R, 4-S, 5-R and 5-S,
showing (a) the signals of selected bornyl ring hydrogens and (b) tBu
groups/Me groups as indicated in (c). Chem. Sci., 2022, 13, 5398–5412 | 5403 © 2022 The Author(s). Published by the Royal Society of Chemistry Fig. 8
Expanded regions of (a) 31P{1H} NMR spectra of 5-R and 5-S; (b)
31P NMR spectra of 5-R and 5-S; (c) an aliquot from a reaction mixture
containing roughly equal portions of (R)- and (S)-1-phenylethylamine
and (d) the product of this reaction following recrystallization. Chemical Science Having established the ability to distinguish 5-R and 5-S in
their isolated form using 31P NMR spectroscopy, we developed
compositions, with the main error in the measurement of the ee
being due to the sensitivity of 31P NMR as well as the presence of
trace amounts of either isomer in the commercially-supplied
samples of the R- and S-isomers. Fig. 8
Expanded regions of (a) 31P{1H} NMR spectra of 5-R and 5-S; (b)
31P NMR spectra of 5-R and 5-S; (c) an aliquot from a reaction mixture
containing roughly equal portions of (R)- and (S)-1-phenylethylamine
and (d) the product of this reaction following recrystallization. Fig. 9
Method for the in situ derivatisation and analysis of a non-
enantiopure amine (in this case 1-phenylethylamine) using 1. Condi-
tions: (A) Et3N (2 eq.), THF, 4 h at RT; (B) methyl iodide (3 eq.), THF, 3 h
at RT. The reaction was stopped after stages (A) and (B) by removal of
the volatiles. Below are shown expansions of the 31P NMR spectra for
scalemic mixtures of 1-phenylethylamine. The determined %ee's are
given on the right of the spectra, and their predicted ee's based on the
ratios of the enantiomers added are shown in parentheses. The ee's
predicted for the pure enantiomers are those stated by the supplier
(Acros Organics). Chemical Science
Edge Article Chemical Science
Edge Article Chemical Science Chemical Science Edge Article Fig. 9
Method for the in situ derivatisation and analysis of a non-
enantiopure amine (in this case 1-phenylethylamine) using 1. Edge Article Chemical Science Chemical Science Table 1
A comparison of the distances between the hydrogen atoms
of 6-R/6-S indicated in Fig. 10, based upon the calculated positions in
their crystal structures, normalised to average neutron C–H distances Table 1
A comparison of the distances between the hydrogen atoms
of 6-R/6-S indicated in Fig. 10, based upon the calculated positions in
their crystal structures, normalised to average neutron C–H distances offering a slightly more sterically hindered chiral centre. Though the intermediate phosphazane analogous to 4-R/4-S
could not be isolated in pure form from the reactions of R- and
(S)-1-phenylpropylamine with 1, in situ reactions of them with
methyl iodide proceeded smoothly to give diffraction-quality
crystals
of
[{(R)-PhCH(Et)NH}P(Me)(m-NtBu)2POBorn]I
(6-R)
and
[{(S)-PhCH(Et)NH}P(Me)(m-NtBu)2POBorn]I
(6-S). X-ray
crystallography demonstrated that 6-R/6-S retain effectively
identical conformations to the related salts 5-R/5-S (see ESI
Fig. S57 and S58†), with the aromatic group sitting above
opposite tBu groups within the pair of diastereomers and hence
the C–H group pointing towards the P2N2 ring units. Bearing in
mind this similarity it is not surprising that many spectro-
scopic features found in 5-R/5-S are also seen in the NMR
spectra of 6-R/6-S in CDCl3. Most importantly, two well-
separated low-eld multiplets are observed in their 31P NMR
spectra, at d 122.6 ppm for 6-R and d 123.1 ppm for 6-S, showing
that assessment of the ee will be possible here also. Ph-Hortho/H
contact
Distance
6-R (˚A)
6-S (˚A)
H6
3.91
2.59
H8
2.88
2.11
H9
2.10
2.32 solution, in which the phenyl group and endo face of the bornyl
group are next to each other. This is also consistent with the
crystallographically
determined
H/H
distances
involved Several features of the 1H NMR spectra of diastereomers 6-R
and 6-S provide greater insight into their solution-state struc-
tures (than was possible for 5-R and 5-S). The rst of these is the
emergence of well-dened multiplets for the diastereotopic
CH2-hydrogens of the Et group within the chiral Ph(Et)CH-
substituent. In the parent amine (R)/(S)-Ph(Et)CHNH2, only
one multiplet (a doublet of quintets, at d 1.69 ppm) is seen. However, incorporation into 6-R/6-S results in resolution into
two signals, at d 2.30 and 2.09 ppm, suggesting conformational
stability. NOESY experiments in CDCl3 reveal weak NOE's for 6-
R/6-S between the phenyl group ortho protons and bornyl ring
protons (see ESI Fig. S9e†). Solution studies and detection limit Although we had established the use of 1 as a CDA, there were
two important questions which we wanted to address, namely,
(i) the relationship between solid-state and solution structure,
and (ii) the detection limit of our methodology. The rst of these was explored (somewhat fortuitously) using
the related amine 1-phenylpropylamine in which the methyl
group of 1-phenylethylamine is replaced by an ethyl group, © 2022 The Author(s). Published by the Royal Society of Chemistry 5404 | Chem. Sci., 2022, 13, 5398–5412 Edge Article A cross peak to H6endo is revealed in
each case and while for 6-R overlap of H8endo and H9exo
precludes rm conclusions regarding the spatial relationship to
the amino group, H8endo and H9exo are well-separated for 6-S
and there is a clear correlation of H8endo with the phenyl group
ortho protons. Taken together with the visible NOEs between
H9endo and the ortho-phenyl protons in both 6-R and 6-S, we
conclude that their solid-state structures are preserved in Fig. 10
Diagram showing the hydrogen atoms for which NOEs are
observed in the cations of 6-R and 6-S. The red-dotted line indicates
a likely NOE Fig. 11
The structure of a representative molecule from the asym-
metric unit of 8-R (ellipsoids at 30% probability). H atoms on C are
omitted for clarity. Selected bond lengths (˚A) and angles (): P1B–N1B
1.635(6), N1B–P2B 1.748(5), P2B–N2B 1.721(6), N2B–P1B 1.642(5),
P1B–N3B 1.609(6), P2B–O1B 1.613(5), P1B–C28B 1.780(7); P1B–N1B–
P2B 96.5(3), N1B–P2B–N2B 80.2(3), P2B–N2B–P1B 97.3(3), N2B–
P1B–N1B 85.9(3), N1B–P1B–N3B 116.3(3), N2B–P1B–N3B 117.0(3),
N1B–P1B–C28B 116.2(3), N2B–P1B–C28B 115.7(3), N3B–P1B–C28B
105.5(3), N1B–P2B–O1B 104.3(2), N2B–P2B–O1B 100.4(3). (b) The
structure of a representative molecule from the asymmetric unit of 8-S
(ellipsoids at 30% probability). H atoms on C are omitted for clarity. Selected bond lengths (˚A) and angles (): P1B–N1B 1.639(6), N1B–P2B
1.764(6), P2B–N2B 1.733(6), N2B–P1B 1.637(6), P1B–N3B 1.608(6),
P2B–O1B 1.614(6), P1B–C28B 1.781(8); P1B–N1B–P2B 95.9(3), N1B–
P2B–N2B 80.0(3), P2B–N2B–P1B 97.2(3), N1B–P1B–N3B 115.3(4),
N2B–P1B–N3B 117.0(4), N1B–P1B–C28B 116.9(4), N2B–P1B–C28B
115.4(4), N3B–P1B–C28B 105.4(4), N1B–P2B–O1B 104.2(3), N2B–
P2B–O1B 100.3(4). Fig. 10
Diagram showing the hydrogen atoms for which NOEs are
observed in the cations of 6-R and 6-S. The red-dotted line indicates
a likely NOE. Fig. 10
Diagram showing the hydrogen atoms for which NOEs are
observed in the cations of 6-R and 6-S. The red-dotted line indicates
a likely NOE. Fig. 10
Diagram showing the hydrogen atoms for which NOEs are
observed in the cations of 6-R and 6-S. The red-dotted line indicates
a likely NOE. © 2022 The Author(s). Published by the Royal Society of Chemistry Chem. Sci., 2022, 13, 5398–5412 | 5405 Chemical Science Edge Article (Fig. 10 and Table 1). This conclusion is important because it
shows that there are no signicant uxional processes occur-
ring which would alter the spatial arrangement between the
OBorn and chiral substituents. explicitly by the crystal structures of the racemic compounds 7-
R/S and 8-R/S, which show disorder indicative of a solid solution
of the two diastereomers. If this situation should be retained in
solution, we would expect the NMR spectra of the diastereomers
of 8-R and 8-S to show much smaller differences compared to
the previous examples 5 and 6. Ultimately, the utility of our protocol will be limited by the
peak separation of the 31P NMR resonances of R- and S- analytes
and therefore the ability to integrate accurately. In order to
explore
this,
we
next
considered
2-phenylpropylamine,
PhCH(Me)CH2NH2, in which the chiral centre is separated by
a CH2 group from the amine functionality (anticipating
a reduction in peak separation). The previous protocol was
used, in which 1 was prepared without purication and subse-
quently reacted with 2-phenylpropylamine in the presence of
a Brønsted base (NEt3) to give the cyclodiphosph(III)azane
intermediates
(R)-PhCH(Me)CH2NHP(m-tBuN)2POBorn
(7-R)
and (S)-PhCH(Me)CH2NHP(m-tBuN)2POBorn (7-S) with the pure
enantiomers and (R/S)-PhCH(Me)CH2NHP(m-tBuN)2POBorn (7-
R/S) with racemic 2-phenylpropylamine. Further treatment of
crude intermediates 7-R, 7-S, 7-R/S with methyl iodide and
recrystallization from toluene afforded excellent crystals of [{(R)-
PhCH(Me)CH2NH}(Me)P(m-tBuN)2POBorn]I
(8-R),
[{(S)-
PhCH(Me)CH2NH}(Me)P(m-tBuN)2POBorn]I (8-S) and [{(R/S)-
PhCH(Me)CH2NH}(Me)P(m-tBuN)2POBorn]I (8-R/S). The solid-
state structures of 7-R, 7-S, 7-R/S, 8-R, 8-S and 8-R/S (ESI
Fig. S60–S64†) were obtained. The structures of 8-R and 8-S (one
of the crystallographically independent molecules) are shown in
Fig. 11. The 1H NMR spectra of 8-R and 8-S are indeed very similar
(see ESI Fig. S13a, S14a and S15a†). Importantly, however, very
sharp 31P{1H} NMR signals are still observed (at d 124.3 and
45.7 ppm for 8-R and d 124.2 and 45.7 ppm for 8-S; Fig. 12). This
can be compared to 7-R and 7-S which show two broad peaks at
d 138 and 103 ppm for both. 31P{1H} NMR spectroscopy gave
sufficient line separation for integration of the PN peaks at ca. d 46 ppm for the two diastereomers in racemic 8-R/S, with the
best peak resolution obtained by inverse-gated decoupling (with
recycle delays $30 s, 30 pulse) and this was additionally
required in order to obtain reliable integration values. Fig. Chemical Science 13 shows the determination of the ee's of mixtures of the
R- and S-isomers of 2-phenylpropylamine using our in situ
protocol. It is clear from these data that the measured and
predicted values are reasonably close to the values determined Fig. 13
Determination of the ee's for mixture of 2-phenylpropylamine
using 1 The conditions are the same as those given in the caption to
Fig. 9. The determined %ee's are shown on the right, and their pre-
dicted ee's based on the ratios of the enantiomers added are given in
parentheses. The ee's of the pure enantiomers and racemic amine
were determined independently by chiral HPLC (since these were not
stated by the supplier, Sigma). The amines were converted into amides
by reaction with acetic anhydride for this purpose. There is an esti-
mated error of up to 2% in the determined ee's due to poor separation
on the column. The other predicted values (1 : 3 and 3 : 1) were
calculated based on the ee's of the ‘pure’ enantiomers determined
from HPLC (so an error of up to ca 2% applies to these values too). Notably, the crystal structures of the diastereomers are iso-
structural for both 7-R/7-S and 8-R/8-S, showing that the
molecular conformation (in the solid state) is effectively
invariant for the two diastereomers in each case. The R or S
conguration at the chiral centre of the amine is accommo-
dated with no change in conformation for the remainder of the
molecule, and in particular no change to the orientation of the
bornyl
group. The
molecular
similarity
is
demonstrated Fig. 12
Expansions of the 31P{1H} NMR spectra (CDCl3) of 8-R and 8-S. Fig. 13
Determination of the ee's for mixture of 2-phenylpropylamine
using 1 The conditions are the same as those given in the caption to
Fig. 9. The determined %ee's are shown on the right, and their pre-
dicted ee's based on the ratios of the enantiomers added are given in
parentheses. The ee's of the pure enantiomers and racemic amine
were determined independently by chiral HPLC (since these were not
stated by the supplier, Sigma). The amines were converted into amides
by reaction with acetic anhydride for this purpose. There is an esti-
mated error of up to 2% in the determined ee's due to poor separation
on the column. Substrate scope This is particularly noticeable in the case of 13 in which the
conformational exibility present in the n-butyl group results in
the
smallest
PO
and
PN
peak
separation
between
the
diastereomers. (ii) The poorest peak separations are seen in 8, 12, 13 and 17
in which there is apparently the greatest degree of conforma-
tional exibility in the aliphatic groups of the primary amines. This is particularly noticeable in the case of 13 in which the
conformational exibility present in the n-butyl group results in
the
smallest
PO
and
PN
peak
separation
between
the
diastereomers. (iii) Perhaps unsurprisingly bearing in mind the mild
conditions and reagents involved, there appears to be good
functional group tolerance across the spectrum of primary
amines investigated, especially in the case of amino acid esters
which bear greater chemical functionality (15–17). (iv) There is a noticeably strong correlation between the PO
chemical shiand the absolute conguration for the eleven a-
amines explored (i.e. 5, 6, 9–17). With the exception of 13 (where
dS z dR), the PO chemical shiof the S-isomer is always higher
than for the R-isomer (dS > dR). It can be noted that the b-amine
2-phenylpropylamine (8) does not t the trend. In contrast, the
PN resonances do not exhibit any such clear correlation; 5, 8, 9,
11, 12, 13 have higher PN chemical shis for the R-isomer (dR >
dS) whereas the opposite holds for 6, 14, 15, 16, 17 (dS > dR), and
only for 10 dS z dR. (iv) There is a noticeably strong correlation between the PO
chemical shiand the absolute conguration for the eleven a-
amines explored (i.e. 5, 6, 9–17). With the exception of 13 (where
dS z dR), the PO chemical shiof the S-isomer is always higher
than for the R-isomer (dS > dR). It can be noted that the b-amine
2-phenylpropylamine (8) does not t the trend. In contrast, the
PN resonances do not exhibit any such clear correlation; 5, 8, 9,
11, 12, 13 have higher PN chemical shis for the R-isomer (dR >
dS) whereas the opposite holds for 6, 14, 15, 16, 17 (dS > dR), and
only for 10 dS z dR. Our reagent 1 can therefore be used to determine the abso-
lute conguration of chiral a-amines, with the PO chemical shi
being a reliable indicator. Edge Article discussed 5, 6 and 8) are summarised in Table 2 which also
indicates whether 31P or 31P{1H} NMR provide sufficient sepa-
ration of the resonances for measurement of the ee. by chiral HPLC (bearing in mind the estimated ca 2% error in
these values due to poor separation of the acetyl amide on the
column). However, the extent to which the overall molecular
shape is affected by changing the chiral centre in the amine
from R to S probably makes our method less suitable for amines
in which the chiral substituent is remote from the amine
functionality. Products 9–11 were also prepared using our in situ method
outlined in Fig. 8 (see Scheme 7) to conrm its applicability over
a larger number of substrates. The resulting 31P NMR spectra
starting from racemic and 2 : 1 R : S scalemic mixtures of 9–11
are well resolved and appended in the ESI (Fig. S37–S39, S52†). The assignment thereof is supported by the spectra of the cor-
responding enantiopure compounds prepared individually. Substrate scope The most important condition that must be met to apply our
methodology for measuring ee is adequate line separation of
the 31P NMR signals. In order to establish the scope of the
method, a greatly expanded range of commercially available
chiral a-primary amines incorporating a variety of functional
groups and steric characteristics was explored to establish if
there are sufficiently large chemical shiseparations of the 31P
NMR resonances (Scheme 6). The eighteen new compounds (9–
17, -R and -S) were prepared starting from enantiopure amines
and fully characterised, including the single-crystal X-ray
structures of 10-R, 10-S, 12-R, 12-S, 13-R and 13-S. Full details
of the spectroscopic analyses and characterisation of 9–17 are
included in the ESI. Of particular interest are the expansions of
31P{1H} NMR spectra showing the peak separation between
products with the pairs of enantiomeric substrates (ESI
Fig. S43–S51†). The results (including those for previously The major conclusion that can be drawn from the data
shown in Table 2 is that 1 can be used to discriminate between
all of the chiral amines studied, although the small values of Dd
for both PO and PN in the cases of 8, 12 and 13 would lead to less
accurate determination of ee. In these cases the use of an
inverse gated 31P{1H} decoupling routine is, however, found to
provide sufficient peak resolution for these substrates. While
the inherent difficulties in obtaining accurate ee's using the
integration method limit the sensitivity of the method for 31P
NMR spectroscopy, the sensitivity is clearly more than adequate
to provide a quick assessment of chiral purity on a convenient
scale and in a straightforward manner (using the PO or PN
signals). There are four other conclusions that we can draw from
these data: (i) the presence of aromatic substituents on the primary
amine is not required to promote signicant chiral inequiva-
lence; rather, the origin of the separation of the PO and PN
resonances of the R and S diastereomers is probably steric/
spatial in origin. S h
6
S
th
i
f 9 17 R/S Chi
l
t
i
th
i (ii) The poorest peak separations are seen in 8, 12, 13 and 17
in which there is apparently the greatest degree of conforma-
tional exibility in the aliphatic groups of the primary amines. Chemical Science The other predicted values (1 : 3 and 3 : 1) were
calculated based on the ee's of the ‘pure’ enantiomers determined
from HPLC (so an error of up to ca 2% applies to these values too). Fig. 12
Expansions of the 31P{1H} NMR spectra (CDCl3) of 8-R and 8-S. 5406 | Chem. Sci., 2022, 13, 5398–5412 © 2022 The Author(s). Published by the Royal Society of Chemistry Chemical Science Chemical Science Substrate scope This is important because, uniquely,
this can be done at the same time as the determination of the ee
in a single 31P NMR experiment of a scalemic mixture. Scheme 6
Synthesis of 9–17-R/S. Chiral centre in the amino groups
marked *. © 2022 The Author(s). Published by the Royal Society of Chemistry Chem. Sci., 2022, 13, 5398–5412 | 5407 Chemical Science Edge Article Edge Article Edge Article Table 2
A summary of the 31P NMR data, separation of low-field (PO) and high-field (PN) signals, and an indication of the suitability of 31P NMR for
measuring ee (with and without decoupling) Table 2
A summary of the 31P NMR data, separation of low-field (PO) and high-field (PN) signals, and an indication of the suitability of 31P NMR for
measuring ee (with and without decoupling) Substrate (compound
number)
PO (d ppm)
PN (d ppm)
DPO
DPN
Suitability for ee
measurement
31P
31P{1H}
123.0 (S)
38.8 (R/S)
0.33
0.03
Good
Good
122.7 (R)
dR > dS
123.1 (S)
38.9 (S)
0.47
0.04
Good
Good
122.6 (R)
38.8 (R)
124.3 (R/S)
45.7 (R/S)
0.09
0.09
Poor
Medium
dR > dS
dR > dS
122.9 (S)
39.2 (R/S)
0.34
0.07
Good
Good
122.6 (R)
dR > dS
122.5 (S)
41.2 (R/S)
0.22
0
Poor
Good
122.2 (R)
dR z dS
123.7 (S)
41.9 (S)
0.16
0.10
Poor
Good
123.5 (R)
42.0 (R)
122.4 (R/S)
41.2 (R/S)
0.01
0.04
Poor
Medium
dS > dR
dR > dS
122.2 (R/S)
40.7 (R/S)
0
0.07
Poor
Good
dS z dR
dR > dS
121.8 (S)
39.2 (R/S)
0.52
0.02
Good
Good
121.3 (R)
dS > dR Substrate (compound
number) 5408 | Chem. Sci., 2022, 13, 5398–5412 © 2022 The Author(s). Published by the Royal Society of Chemistry © 2022 The Author(s). Published by the Royal Society of Chemistry Table 2
(Contd.)
Edge Article
Chemical Science Edge Article Chemical Science Chemical Science Substrate (compound
number)
PO (d ppm)
PN (d ppm)
DPO
DPN
Suitability for ee
measurement
31P
31P{1H}
124.1 (S)
44.2 (S)
0.39
0.28
Good
Good
123.7 (R)
43.9 (R)
123.4 (S)
42.3 (S)
0.30
0.67
Good
Good
123.1 (R)
41.6 (R)
123.9 (R/S)
43.5 (S)
0.02
0.16
Poor
Good
dS > dR
43.4 (R) A clue to the probable feature inuencing the PO chemical
shis is provided by the numerous snapshots of the molecular
conformation established within the crystal structures. Substrate scope Since
several of the structures show more than one independent
molecule and/or disorder of the bornyl group, more than 30
such snapshots are available (see ESI Section 4†). The orienta-
tion of the bornyl group with respect to the P2N2 ring is
described by the P/P–O–CBorn (s1) and P–O–CBorn–C(Me) (s2) torsion angles, as shown in Fig. 14a. A scatterplot of s1 vs. s2
(ESI, Fig. S73†) shows an overall broad spread of points (indi-
cating that the bornyl conformation is not inherently “locked”),
but with a prominent cluster comprising 14 points centred at s1
z 157, s2 z 120, which corresponds to the bornyl confor-
mation shown in Fig. 2b and 6a. The cluster includes at least
one molecule from all characterised compounds showing the
Oexo conformation, which indicates that it is likely to represent
(at least a local) energy minimum for the orientation of the
bornyl group. Although the snapshots are taken from the crystal
structures, while the PO resonances are measured in solution,
any conformation observed in the solid state must be intrinsi-
cally accessible in solution, while a conformation not observed
in the solid state is not proven to be. Considering the quater-
nised compounds, both the R- and S- diastereomers are seen
within the cluster for 8, 12 and 13, and little difference is seen
between dS and dR in the 31P NMR. For 5, 6 and 10, however, the
R-diastereomer is seen within the cluster but the S-diasteromer
is not, and there is a clear distinction between dS and dR in the
31P NMR. Hence, we conclude that discrimination between dS
and dR for the PO resonance arises when the S-substrate blocks
access to the low-energy bornyl conformation. The sharpness of
the signals results from conformational locking of the bornyl
group, while the difference between dS and dR indicates the
extent of the difference between the two locked conformations. For amines where both the R- and the S-substrate permit access
to the same bornyl conformation, the discrimination will be
reduced. The observed conformational differences are interpreted to
be steric in origin. The ‘Me-side’ of the bornyl group appears to
have a large steric inuence, with the most sterically demanding
group of the amine (R2 in Fig. 14b) being diagonally opposite to Scheme 7
In situ formation of 9–11-R/S. Chiral centre in the amines
marked *. © 2022 The Author(s). Conclusions 14
(a) Defining the P/P–O–CBorn (s1) and P–O–CBorn–C(Me) (s2)
torsion angles; (b) diagrammatic representation of the solid-state
structures of R- and S-isomers (in which the O atoms are all sp2
hybridised, consistent with the X-ray structures which show C–O–P
angles close to 120), showing the orientation of the lone pair in p and
sp2 orbitals on the O atom. The blue circles are used to highlight the
relative steric sizes of the groups. The steric interactions between the
two ends of the molecule are mediated through the t-butyl groups,
which show rotational variation. the Me-side in the S-isomer, but adjacent in the R-isomer. The
distance between bornyl and amine is too large to have a direct
effect, but the steric interaction is mediated by the tert-butyl
group, which changes its orientation in the R- and S- diaste-
reomers. The effect is clearest in 5 and 6, where the tert-butyl
groups show an eclipsed conformation in the R- but a staggered
conformation in the S-diastereomer. Substrate scope Published by the Royal Society of Chemistry © 2022 The Author(s). Published by the Royal Society of Chemistry Chem. Sci., 2022, 13, 5398–5412 | 5409 Edge Article Chemical Science Fig. 14
(a) Defining the P/P–O–CBorn (s1) and P–O–CBorn–C(Me) (s2 Conclusions In summary, we have developed a simple inorganic chiral
derivatisation agent based on a cyclodiphosphazane, which can
be used to detect chirality and measure enantiomeric excess in
chiral amines and employs a cheap chiral auxiliary. A two-step
process, involving the substitution of the P–Cl group with the
amine, followed by selective quaternisation of one of the P
centres
gives
diastereomeric
iodide
salts
of
the
type
[(R*NH)(Me)P(m-NtBu)2POBorn]I, where R* is a chiral group and
OBorn is the chiral ancillary. 31P NMR spectroscopy proves to be
particularly useful for measuring the ratio of diastereomers
produced from scalemic mixtures, with the small but useable
separations in chemical shis of diastereomeric pairs. The
origin of the effect is the steric inuence of the chiral amine on
the orientation of the bornyl group. Though the method does
not give highly accurate measures of enantiomeric excess, it is
nonetheless sensitive enough to provide a rapid assay of chiral
composition. Screening a range of commercial chiral amines
indicates that measurement of ee is possible by integration of
the PO and/or PN
31P/31P{1H} NMR resonances. The method is
most suited to a-chiral amines in which greatest separation of
the PO and PN resonances occurs, but can also be applied to b-
chiral amines. Using 31P NMR spectroscopy allows detection of
chiral derivatives in relatively small amounts (suitable for NMR
assays) without any signicant interference of side products
with the signals of interest. Finally, the relative values of the PO
chemical shis are diagnostic of the absolute conguration of
a-alkyl-amines, so that our CDA performs all three tasks;
detection of chirality, measurement of ee and determination of
absolute
conguration;
in
one
simple
experiment. This
contrasts
with
previously
developed
organic
CDAs
(like
Mosher's acid) where two separate experiments are required to
determine ee's and absolute conguration.11a Clearly, our CDA
design concept is general, and better selectivity and more
accurate measurement of ee's is likely to be possible using other
chiral auxiliaries and/or P2N2 ring substituents. Work on
rening these systems and on extending the scope to chiral
alcohols and carboxylic acids is underway. Fig. Conflicts of interest There are no conicts to declare. Conflicting interests DFT (B3LYP/TZVP) geometry optimisations of 4-R, 4-S and
5-R in CHCl3 reproduce their solid-state structures closely
(Fig. S74, S82 and S84, ESI†), with calculated CBorn–O–P/P
dihedrals being very similar to those found experimentally. A
survey of the frontier orbitals of 5-R and 5-S does not indicate
any donation of the O-lone pairs into the s* orbital of the ring
P–N bonds, and NBO analysis shows that the charges on the
principal atoms of both are almost identical (ESI, Table S8†). Given the above, we conclude that the most likely origin of the
difference in the chemical shiarising from a change in
orientation of the bornyl group is the relative orientation of the
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graphic data for 1, 2, 3, 4-R, 4-S, 5-R, 5-S, 6-R, 6-S, 7-R, 7-S, 7-R/S,
8-R, 8-S, 8-R/S, 10-R, 10-S, 12-R, 12-S, 13-R, 13-S have been
deposited at the CCDC (deposition numbers 2105705, 2105711,
2105707,
2105709,
2105718,
2105714,
2105706,
2105708,
2105717,
2105710,
2105715,
2105712,
2105716,
2105713, O- and P-lone pairs.35 5410 | Chem. Sci., 2022, 13, 5398–5412 © 2022 The Author(s). Published by the Royal Society of Chemistry Chemical Science Edge Article 2105719,
2157896,
2157893,
2157895,
2157894,
2157891,
2157892, respectively) and can be obtained on request. Full
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funding. R. B. Jethwa would like to thank the U.K. EPSRC and
the Royal Dutch Shell plc. for an I-Case (EP/R511870/1). J. Slaughter thanks the Faraday Institution (grant numbers
FIRG001 and FIRG024) for funding. We thank Duncan Howe for
assistance with NMR spectroscopy. Calculations were carried
out on the in-house Odyssey HPC cluster and the authors are
grateful for the calculation time used therein. We also thank Dr
Robert Phipps and Robert Pearce-Higgins for running chiral
HPLC on one of the amines. 17 A. J. Plajer,
R. Garc´ıa-Rodr´ıguez,
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A. J. P. conceived the original idea. All authors were involved in
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American Chemical Society, Washington, DC, 1981. 35 Few 31P NMR studies have identied lone pair orientation as
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Chemistry: Proceedings of the 1981 International Conference,
ACS Symp. Ser. 171, ed. L. D. Quin and J. G. Verkade,
American Chemical Society, Washington, DC, 1981. 35 Few 31P NMR studies have identied lone pair orientation as
an inuence on chemical shi; a notable example is that of
diphosphanes—vide J. P. Albrand and C. Ta¨ıeb, Phosphorus
Chemistry: Proceedings of the 1981 International Conference,
ACS Symp. Ser. 171, ed. L. D. Quin and J. G. Verkade,
American Chemical Society, Washington, DC, 1981. 31 E. L. Doyle, F. Garc´ıa, S. M. Humphrey, R. A. Kowenicki,
L. R´ıera, A. D. Woods and D. S. Wright, Dalton Trans.,
2004, 807–812. 5412 | Chem. Sci., 2022, 13, 5398–5412 © 2022 The Author(s). Published by the Royal Society of Chemistry 34 T. Steiner, Acta Crystallogr., Sect. B: Struct. Sci., 1998, 54, 456–
463. 33 A. Tarrasoli, M. L. Thompson, R. C. Haltiwanger, T. G. Hill
and A. D. Norman, Inorg. Chem., 1988, 27, 3382–3386.
34 T. Steiner, Acta Crystallogr., Sect. B: Struct. Sci., 1998, 54, 456–
463.
35 Few 31P NMR studies have identied lone pair orientation as
an inuence on chemical shi; a notable example is that of
diphosphanes—vide J. P. Albrand and C. Ta¨ıeb, Phosphorus
Chemistry: Proceedings of the 1981 International Conference,
ACS Symp. Ser. 171, ed. L. D. Quin and J. G. Verkade,
American Chemical Society, Washington, DC, 1981. References 32 This was identied by the presence of a doublet of doublets
in the 31P NMR spectrum (in CDCl3) at, d ¼ 7.0 ppm, with
1JP-H ¼ 583 Hz and 2JP-P ¼ 10 Hz. 5412 | Chem. Sci., 2022, 13, 5398–5412
|
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https://discovery.ucl.ac.uk/id/eprint/1378611/1/1378611.pdf
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English
| null |
A beta-mixture quantile normalization method for correcting probe design bias in Illumina Infinium 450 k DNA methylation data
|
Bioinformatics
| 2,012
|
cc-by
| 8,480
|
ABSTRACT Motivation: The Illumina Infinium 450 k DNA Methylation Beadchip is a
prime candidate technology for Epigenome-Wide Association Studies
(EWAS). However, a difficulty associated with these beadarrays is that
probes come in two different designs, characterized by widely differ-
ent DNA methylation distributions and dynamic range, which may bias
downstream analyses. A key statistical issue is therefore how best to
adjust for the two different probe designs. Results: Here we propose a novel model-based intra-array normal-
ization strategy for 450 k data, called BMIQ (Beta MIxture Quantile
dilation), to adjust the beta-values of type2 design probes into a stat-
istical distribution characteristic of type1 probes. The strategy involves
application of a three-state beta-mixture model to assign probes to
methylation states, subsequent transformation of probabilities into
quantiles and finally a methylation-dependent dilation transformation
to preserve the monotonicity and continuity of the data. We validate
our method on cell-line data, fresh frozen and paraffin-embedded
tumour tissue samples and demonstrate that BMIQ compares favour-
ably with two competing methods. Specifically, we show that BMIQ
improves the robustness of the normalization procedure, reduces the
technical variation and bias of type2 probe values and successfully
eliminates the type1 enrichment bias caused by the lower dynamic
range of type2 probes. BMIQ will be useful as a preprocessing step for
any study using the Illumina Infinium 450 k platform. Motivation: The Illumina Infinium 450 k DNA Methylation Beadchip is a
prime candidate technology for Epigenome-Wide Association Studies
(EWAS). However, a difficulty associated with these beadarrays is that
probes come in two different designs, characterized by widely differ-
ent DNA methylation distributions and dynamic range, which may bias
downstream analyses. A key statistical issue is therefore how best to
adjust for the two different probe designs. Results: Here we propose a novel model-based intra-array normal-
ization strategy for 450 k data, called BMIQ (Beta MIxture Quantile
dilation), to adjust the beta-values of type2 design probes into a stat-
istical distribution characteristic of type1 probes. The strategy involves
application of a three-state beta-mixture model to assign probes to
methylation states, subsequent transformation of probabilities into
quantiles and finally a methylation-dependent dilation transformation
to preserve the monotonicity and continuity of the data. We validate
our method on cell-line data, fresh frozen and paraffin-embedded
tumour tissue samples and demonstrate that BMIQ compares favour-
ably with two competing methods. *To whom correspondence should be addressed ABSTRACT Specifically, we show that BMIQ
improves the robustness of the normalization procedure, reduces the
technical variation and bias of type2 probe values and successfully
eliminates the type1 enrichment bias caused by the lower dynamic
range of type2 probes. BMIQ will be useful as a preprocessing step for
any study using the Illumina Infinium 450 k platform. A key statistical issue with the Illumina 450k beadchip is that
probes come in two different designs, which causes the methyla-
tion values derived from these two designs to exhibit widely dif-
ferent distributions (Dedeurwaerder et al., 2011). Indeed, type2
probes are typically characterized by a much lower dynamic
range compared with type1 probes, even after adjustment for dif-
ferences in biological characteristics such as CpG density
(Dedeurwaerder et al., 2011). Comparison with bisulphite pyrose-
quencing data further showed that type2 probe values are biased
and generally less reproducible (Dedeurwaerder et al., 2011). To
correct for this bias, a peak-based correction (PBC) method was
proposed (Dedeurwaerder et al., 2011) which normalises type2
design probes so as to render them comparable with type1
probes. Making the statistical distributions of type1 and type2
probes comparable is important for several reasons. Not doing
so may introduce an enrichment bias towards type1 probes when
ranking probes in supervised analyses, as the dynamic range of
type1 probes is significantly higher. Moreover, methods that seek
to determine differentially methylated regions (Jaffe et al., 2012)
also assume that probes within these regions are comparable and
thus one would want to avoid any sources of technical variation
within them. Finally, one would wish to apply unsupervised di-
mensional reduction algorithms (Houseman et al., 2008; Koestler
et al., 2010) and classification algorithms (Zhuang et al., 2012) to
one single dataset, and not separately to two different assays. Availability:
BMIQ
is
freely
available
from
http://code.google. com/p/bmiq/. Contact: a.teschendorff@ucl.ac.uk Supplementary information: Supplementary data are available at
Bioinformatics online Supplementary information: Supplementary data are available at
Bioinformatics online Received on June 27, 2012; revised on October 9, 2012; accepted on
November 16, 2012 Received on June 27, 2012; revised on October 9, 2012; accepted on
November 16, 2012 The Author 2012. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. A beta-mixture quantile normalization method for correcting
probe design bias in Illumina Infinium 450 k DNA methylation data
Andrew E. Teschendorff1,*, Francesco Marabita2, Matthias Lechner3, Thomas Bartlett1,
Jesper Tegner2, David Gomez-Cabrero2 and Stephan Beck3 A beta-mixture quantile normalization method for correcting
probe design bias in Illumina Infinium 450 k DNA methylation data
Andrew E. Teschendorff1,*, Francesco Marabita2, Matthias Lechner3, Thomas Bartlett1,
Jesper Tegner2, David Gomez-Cabrero2 and Stephan Beck3 p
g
,
p
1Statistical Genomics Group, UCL Cancer Institute, University College London, London WC1E 6BT, UK, 2Department of
Medicine, Unit of Computational Medicine, Centre for Molecular Medicine, Karolinska Institute, Solna 171 76, Stockholm,
Sweden and 3Medical Genomics Group, UCL Cancer Institute, University College London, London WC1E 6BT, UK Associate Editor: Olga Troyanskaya (Baylin and Ohm, 2006; Feinberg et al., 2006; Jones and Baylin,
2007). One particular epigenetic mark of interest is DNA methy-
lation. Indeed, DNA methylation markers have been proposed as
early detection, diagnostic and prognostic markers in a wide range
of different diseases (Rakyan et al., 2011). Underpinning this
increased interest in epigenomics are significant advances in bea-
darray technology, which now allow routine measurement of
DNA methylation at over thousands of CpG dinucleotides
(Bibikova et al., 2009, 2011; Sandoval et al., 2011). Among
these,
the
Illumina
Infinium
450 k
Human
Methylation
Beadchip offers both scalability and coverage (4480 000 probes)
and is thus suitable for Epigenome-Wide Association Studies
(EWAS) (Dedeurwaerder et al., 2011; Rakyan et al., 2011;
Sandoval et al., 2011). Gene expression Advance Access publication November 21, 2012 Advance Access publication November 21, 2012 A beta-mixture quantile normalization method for correcting
probe design bias in Illumina Infinium 450 k DNA methylation data
Andrew E. Teschendorff1,*, Francesco Marabita2, Matthias Lechner3, Thomas Bartlett1,
Jesper Tegner2, David Gomez-Cabrero2 and Stephan Beck3
1Statistical Genomics Group, UCL Cancer Institute, University College London, London WC1E 6BT, UK, 2Department of
Medicine, Unit of Computational Medicine, Centre for Molecular Medicine, Karolinska Institute, Solna 171 76, Stockholm,
Sweden and 3Medical Genomics Group, UCL Cancer Institute, University College London, London WC1E 6BT, UK
Associate Editor: Olga Troyanskaya BIOINFORMATICS
ORIGINAL PAPER BIOINFORMATICS
ORIGINAL PAPER ORIGINAL PAPER Vol. 29 no. 2 2013, pages 189–196
doi:10.1093/bioinformatics/bts680 1
INTRODUCTION In the past few years, the field of epigenomics has risen to prom-
inence (Feinberg, 2010; Petronis, 2010). Epigenomics not only
offers an improved understanding of fundamental biological
processes such as cellular differentiation and early embryogenesis,
but is also widely recognized to be key in understanding
the pathogenesis of
complex
genetic diseases
like cancer Although the PBC method was validated in one dataset
(Dedeurwaerder et al., 2011) and has now been implemented in
a pipeline for 450 k data (Wang et al., 2012), two recent studies
have exposed potential problems with PBC, specially when The Author 2012. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. A.E.Teschendorff et al. (HNCs), of which 18 were HPVþ and 14 HPV, as well as five fresh
frozen HNCs (FF), of which 2 were HPVþ and 3 HPV. The data are
available from GEO under accession number GSE38271. (HNCs), of which 18 were HPVþ and 14 HPV, as well as five fresh
frozen HNCs (FF), of which 2 were HPVþ and 3 HPV. The data are
available from GEO under accession number GSE38271. applied to tissue samples (Maksimovic et al., 2012; Touleimat
and Tost, 2012). In fact, as noted in these studies, PBC breaks
down when the methylation density distribution does not exhibit
well-defined peaks/modes. Hence, both studies proposed subset
quantile normalization methods (SQN and SWAN) to correct
for the type2 bias and which avoid the pitfalls of PBC
(Maksimovic et al., 2012; Touleimat and Tost, 2012). In this
work, we show that PBC often leads to discontinuities (‘holes’)
in the type2 density distribution. To address this problem, we
here propose a novel mixture model-based normalization algo-
rithm, called Beta MIxture Quantile dilation (BMIQ). We sub-
ject BMIQ to a rigorous evaluation using numerous independent
datasets and using a number of different evaluation criteria to
assess its robustness and performance. Specifically, we assess
BMIQ in terms of reducing (i) the technical variance, (ii) the
type2 bias, (iii) and the above-mentioned type1 enrichment
bias. We further benchmark BMIQ against PBC and SWAN. 1
INTRODUCTION For assessing technical variance and to allow a comprehensive
comparison of BMIQ to PBC/SWAN across many datasets, we
use in addition to replicates, a novel evaluation framework based
on using adjacent type1–type2 probe pairs within probe clusters,
a framework which we show leads to consistent and robust con-
clusions across 10 independent datasets. We demonstrate that,
overall, BMIQ compares favourably to PBC and SWAN. Dataset
5: (GBM)
This 450 k dataset consists of 81 glioblastoma
multiformes (GBMs) (Turcan et al., 2012), 49 of which were categorized as
CpG island methylator positive (CIMPþ) and 32 as CIMP. Datasets
6–10: TCGA, LIV, LC, BLDC, HCC
These 450k
samples are all from the TCGA: Specifically, Dataset6 (TCGA) consists
of
10
samples
as
provided
in
the
Bioconductor
data
package
TCGAmethylation 450 k, Dataset7 (LIV) consists of nine normal liver
tissue samples from Batch203 in the TCGA data portal, Dataset8 (LC)
consists of 22 lung cancer samples from Batch196, Dataset9 (BLDC)
consists of 12 bladder cancer samples from Batch86 and Dataset10
(HCC) consists of 10 hepatocellular carcinoma samples from Batch153. 2.2
BMIQ: Beta MIxture Quantile dilation
normalization strategy The normalization of type2 probe values into type1 must satisfy the
following criteria. (i) It must allow for the different biological character-
istics of type1 and type2 probes, i.e type1 probes are significantly more
likely to map to CpG islands than type2 probes, and hence the relative
proportion of methylated and unmethylated probes will vary between the
two designs. In the case of the type2 probes, this means that these pro-
portions must be invariant under the normalization transformation. (ii)
The transformation of the type2 probe values should reduce the bias,
which amounts to matching of the density distributions of the two
design types, specially at the unmethylated and methylated extremes. (iii) The transformation must be monotonic, that is, the relative ranking
of beta values of the type2 probes must be invariant under the transform-
ation. Next, we propose a normalization strategy for the type2 probes
satisfying the above properties and which is based on three steps: 2.1
Biological data: DNA methylation Illumina
Infinium 450k DNAm assay
The DNA methylation
data considered in this work were all generated using Illumina’s
Infinium Human Methylation 450 k beadchip. Full details of this tech-
nology are described in Bibikova et al. (2011) and Sandoval et al. (2011). Briefly, the methylation value of each probe follows an approximate -
valued distribution, with constrained to lie between 0 (unmethylated
locus) and 1 (methylated). This follows from the definition of as the
ratio of methylated to combined intensity values, i.e Fitting of a three-state (unmethylated-U, hemimethylated-H, fully
methylated-M) beta mixture model to the type1 and type2 probes
separately. For sake of convenience we refer to intermediate allelic
methylation as hemimethylation even though hemimethylation
is most often used in the context of strand-specific methylation. Let
aI
U, bI
U
, aI
H, bI
H
, aI
M, bI
M
denote the parameters of the
three beta distributions for the type1 probes, and similarly let
aII
U, bII
U
, aII
H, bII
H
, aII
M, bII
M
describe the estimated parameters of
the three beta components for the type2 probes. State membership
of individual probes is determined by the maximum probability
criterion. ¼
M
U þ M þ e
ð1Þ ð1Þ where U and M are the unmethylated and methylated intensity values of
the probe (averaged over bead replicates) and e ¼ 100 is a small correc-
tion term to regularize probes of low total signal intensity (i.e. probes
with U þ M 0 after background subtraction). Throughout we used
non–background-corrected DNAm data. Of the 485 577 probes, 72%
are of a type2 design in which the U and M measurements are obtained
in different colour channels, while the rest (28%) of the probes are of the
old type1 design in which both U and M measurements are obtained in
the same colour channel. Importantly, type1 and type2 probes differ sig-
nificantly in terms of CpG density, with CpGs mapping to CpGs islands
overrepresented among type1 probes (Bibikova et al., 2011; Sandoval
et al., 2011). where U and M are the unmethylated and methylated intensity values of
the probe (averaged over bead replicates) and e ¼ 100 is a small correc-
tion term to regularize probes of low total signal intensity (i.e. 2.1
Biological data: DNA methylation probes
with U þ M 0 after background subtraction). Throughout we used
non–background-corrected DNAm data. Of the 485 577 probes, 72%
are of a type2 design in which the U and M measurements are obtained
in different colour channels, while the rest (28%) of the probes are of the
old type1 design in which both U and M measurements are obtained in
the same colour channel. Importantly, type1 and type2 probes differ sig-
nificantly in terms of CpG density, with CpGs mapping to CpGs islands
overrepresented among type1 probes (Bibikova et al., 2011; Sandoval
et al., 2011). For those type2 probes assigned to the U-state, transform their
probabilities of belonging to the U-state to quantiles using the in-
verse of the cumulative beta distribution with beta parameters
aI
U, bI
U
estimated from the type1 U component. Let II
U denote
the normalized values of the type2 U-probes. For those type2 probes assigned to the M-state, transform their
probabilities of belonging to the M-state to quantiles using the in-
verse of the cumulative beta distribution with beta parameters
aI
M, bI
M
estimated from the type1 M component. Let II
M denote
the normalized values of the type2 M-probes. Datasets
1 and 2: (BT) and (CL)
This is a subset of the 450 k
dataset considered in Dedeurwaerder et al. (2011). We used the data from
eight fresh frozen (FF) breast tumours and eight normal breast tissue
specimens [hereafter refered to as (BT)], as well as the three replicates
from the HCT116 WT cell-line [hereafer refered to as (CL)]. For these
cell-lines, matched bisulphite pyrosequencing (BPS) data were available
for nine type2 probes. For the type2 probes assigned to the H-state, we perform a dilation
(scale) transformation to ‘fit’ the data into the ‘gap’ with endpoints
defined by max II
U
and min II
M
. We next describe each of the above steps in detail. We first model each
beta value as, We next describe each of the above steps in detail. 2.1
Biological data: DNA methylation S1) to then obtain type2-specific thresholds using a
simple correction reflecting the difference in the modes between the type1
and type2 distributions. Specifically, if tðIÞ
U
is the lower threshold
(i.e. type1 values less than tðIÞ
U are called unmethylated) and UMðIÞ
and UMðIIÞ are the estimated modes of the unmethylated type1 and
type2
components,
the
intial
prior
estimate
for
tðIIÞ
U
would
be tðIIÞ
U ¼ tðIÞ
U þ ðUMðIIÞ UMðIÞÞ. Similarly, the threshold for calling
probes
fully
methylated
or
just
hemi-methylated
would
be
tðIIÞ
M ¼ tðIÞ
M þ ðMMðIIÞ MMðIÞÞ where MM denotes the mode of the
methylated state. We note that resulting thresholds would normally
fall within the ranges 0.2–0.3 and 0.60–0.8, respectively. Having thus
identified reasonable initial estimates for the weights fðIIÞ
U , ðIIÞ
H , ðIIÞ
M g,
the algorithm will then automatically determine the unmethylated, hemi-
methylated and methylated fractions for each sample individually. (ii) A second important observation is the overall robustness of BMIQ
to the goodness of the type2 EM-fit. This is important, since we consis-
tenly observe that the methylated type2 distribution is not well described
by a beta function (Supplementary Fig. S1). In this regard we have
also verified that using a beta mixture model with more than three
states does not improve the overall type2 fit. Fortunately however, as
explained above, the goodness of fit problem associated with the H
and M-probes can be easily circumvented by modelling only the right
tail of the methylated component as the corresponding tail of a beta
distribution. In this case, the left tail is modelled together with the H-
probes using the observed empirical distribution. Hence, the probe values
that are not well described by a beta distribution are not normalized using
estimated beta parameters, which means that their normalization is in-
sensitive to the goodness of fit. mt
s ¼
at
s
at
s þ bt
s
ð3Þ ð3Þ Further, let UII, HII, MII denote the set of type2 probes assigned to
unmethylated, hemimethylated or fully methylated states (using the max-
imum probability criterion), and let UL
II (UR
II) denote the set of UII probes
with -values smaller (larger) than mII
U. Similarly, let ML
II (MR
II) denote the
set of MII probes with -values smaller (larger) than mII
M. 2.1
Biological data: DNA methylation We first model each
beta value as, Datasets
3 and 4: (FFPE) and (FF)
This 450k dataset consists
of 32 formalin-fixed paraffin-embedded (FFPE) head and neck cancers Datasets
3 and 4: (FFPE) and (FF)
This 450k dataset consists
of 32 formalin-fixed paraffin-embedded (FFPE) head and neck cancers ð2Þ pðtÞ ¼ t
UBðjat
U, bt
UÞ þ t
HBðjat
H, bt
HÞ þ t
MBðjat
M, bt
MÞ 190 Adjusting design bias in 450 k studies when performing the conformal transformation. This is because we
observed that it is the left tail end of the methylated type2 distribution
that is generally not well described by a beta-distribution (Supplementary
Fig. S1), presumably as a result of the dye bias, which is specific to the
type2 distribution. where B denotes the beta probability density function and t denotes the
design type t ¼ ðI, IIÞ. We infer the parameters ð, a, bÞ using an
Expectation Maximization (EM) algorithm as described in (Ji et al.,
2005). The estimated parameters we denote again by ðt
s, at
s, bt
sÞ where t
labels the design and s one of the three states (U, H, M). The resulting
means of the estimated beta-distributions are denoted by mt
s where There are a number of other important points to note about BMIQ: (i)
First, it is important to choose reasonable initial weight parameters
fðIIÞ
U , ðIIÞ
H , ðIIÞ
M g in the EM-algorithm. As these fractions can vary signifi-
cantly from study to study, or even sample to sample depending on the
nature of the samples assayed, it is important to choose reasonable initial
values on a per-sample basis. Not doing so may result in mild disconti-
nuities in the type2 density distribution. To obtain estimates for these
prior weight parameters, we first note that their estimation only requires
estimates for the two thresholds used for calling the three states, since the
weights for a given sample are determined given a choice of thresholds. Moreover, although the thresholds will show little inter-sample variabil-
ity, the weights may not, reflecting the biological differences in the
number of probes that are unmethylated, hemimethylated or fully methy-
lated. In BMIQ, the estimation of the initial thresholds proceeds in an
automatic fashion on a per-sample basis: in detail, we use the estimated
thresholds from the type1 distribution (which always gives an excellent fit,
Supplementary Fig. HM ¼ maxH þ minM We want the new normalized maximum and minimum values of
H-probes to satisfy We want the new normalized maximum and minimum values of
H-probes to satisfy nmaxH ¼ minfII
Mg HM nminH ¼ maxfII
Ug þ UH so that ðÞ
H ¼ nmaxH nminH. The normalized -values for the
H-probes is then given by the conformal (shiftþ dilation) transformation 2.1
Biological data: DNA methylation This subdivision
into values which fall left (L) or right (R) of the mean are necessary since
the state membership probabilities estimated from the EM algorithm are
two tailed. Next, for the UL
II probes we estimate their type2 tail probabil-
ities of belonging to the U-state, i.e p ¼ PðUjUL
IIÞ ¼ FðUL
IIjaII
U, bII
UÞ where
F denotes the cumulative distribution beta function. We then transform
these probabilities back to quantiles (i.e -values), but using the type1
parameters, i.e q ¼ F1ðpjaI
U, bI
UÞ
ð4Þ ð4Þ and finally set the normalized -value, UL
II ¼ q. An identical transform-
ation (using 1 F instead of F) is performed for the UR
II probes. Next,
we perform the analogous operation for the ML
II and MR
II probes. This
therefore yields normalized type2 values for all type2 U and M probes. Finally, it remains to normalize the type2 H probes. Since the type2 H
probe value distribution is sandwiched between the U and M probe dis-
tributions, we can use an empirical approach to normalize these values,
thus also bypassing the difficulty that type2 H probe values are not well
described by a beta distribution (Supplementary Fig. S1). Specifically, we
first
identify
the
minima
and
maxima
of
the
type2
H-probes,
maxH ¼ maxfII
Hg and minH ¼ minfII
Hg, and let ðÞ
H ¼ maxH minH. We also find the minimum of the M-probes, i.e minM ¼ minfII
Mg and the
maximum of the U-probes, i.e. maxU ¼ maxfII
Ug. We point out that in
fact all of these extrema represent robust values, because they do not
represent extrema on the bounded (0,1) support, i.e. the values
maxU, minH, maxH and minM are not close to 0 or 1. Next, we define
distances College London on June 18, 2013 UH ¼ minH maxU
HM ¼ maxH þ minM UH ¼ minH maxU HM ¼ maxH þ minM 3.1
Improved robustness of BMIQ II
H ¼ nminH þ dfðII
H minHÞ II
H ¼ nminH þ dfðII
H minHÞ
ð5Þ ð5Þ To validate BMIQ, we first applied it to data where the PBC
method has been shown to work reasonably well. Thus, we
applied it to a fresh frozen breast tumour sample from
Dataset1 (Dedeurwaerder et al., 2011) (Fig. 1A). We can see
that for this particular sample, the methylated type1 peak is
well defined and as a result both PBC and BMIQ appear to do
well in generating smooth density distributions for the type2
probes, which at the methylation extremes are also reasonably
well matched to the type1 density distribution. Next, we applied
both PBC and BMIQ to the FFPE tumour samples from where df ¼ ðÞ
H =ðÞ
H is the dilation factor. It is important to observe that
the conformal transformation involves a non-uniform rescaling of the
H probe beta values since it depends on the beta-value of the probe. This is absolutely key in order to avoid gaps or holes from emerging in
the normalized distribution. This algorithm is flexible in that the dilation can be performed also
including the ML
II (and/or the URÞIIÞ probes, which means that the match-
ing of the density distributions is only done on the respective tails (i.e. the
MR
II and UL
II probes). We point that in practice we find that the optimal
performance is attained by including the ML
II probes with the H-probes 191 A.E.Teschendorff et al. Dataset3, for which the type1 methylated peak was not well
defined (Fig. 1B). In these samples, PBC generated a type2 dens-
ity distribution that exhibited relatively sharp changes (‘holes’)
(Fig. 1B), suggestive of a non-optimal adjustment and indicating
that in such cases PBC breaks down. This is not suprising since
PBC relies heavily on the ability to detect clear unmethylated and methylated modes in the type1 density distribution in order to
then adjust the type2 distribution accordingly. Importantly,
BMIQ does not use the type1 modes to adjust the type2 data,
and hence BMIQ normalization of the type2 probes generated a
much smoother density distribution, suggestive of an improved
normalization framework (Fig. 1B). Moreover, the tail ends of
the BMIQ type2 distribution better matched those of the type1
distribution without affecting the fractions of unmethylated,
hemimethylated and fully methylated probes, which are pre-
served by the BMIQ transformation. Fig. 1. 3.3
BMIQ reduces bias of type2 methylation values Fig. 1. (A) Density distributions of beta-values for the type1 probes,
type2 probes (unnormalized) and normalized type2 probes for a breast
tumour sample from Dataset1. (B) Density distributions of beta-values
for the type1 probes, type2 probes (unnormalized) and normalized type2
probes for a head and neck tumour sample from Dataset3. Left panels
are for PBC, right panels for BMIQ Using replicates to evaluate normalization methods assesses the
method in terms of reducing technical variability but does not
evaluate whether the actual values of the replicates are closer to
the true estimate. This requires comparison with a gold-standard,
which is provided by matched BPS data (Dedeurwaerder et al.,
2011). Hence, we compared the methods in terms of the devi-
ations from BPS methylation values for the nine type2 probes in
Dedeurwaerder et al. (2011) for which matched 450k BPS data
were available. Similar to PBC, we observed that BMIQ signifi-
cantly reduced the bias of type2 values (Fig. 3), although there
was no improvement over PBC itself, presumably owing to the ations from BPS methylation values for the nine type2 probes in
Dedeurwaerder et al. (2011) for which matched 450k BPS data
were available. Similar to PBC, we observed that BMIQ signifi-
cantly reduced the bias of type2 values (Fig. 3), although there
was no improvement over PBC itself, presumably owing to the
Fig. 2. (A) Boxplots comparing the standard deviation (y-axis) of type1
(1) and type2 (2) probes over the three HCT116 WT replicates from
(Dedeurwaerder et al., 2011), for the case of no design normalization
(NONE), PBC and BMIQ. (B) As (A) but now comparing the
Manhattan distances over type2 probes only for each pair of replicates
Fig. 3. Barplot comparing the maximum and mean absolute deviation of
450 k values from their corresponding bisulphite pyrosquencing values
over the nine type2 probes considered in Dedeurwaerder et al., (2011). We compare these deviation measures for the case of no design normal-
ization (NONE), PBC normalization and BMIQ Fig. 2. (A) Boxplots comparing the standard deviation (y-axis) of type1
(1) and type2 (2) probes over the three HCT116 WT replicates from
(Dedeurwaerder et al., 2011), for the case of no design normalization
(NONE), PBC and BMIQ. (B) As (A) but now comparing the
Manhattan distances over type2 probes only for each pair of replicates Fig. 3. 3.1
Improved robustness of BMIQ (A) Density distributions of beta-values for the type1 probes,
type2 probes (unnormalized) and normalized type2 probes for a breast
tumour sample from Dataset1. (B) Density distributions of beta-values
for the type1 probes, type2 probes (unnormalized) and normalized type2
probes for a head and neck tumour sample from Dataset3. Left panels
are for PBC, right panels for BMIQ 3.2
BMIQ reduces technical variation To further test BMIQ we applied it to Dataset2 (CL) consisting
of three replicates of a given cell-line, to investigate if reprodu-
cibility is improved. First, we computed for each of the probes its
standard deviation across the three replicates and for each of the
three scenarios: no normalization, PBC and BMIQ. As seen,
BMIQ performed similarly to PBC and led to a significant re-
duction in inter-replicate variability (Fig. 2A). To check this fur-
ther, we compared the normalization methods in terms of the
Euclidean distance between the three possible pairs of replicates
across the type2 probes (Fig. 2B). Using this measure, BMIQ not
only led to a significant improvement, but was also marginally
better than PBC (Fig. 2B). 3.5
Reduced technical variability within probe clusters 3.5
Reduced technical variability within probe clusters
To further assess BMIQ, we devised an evaluation framework
which exploits the well known spatial correlation of DNA
methylation
at
scales 5500bp
(Eckhardt
et
al.,
2006). Approximately 27% of the 450k probes fall into 12 501 probe
clusters, defined as contiguous regions containing at least seven
probes with no two adjacent probes separated by4300 bp (Jaffe
et al., 2012). Within these probe clusters, we posited that pairs of
adjacent probes, one from each design and within 200 bp of each
other, should have similar methylation values. Among the 12 501
probe clusters we identified on the order of 30 000 of such
adjacent type1–type2 probe pairs. Thus, to evaluate the normal-
ization algorithms, we asked which one minimizes the absolute
difference in methylation between such closely adjacent type1–
type2 pairs. We considered a total of 10 independent datasets,
seven of which had idat files, thus allowing also for a direct
comparison with SWAN (Maksimovic et al., 2012). For each
dataset, we computed the mean of the absolute deviations over
probe pairs and samples. Comparison of these average deviations
revealed that BMIQ consistently reduced the technical variation,
while also outperforming PBC and SWAN (Table 1). In fact, in
9 of 10 datasets, BMIQ was substantially better as assessed using
a paired Wilcoxon rank sum test over all probe pairs and samples
(Table 1). Example methylation profiles within these probe
clusters confirmed that BMIQ successfully reduces the technical
variability, while PBC can break down either overcorrecting
or suppressing the type2 data values, leading to substantial dif-
ferences in methylation between neighbouring probes, even at
scales of5100bp (Fig. 5 and Supplementary Table S1). Fig. 4. OR of relative enrichment of type1 probes over type2 probes among the top 100, top 250, top 500 and top 1000 ranked probes, where probes were
ranked according to the absolute difference in methylation (beta-values). ORs are shown for the case of no design normalization (NONE), PBC and BMIQ
normalization. Supervised analysis and ranking was performed only on probes mapping to CpG islands and within 200bp upstream of transcription start
site to correct for biological differences between type1 and type2 probes. The line OR¼ 1 represents the ideal scenario of no relative enrichment of type1
versus type2 probes. The 95% confidence envelope around OR¼ 1 is shown to assess significant deviations from OR¼ 1. 3.3
BMIQ reduces bias of type2 methylation values Barplot comparing the maximum and mean absolute deviation of
450 k values from their corresponding bisulphite pyrosquencing values
over the nine type2 probes considered in Dedeurwaerder et al., (2011). We compare these deviation measures for the case of no design normal-
ization (NONE), PBC normalization and BMIQ Fig. 3. Barplot comparing the maximum and mean absolute deviation of
450 k values from their corresponding bisulphite pyrosquencing values
over the nine type2 probes considered in Dedeurwaerder et al., (2011). We compare these deviation measures for the case of no design normal-
ization (NONE), PBC normalization and BMIQ Fig. 2. (A) Boxplots comparing the standard deviation (y-axis) of type1
(1) and type2 (2) probes over the three HCT116 WT replicates from
(Dedeurwaerder et al., 2011), for the case of no design normalization
(NONE), PBC and BMIQ. (B) As (A) but now comparing the
Manhattan distances over type2 probes only for each pair of replicates 192 Adjusting design bias in 450 k studies fact that in these specific samples the methylated type1 peak was
well defined, a scenario in which PBC works well. fact that in these specific samples the methylated type1 peak was
well defined, a scenario in which PBC works well. probes. In contrast, BMIQ eliminated the type1 enrichment
bias in all three datasets (the resulting OR was always close
to 1) without overcorrecting the data and avoiding the type2
enrichment bias seen for PBC. 3.4
BMIQ eliminates the type1 enrichment bias To further test BMIQ, we considered the supervised context, in
which a ranked list of probes correlating with a phenotype of
interest is derived. Given the higher dynamic range of type1
probes, one expects that this would favour type1 probes and
that therefore there would be a relative over enrichment of
type1 over type2 probes in a top ranked list of probes. However, one key difficulty when assessing whether there is a
bias towards type1 probes is that type1 and type2 probes differ
significantly in terms of their biological characteristics, in par-
ticular in terms of CpG density. Hence, in order to avoid con-
founding by CpG density, we only selected probes that mapped
to CpG islands and to 200 bp upstream of the TSS, thus allowing
a sensible comparison between type1 and type2 probes. We con-
sidered three different datasets and derived for each a ranked list
of probes associated with a phenotype of interest: breast cancer
versus normal breast [Dataset1 (BT)], HPVþ versus HPVHNC
[Dataset3
(FFPE)],
and
CIMPþ
versus
CIMP
(GBM)
(Dataset5). The ranking was performed using the magnitude of
differential methylation. Although this ranking does not take the
within-phenotype variability into account, it remains a popular
method (Dedeurwaerder et al., 2011; Du et al., 2010), and for our
purposes, using the absolute difference in beta-values allows us
to better interpret the performance of the different normalization
methods. To assess any potential bias towards type1 probes, we
computed for a given number of top ranked probes the odds
ratio (OR) of relative enrichment of type1 over type2 probes. Across all three datasets, we indeed observed a bias towards
type1 probes, although the severity of this bias varied substan-
tially from study to study (Fig. 4). Using PBC, in one dataset this
bias was eliminated; however, in the other two datasets, PBC
overcorrected the data leading to a bias favouring type2 3.5
Reduced technical variability within probe clusters (A) Eight breast cancers versus
eight normal breast (Dataset1), (B) 18 HPVþ HNCs versus 14 HPV HNCs (Dataset3), (C) 49 CIMPþ GBMs versus 32 CIMP GBMs (Dataset5) Fig. 4. OR of relative enrichment of type1 probes over type2 probes among the top 100, top 250, top 500 and top 1000 ranked probes, where probes were
ranked according to the absolute difference in methylation (beta-values). ORs are shown for the case of no design normalization (NONE), PBC and BMIQ
normalization. Supervised analysis and ranking was performed only on probes mapping to CpG islands and within 200bp upstream of transcription start
site to correct for biological differences between type1 and type2 probes. The line OR¼ 1 represents the ideal scenario of no relative enrichment of type1
versus type2 probes. The 95% confidence envelope around OR¼ 1 is shown to assess significant deviations from OR¼ 1. (A) Eight breast cancers versus
eight normal breast (Dataset1), (B) 18 HPVþ HNCs versus 14 HPV HNCs (Dataset3), (C) 49 CIMPþ GBMs versus 32 CIMP GBMs (Dataset5) 193 A.E.Teschendorff et al. 3.6
BMIQ robustly identifies features associated
with HPV status of sensitivity and positive predictive value (PPV) between the
different normalization methods. To perform this analysis,
we used Dataset4(FF) consisting of 2 HPVþ and 3 HPV
fresh frozen head and neck cancers to derive features associated
with HPV status. As test set we used Dataset3(FFPE) consisting
of 18 HPVþ and 14 HPV head and neck cancers (FFPE
tissue). Using limma (Smyth, 2005) and an FDR threshold
of 0.35, we observed that BMIQ identified substantially more
differentially
methylated
features
than
PBC
or
SWAN
(Table 2). Importantly, this was not at the expense of a smaller
PPV, and so, overall, BMIQ identified substantially more
true positives (Table 2). Finally, it must be verified that the reduction in technical vari-
ance obtained with BMIQ is not at the expense of a reduced
biological signal. Since it is difficult to establish what constitutes
a true positive, we used a training test set strategy, to identify
features in a training set and calling them true positives if vali-
dated in a test set. This strategy thus allows for a comparison Table 1. For each dataset we compare the absolute deviation in methy-
lation between adjacent type1–type2 probe pairs (probes within 200 bp of
each other), averaged over probe pairs and samples, for four different
normalisation methods 4
DISCUSSION Dataset
NONE
(%)
PBC
(%)
SWAN
(%)
BMIQ
(%)
P
BT
7.8
6.3
NA
6.2
51010
CL
8.6
18.4
NA
7.2
51010
FFPE
9.2
8.0
8.5
7.8
51010
FF
8.5
8.1
7.6
7.3
51010
GBM
9.2
7.6
NA
7.5
51010
TCGA
9.4
7.8
8.3
7.4
51010
LIV
10.0
6.3
7.4
6.4
1
LC
10.3
7.0
7.7
6.7
51010
BLDC
11.0
8.0
7.9
7.6
51010
HCC
12.0
8.5
8.7
8.1
51010
NONE refers to the case of no adjustment for probe design type. The last column
give the paired Wilcoxon rank sum test P-value (treating each probe-pair deviation in
each sample as a separate value), assessing the statistical significance that the absolute
deviation for BMIQ is smaller than the next best competing method. NA indicates
non-available owing to lack of access to idat files needed for processing by SWAN. In bold-face we show the smallest deviation across methods. Dataset
NONE
(%)
PBC
(%)
SWAN
(%)
BMIQ
(%)
P
BT
7.8
6.3
NA
6.2
51010
CL
8.6
18.4
NA
7.2
51010
FFPE
9.2
8.0
8.5
7.8
51010
FF
8.5
8.1
7.6
7.3
51010
GBM
9.2
7.6
NA
7.5
51010
TCGA
9.4
7.8
8.3
7.4
51010
LIV
10.0
6.3
7.4
6.4
1
LC
10.3
7.0
7.7
6.7
51010
BLDC
11.0
8.0
7.9
7.6
51010
10 In this work we have presented a novel mixture-model-based
algorithm (BMIQ) for correcting the bias associated with
type2 probe values in 450k studies. Confirming the observations Table 2. Table listing the number of differentially methylated probes
(nDMPs) associated with HPV status in Dataset4 (FF), and the corres-
ponding estimates for the positive predictive value (PPV) and number of
true positives (nTPs) estimated using Dataset3 (FFPE) as test set Metric
NONE
PBC
SWAN
BMIQ
nDMP
51
70
41
252
PPV
0.25
0.18
0.19
0.20
nTP
13
13
8
51
DMPs were defined at an FDR threshold of 0.35, and those with the same sign of
limma t-statistic in the two sets and with a corresponding P-value50.01 in the test
set were deemed true positives. NONE refers to the case of no adjustment for probe design type. The last column
give the paired Wilcoxon rank sum test P-value (treating each probe-pair deviation in
each sample as a separate value), assessing the statistical significance that the absolute
deviation for BMIQ is smaller than the next best competing method. 4
DISCUSSION NA indicates
non-available owing to lack of access to idat files needed for processing by SWAN. In bold-face we show the smallest deviation across methods. DMPs were defined at an FDR threshold of 0.35, and those with the same sign of
limma t-statistic in the two sets and with a corresponding P-value50.01 in the test
set were deemed true positives. Fig. 5. Examples of methylation profiles, from Dataset4(FF), of three probe clusters on chromosomes 19, 1 and 15, respectively. The design type of each
probe is indicated with 1 and 2. The non design normalized data (NONE), PBC, SWAN and BMIQ corrected data are superimposed. Observe how
across the three loci, BMIQ generally corrects the data in a way which is more consistent with the neighbouring type1 values. In the left panel PBC
overcorrects the data, in the right panel there is suppression, while in the middle panel PBC both overcorrects and suppresses beta values. The data values
can be found in Supplementary Table S1 Fig. 5. Examples of methylation profiles, from Dataset4(FF), of three probe clusters on chromosomes 19, 1 and 15, respectively. The design type of each
probe is indicated with 1 and 2. The non design normalized data (NONE), PBC, SWAN and BMIQ corrected data are superimposed. Observe how
across the three loci, BMIQ generally corrects the data in a way which is more consistent with the neighbouring type1 values. In the left panel PBC
overcorrects the data, in the right panel there is suppression, while in the middle panel PBC both overcorrects and suppresses beta values. The data values
can be found in Supplementary Table S1 194 Adjusting design bias in 450 k studies made in Touleimat and Tost (2012) and Maksimovic et al. (2012), we have seen that PBC can break down in samples
with ill-defined type1 methylation peaks, causing sharp, almost
discontinuous changes (which we call ‘holes’) in the density dis-
tributions (Fig. 1B), which motivated our quest to find a more
robust algorithm. We have shown that BMIQ improves the
robustness and can successfully normalize the type2 distribution,
avoiding the appearance of such ‘holes’ (Fig. 1B). Moreover,
BMIQ successfully matches the tail-ends of the type1 and
type2 distributions, while faithfully preserving the proportions
of unmethylated and methylated probes within each of the
two designs. 4
DISCUSSION In summary, using a number of different evaluation criteria
and numerous datasets, we have seen that BMIQ compares fa-
vourably with both PBC and SWAN. Although we did not com-
pare BMIQ to SQN (Touleimat and Tost, 2012), this latter
method is very similar to SWAN, as they both rely on a probe
subset quantile normalization. Like SQN/SWAN, BMIQ uses
quantiles to normalize the type2 probe values into a distribution
that is comparable with that of type1 probes. However, unlike
SQN and SWAN, BMIQ is based on an explicit beta-mixture
modeling framework, and uses state-membership probabilities
under this beta mixture model to reassign the quantiles of
the type2 probes according to the type1 distribution. Thus,
BMIQ is assumption-free, as it does not require a separate
normalization to be performed on selected subsets of probes
that are matched for biological characteristics (e.g. CpG density),
as done in SQN and SWAN. In fact, under the BMIQ frame-
work, all the biological differences (including CpG density)
between the type1 and type2 probes are captured by the esti-
mated fractions of unmethylated, hemimethylated and methy-
lated probes, which will be different between the two assays. Thus, BMIQ does not depend on a priori and somewhat
arbitrary choices of which biological characteristics to use
when matching the type1 and type2 distributions. For instance,
in SQN the normalization is performed on probe subsets defined
by specific CpG characteristics (e.g. shelves, shores, CpG is-
lands); however, multiple different definitions for say CpG is-
lands exist (Gardiner-Garden and Frommer, 1987; Takai and
Jones, 2002; Wu et al., 2010; Zhao et al., 2009). Similarly, in
SWAN the number of CpGs in the probe body, even if they
differ by one, is used to define probe normalization categories,
and thus it is unclear whether these probe categories represent an
optimal way of dividing the probes up. Therefore, we see the
beta-mixture model framework of BMIQ as an important con-
ceptual advantage over SQN/SWAN, since, as demonstrated
here, it successfully normalises type2 probe values, faithfully pre-
serving the numerous and complex biological differences that
exist between the two designs without ever needing to define
probe subsets. Nevertheless, it will be interesting to conduct a
comprehensive and detailed comparison of BMIQ, SQN and
SWAN on matched 450k BPS data on a sufficiently large
number of loci and samples. 4
DISCUSSION To further test BMIQ, we used data on technical replicates (to
show that it reduces technical variability) and matched BPS data
(to show that it reduces the bias of type2 probe values). Using
these criteria, we have seen that BMIQ leads to significant im-
provements, similar to the improvements noted for PBC (Figs 2
and 3). In relation to these evaluation criteria, it is worth point-
ing out that BMIQ was compared with PBC on samples with
well-defined type1 methylation peaks, i.e. on data that were used
to develop PBC itself. Hence, it is likely that an evaluation of
technical reproducibility (using replicates) and type2 value bias
(using matched BPS data) on data where the methylated type1
peaks are less well-defined would favour BMIQ over PBC. However, we did not have access to technical replicates or
matched BPS data in the other specific datasets considered
here. Therefore, in order to further assess BMIQ, we devised a
supervised framework across three independent datasets to ob-
jectively compare the algorithms in their ability to reduce the
expected enrichment bias of type1 probes. First, we showed
that if no design normalization is performed then there is
indeed an enrichment bias towards type1 probes, even when ad-
justed for CpG density (Fig. 4). We also showed that in two
datasets, PBC overcorrected the type2 data, leading to an over-
inflated dynamic range, thus favouring type2 probes and causing
an ‘overshooting’ of the enrichment scores, reflected by a signifi-
cant underenrichment of type1 probes (Fig. 4B and C). In con-
trast, BMIQ successfully avoided any type1/type2 enrichment
bias in all three datasets, indicative of an improved normaliza-
tion of type2 values (Fig. 4). We should point out that the over-
correction of type2 values and the associated overinflated
dynamic range caused by PBC is consistent with the presence
of ‘holes’ in the hemimethylated region of the type2 density dis-
tribution. Thus, with PBC there is an artificial expulsion of data
points from the hemimethylated region to the unmethylated and/
or methylated extremes. In a further assessment of BMIQ, we
conducted a detailed spatial analysis of DNA methylation at the
level of probe clusters across 10 independent datasets. 4
DISCUSSION By care-
fully analysing adjacent type1–type2 probe pairs, we observed
that PBC can often overcorrect or suppress the data (in some
cases inducing abnormally large 30% changes in methylation), in
contrast to BMIQ, which normalized type2 values in a way that
rendered them more consistent with the values of neighbouring
type1 probes (Fig. 5 and Table 1). Interestingly, BMIQ also
appeared to outperform SWAN (Table 1), which is part of the
popular and widely used minfi package (Hansen and Aryee,
2012). Of note, the reduction in technical variance achieved
by BMIQ was not at the expense of a lower biological
signal (Table 2). at University College London on June 18, 2013
http://bioinformatics.oxfordjournals.org/
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illumina infinium humanmethylation450 beadchips. Genome Biol., 13, R44. r early oncogenic pathway addiction? Nat. Rev. Cancer, 6, 107–116 lumina infinium humanmethylation450 beadchips. Genome Biol., 13 Bibikova,M. et al. (2011) High density DNA methylation array with single CpG site
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diseases. Nat. Rev. Genet., 12, 529–541. Dedeurwaerder,S. et al. (2011) Evaluation of the infinium methylation 450k tech-
nology. Epigenomics, 3, 771–784. Sandoval,J. et al. (2011) Validation of a DNA methylation microarray for 450,000
CpG sites in the human genome. Epigenetics, 6, 692–702. Sandoval,J. et al. (2011) Validation of a DNA methylation m CpG sites in the human genome. Epigenetics, 6, 692–702 Du,P. et al. (2010) Comparison of Beta-value and M-value methods for quantifying
methylation levels by microarray analysis. BMC Bioinformatics, 11, 587. Smyth,G.K. (2005) Limma: linear models for microarray data. In: Gentleman,R. et al. (eds) Bioinformatics and Computational Biology Solutions using R and
Bioconductor. Springer, New York, pp. 397–420. methylation levels by microarray analysis. BMC Bioinformatics, 11, Eckhardt,F. et al. (2006) DNA methylation profiling of human chromosomes 6,
20 and 22. Nat. Genet., 38, 1378–1385. Takai,D. and Jones,P.A. (2002) Comprehensive analysis of cpg islands in human
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accurate dna methylation estimation. Epigenomics, 4, 325–341. Gardiner-Garden,M. and Frommer,M. (1987) CpG islands in vertebrate genomes. J. Mol. Biol., 196, 261–282. Turcan,S. et al. (2012) IDH1 mutation is sufficient to establish the glioma hyper-
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CONCLUSIONS We have presented a mixture model assumption-free normaliza-
tion algorithm, BMIQ, which will be useful for correcting the
bias associated with the type2 assay in DNA methylation studies
using the Illumina Infinium 450k platform. Funding: A.E.T. is supported by a Heller Research Fellowship. M.L. was supported by a Wellcome Trust Research Training
Fellowship (093855). S.B. was supported by a Royal Society
Wolfson Research Merit Award (WM100023) and grants from
the Wellcome Trust (084071) and EU-FP7 BLUEPRINT
(282510). We also thank FP7 SYNERGY-COPD (F.B., D.G.,
J.T.), BILS (D.G.) and Stockholm County (J.T.). Conflict of Interest: none declared. 195 A.E.Teschendorff et al. REFERENCES and Aryee,M. (2012) Minfi: Analyze Illumina’s 450k Methylation
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na’s 450k infinium methylation data. Bioinformatics, 28, 729–730. Houseman,E.A. et al. (2008) Model-based clustering of dna methylation array data:
a recursive-partitioning algorithm for high-dimensional data arising as a mixture
of beta distributions. BMC Bioinformatics, 9, 365. Wu,H. et al. (2010) Redefining CpG islands using hidden Markov models. Biostatistics, 11, 499–514. of beta distributions. BMC Bioinformatics, 9, 365 Zhao,Q. et
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https://www.nature.com/articles/ncomms7539.pdf
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English
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Aqueous proton transfer across single-layer graphene
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Nature communications
| 2,015
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cc-by
| 8,895
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ARTICLE Received 5 Nov 2014 | Accepted 6 Feb 2015 | Published 17 Mar 2015 & 2015 Macmillan Publishers Limited. All rights reserved. Aqueous proton transfer across single-layer
graphene B
r
n
s charge density, in the duration of the jumps or in the rates of the
jumps when single-layer graphene is present. These findings
indicate that the acid–base chemistry at the fused silica/water
interface occurs in an unimpeded fashion in the presence of
single-layer graphene. Importance of macroscopic defects ruled out. Scanning electron
microscopy (SEM, Methods) images of graphene single layers
deposited on fused silica windows show a low density of macro-
scopic pinholes and that the graphene is free of cracks or folds
(Fig. 1c). Two-dimensional (2D) diffusion from those locations to
the location of the laser beam is considered by calculating, for a
given proton diffusion coefficient D, the mean-square displace-
ment, hDr2i, according to hDr2i ¼ z t D, where t is time and z is
the number of neighbouring sites to which the proton can hop23
(six in for the case of the hexagonal graphene lattice). In the
literature, reported theoretically and experimentally determined
proton surface diffusion coefficients range between 1.01 10 7
and 9.00 10 5 cm2 s 1 (refs 24–39). While the bulk diffusion
coefficient for a proton in water is accepted to range between
8 10 5 and 9 10 5 cm2 s 1, there are disagreements in the
literature about whether the surface proton diffusion coefficient is
similar to the bulk coefficient or slower than the bulk coefficient
on hydrophobic and hydrophilic surfaces for a variety of different
systems24,28,38. Reactivity is expected to substantially slow down
the 2D diffusion of the proton (approximately magnitude 20
reduction)30,40,41 when it moves across an amphoteric oxide
whose protonation effectively terminates the diffusion path. Reactive proton diffusion coefficients reported for Nafion42,43 are
similarly low. Indeed, our own reactive force field calculations
containing partially hydroxylated quartz surfaces show the proton
diffusion is quickly terminated by protonation of the surface
SiO groups (Supplementary Note 4 and Supplementary Fig. 6). This result indicates that proton diffusion is significantly slower
in the presence of surface anionic species due to proton trapping
at these sites. Aqueous proton transfer across single-layer
graphene ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7539 B
rick-and-mortar
networks
of
stacked
graphene
oxide
nanosheets can act as effective membranes1–8, while
single-layer
graphene
exhibits
dramatically
lower
permeabilities towards gases4,9. In fact, graphene is thought to
be unfit even for proton transfer, which is associated with
computed gas-phase energy barriers exceeding 1.4 eV (ref. 10)
unless
dopants
or
nanoscale
openings
are
externally
introduced6,7,10,11, or an external potential bias is applied12. To
determine whether graphene is indeed impermeable to protons,
we place well-characterized single-layer graphene13 on top of a
fused silica substrate and cycle, at room temperature and constant
ionic strength, the bulk pH of an aqueous solution above the
graphene layer between basic and acidic. We test for proton
exchange through graphene by probing the underlying silica
surface with an interfacial potential-dependent version of second
harmonic generation (SHG)14,15 using 120 fs input pulses at
energies well below the graphene damage threshold13. With a
detection limit of 10 5 to 10 6 V (ref. 16), the method is
sensitive enough to follow protonation or deprotonation of as
little as 1% of the available silanol groups present in the area
probed by SHG. The interfacial potential vanishes at the point of
zero charge (PZC of fused silica B2.5) (ref. 17), and the SHG
signal intensity is small14,18,19. Increasing the pH at constant
ionic strength shifts the relevant interfacial acid–base equilibria
SiOH2þ þ OH "SiOH þ H2O
and
SiOH þ OH "SiO
þ H2O (pKa B4.5 and B8.5)14,18,20 to the right, and the
resulting interfacial potential polarizes the interfacial water
molecules such that the SHG signal intensity increases14,18. Intuitively, the close proximity of the graphene layer and the
charged fused silica surface, combined with the sensitivity of the
method, make our approach akin to an Å-scale voltmeter for
detecting even rare occurrences of proton exchange. We find no
significant difference between the SHG versus time traces
recorded in the presence and absence of graphene. After ruling
out diffusion through macroscopic pinholes, the protons are
found to transfer through rare, naturally occurring atomic defect
sites in the graphene layer. Computer simulations reveal low
energy processes for water-mediated proton transfer across
hydroxyl-terminated atomic defect sites that participate in a
Grotthuss-type relay, while defects terminated by pyrylium-like
ether bridges shut down proton exchange. Aqueous proton transfer across single-layer
graphene In our experiments, the continuous proton supply from the
aqueous bulk is expected to form a propagating reaction front:
our calculations show a drastically increased proton diffusion
coefficient of 4.944 10 5 cm2 s 1, or just half of that of bulk
water, once protons arriving through any opening within the
graphene sheet interact with the hydroxylated portion of the
surface that is located behind the reaction front. To conservatively
assess an upper bound limit for our estimations, we calculated the
proton mean-square displacement using a D-value of 1 10 6
cm2 s 1. The probability of placing our laser beam within the
propagating reaction front emanating from a given macroscopic
pinhole was then estimated to be 4% and 21% for 1 and 10 s SHG
jump times, respectively (Supplementary Note 5, Supplementary
Figs. 7–10 and Supplementary Table 3). Given that the pH jumps
were repeated on at least 18 different days with eight different
graphene samples and delays in changes of the SHG response
were not observed with statistical significance; so we conclude
that the diffusion of protons from the few macroscopic pinholes
that are present in our samples, or, alternatively, from the sample
edge, to the area probed by the laser cannot explain our
observations of proton transfer through graphene. Aqueous proton transfer across single-layer
graphene Jennifer L. Achtyl1, Raymond R. Unocic2, Lijun Xu3, Yu Cai3, Muralikrishna Raju5, Weiwei Zhang6,
Robert L. Sacci7, Ivan V. Vlassiouk7, Pasquale F. Fulvio8,9, Panchapakesan Ganesh2, David J. Wesolowski9,
Sheng Dai9, Adri C.T. van Duin6, Matthew Neurock3,4 & Franz M. Geiger1 Jennifer L. Achtyl1, Raymond R. Unocic2, Lijun Xu3, Yu Cai3, Muralikrishna Raju5, Weiwei Zhang6,
Robert L. Sacci7, Ivan V. Vlassiouk7, Pasquale F. Fulvio8,9, Panchapakesan Ganesh2, David J. Wesolowski9,
Sheng Dai9, Adri C.T. van Duin6, Matthew Neurock3,4 & Franz M. Geiger1 Proton transfer across single-layer graphene proceeds with large computed energy
barriers and is therefore thought to be unfavourable at room temperature unless nanoscale
holes or dopants are introduced, or a potential bias is applied. Here we subject single-layer
graphene supported on fused silica to cycles of high and low pH, and show that protons
transfer reversibly from the aqueous phase through the graphene to the other side where
they undergo acid–base chemistry with the silica hydroxyl groups. After ruling out diffusion
through macroscopic pinholes, the protons are found to transfer through rare, naturally
occurring atomic defects. Computer simulations reveal low energy barriers of 0.61–0.75 eV
for aqueous proton transfer across hydroxyl-terminated atomic defects that participate in a
Grotthuss-type relay, while pyrylium-like ether terminations shut down proton exchange. Unfavourable energy barriers to helium and hydrogen transfer indicate the process is
selective for aqueous protons. 1 Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60201, USA. 2 Center for Nanophase Materials Sciences, Oak
Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA. 3 Departments of Chemical Engineering and Chemistry, University of Virginia, 102 Engineers’
Way, Charlottesville, Virginia 22904-4741, USA. 4 Department of Chemical Engineering and Materials Science, University of Minnesota, 421 Washington
Avenue SE, Minneapolis, Minnesota 55455, USA. 5 Department of Physics, Pennsylvania State University, University Park, Pennsylvania 16802, USA. 6 Department of Mechanical and Nuclear Engineering, Pennsylvania State University, University Park, Pennsylvania 16801, USA. 7 Measurement Science and
Systems Engineering Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37931, USA. 8 Department of Chemistry, University of Puerto Rico, Rı´o
Piedras Campus; San Juan, Puerto Rico 00931, USA. 9 Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA. Correspondence and requests for materials should be addressed to F.M.G. (email: geigerf@chem.northwestern.edu). 1 NATURE COMMUNICATIONS | 6:6539 | DOI: 10.1038/ncomms7539 | www.nature.com/naturecommunications URE COMMUNICATIONS | 6:6539 | DOI: 10.1038/ncomms7539 | www & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | 6:6539 | DOI: 10.1038/ncomms7539 | www.nature.com/naturecommunications
& 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7539 (b) p-in/all-out polarized SHG response recorded as a
function of time from the fused silica/water interface during pH jumps from 7 to 3 to 10 and subsequent pH cycling between 3 and 10 at a bulk aqueous
flow of 0.9 ml/s and 1 mM NaCl concentration in the absence (crimson, bottom) and presence (black, top, offset for clarity) of single-layer graphene placed
between the fused silica substrate and the flowing bulk aqueous phase. Five-point boxcar indicated by dark lines. (c) Composite of 25 SEM images of
single-layer graphene on a fused silica substrate, showing seven macroscopic pinholes, marked by white circles. (d) High-resolution aberration-corrected
ADF STEM images of defect-free single-layer graphene on a transmission electron microscopy grid and (e) of a rarely imaged atomic defect. Scale, 1 nm. We find that the main restriction for aqueous proton transfer
through pristine, defect-free graphene is the energy required to
push the proton through the center of an aromatic ring in the
hydrophobic graphene layer as shown in Supplementary Fig. 11. While the protons readily migrate in the solution phase above
and below the graphene surface via proton shuttling, they are
unable to pass through the hydrophobic graphene layer. The
energy costs to desolvate the proton from the aqueous layer and
drive it through the center of an intact aromatic ring within the
graphene layer are quite high and result in an activation barrier,
that is, over 3.8 eV. point defects
, , we find, albeit rarely, atomic defects (Fig. 1e). Unless hydrocarbons or heavy metal atoms46 are present in
graphene, defect formation due to electron beam-induced etching
(as opposed to ion bombardment or oxidative etching)47 of
pristine chemical vapour deposition (CVD) graphene at the
energies employed here is unlikely. Rather, the rare defects we
observe on occasion are more likely to originate from the
synthesis process or cosmic rays, as the STEM experiments are
carried out below the knock-on damage threshold for graphene48,
and the femtosecond laser pulses are attenuated below the onset
of processes other than SHG13. Given a lower limit to the
estimated defect-to-defect distance of B0.1 mm (ref. 49) (while
difficult to determine accurately from Raman spectroscopy, the
actual distance is likely to be longer), we assess the probability of
placing our laser beam within the propagating reaction front
emanating from a given atomic defect to be 100%. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7539 Mirror
/2 plate
Lens
Lens
Lens
Fundamental (ω)
600 nm
Mirror
Graphene film
Ring of
water
Teflon flow cell
pH 7
10
10
10
10
10
10
10
3
3
3
3
3
3
3
3
2
Normalized lSHG
1
0
0
1,200
2,400
3,600
4,800
6,000
7,200
8,400
Time (s)
Outlet
Inlet
Solution
#2
PMT
40 × 30 μm2
Solution
#1
Waste
Fused silica hemisphere
Fused silica window
SHG (2ω)
pH
conductivity
Filter
Figure 1 | Experimental approach. (a) Experimental setup using a waveplate (l/2) to prepare 600 nm light plane-polarized parallel to the plane of
incidence (p-in) while a photomultiplier tube (PMT) detects the SHG photons at l ¼ 300 nm. (b) p-in/all-out polarized SHG response recorded as a
function of time from the fused silica/water interface during pH jumps from 7 to 3 to 10 and subsequent pH cycling between 3 and 10 at a bulk aqueous
flow of 0.9 ml/s and 1 mM NaCl concentration in the absence (crimson, bottom) and presence (black, top, offset for clarity) of single-layer graphene placed
between the fused silica substrate and the flowing bulk aqueous phase. Five-point boxcar indicated by dark lines. (c) Composite of 25 SEM images of
single-layer graphene on a fused silica substrate, showing seven macroscopic pinholes, marked by white circles. (d) High-resolution aberration-corrected
ADF STEM images of defect-free single-layer graphene on a transmission electron microscopy grid and (e) of a rarely imaged atomic defect. Scale, 1 nm Mirror
/2 plate
Lens
Lens
Lens
Fundamental (ω)
600 nm
Mirror
Graphene film
Ring of
water
Teflon flow cell
Outlet
Inlet
Solution
#2
PMT
Solution
#1
Waste
Fused silica hemisphere
Fused silica window
SHG (2ω)
pH
conductivity
Filter 40 × 30 μm2 pH 7
10
10
10
10
10
10
10
3
3
3
3
3
3
3
3
2
Normalized lSHG
1
0
0
1,200
2,400
3,600
4,800
6,000
7,200
8,400
Time (s) Figure 1 | Experimental approach. (a) Experimental setup using a waveplate (l/2) to prepare 600 nm light plane-polarized parallel to the plane of
incidence (p-in) while a photomultiplier tube (PMT) detects the SHG photons at l ¼ 300 nm. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7539 The basal planes of pristine graphene can, and do, contain rare
atomic-scale defect sites comprised of carbon atom vacancies, as
shown in Fig. 1e. Our calculations indicate that while the
activation barrier for proton transfer through a single-vacancy
site is over 1.9 eV lower than that for transfer through the pristine
graphene surface, it is still nearly 2.0 eV due to the small size of
the vacancy and the hydrophobicity of the surface. The formation
of di- and tri-vacancy sites increases the diameter of the opening
in the graphene layer and reduces the barrier further to B1.5 eV,
but this barrier is still too high to permit aqueous proton transfer
at room temperature. Results
l
l Silanol protonation and deprotonation unimpeded by graphene. Using a dual-pump flow system (Fig. 1a) at a flow rate of at
B0.9 ml s 1, we varied the bulk solution pH between 3 and 10
while maintaining constant 1 mM ionic strength (see Methods). As shown in Fig. 1b, we find no significant difference between the
SHG versus time traces recorded in the presence and absence of
graphene, and no statistically significant differences in the kinetic
rates and jump durations (Supplementary Note 1, Supplementary
Figs 1–2 and Supplementary Tables 1–2). The SHG responses to
pH changes are consistent with the acid–base equilibria of the
fused silica/water interface14,15,19,21, yielding effective pKa,eff
values of 3.5(1) and 8.3(2), which fall within the reported
literature values (Supplementary Note 2 and Supplementary
Fig. 3)22. This finding indicates that the SHG experiments do not
track merely ion adsorption to the graphene/water interface but
acid–base chemistry at the fused silica surface underneath it,
for which proton transfer across the membrane is a necessary
condition. As
expected
from
refs
1–5,
porous
graphene
multilayers
do
not
inhibit
proton
transfer
either
(Supplementary Note 3 and Supplementary Figs 4–5). On the
basis of these results we conclude that the fused silica/water
interface does not behave differently in terms of relative surface Imaging rare atomic defects. Scanning transmission electron
microscopy (STEM) was then used to search for atomic defects
using annular dark field (ADF) STEM imaging at 60 kV (see
Methods). The majority of the images show perfect six-fold
symmetry in the position of the carbon atoms and vast areas that
lack grain boundaries and atomic, or vacancy, defects (Fig. 1d). Nevertheless, similar to prior reports of atomic-scale vacancy NATURE COMMUNICATIONS | 6:6539 | DOI: 10.1038/ncomms7539 | www.nature.com/naturecommunications
& 2015 Macmillan Publishers Limited. All rights reserved. 2 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7539 ARTICLE & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | 6:6539 | DOI: 10.1038/ncomms7539 | www.nature.com/naturecommunications
& 2015 Macmillan Publishers Limited. All rights reserved. Discussion
T
l
d Snapshots (c,d) and energetics (e,f) from the nudged elastic
band calculations for proton transfer through the oxygen- and OH-
terminated defect sites marking (region I) release of proton from H3O þ to
oxygen and OH groups, respectively; (region II) relay of proton between
oxygen and OH groups, respectively; (region III) release of proton from
oxygen and OH groups to H3O þ, respectively. Denotations of spheres:
grey ¼ carbon; red ¼ oxygen; white ¼ hydrogen atoms. Additional ReaxFF simulations show that a proton transfer
channel, consisting o water molecules that transfer the protons
through Grotthuss-type reactions, thins and finally vanishes when
the pairs of OH groups terminating the defect site are successively
replaced with oxygen atoms (Fig. 3a–d). These transfer paths are
selective to aqueous protons as helium and H2 transfer requires
barriers exceeding 1.9 eV (Supplementary Note 7 and Supplemen-
tary Fig. 14). Table 1 gives the comparison of activation barriers
for proton transfer through graphene in water calculated by
ReaxFF and DFT. The barriers given by DFT for the pristine and
single-vacancy case are high (3.9 and 42.0 eV, respectively) and
insurmountable during molecular dynamics (MD) simulations at
300 K. ReaxFF overpredicts the barriers for proton transfer in the
pristine and single vacancy case. Yet, the barriers for the relevant
quad-vacancy cases given by ReaxFF are in good agreement with
DFT. Note that ReaxFF was not specifically trained against any of
these barriers. three oxygen atoms in epoxide-like arrangements reminiscent
of pyrylium cations (different from the crown ethers recently
reported by Guo et al.)52, or with six hydroxide groups. Proton
transfer through the pyrylium-terminated quad-vacancy site
requires 1.7 eV (Fig. 2a), attributed to the protophobicity of
pyrylium cations and their in-plane localization, which leaves a
3.4 Å gap between water and the graphene substrate that prevents
proton transfer. The hydroxyl-terminated site (Fig. 2b), however,
provides hydrogen-bonding networks (Fig. 2d) that interconnect
the graphene surface to the water layers above and below it. DFT
and ReaxFF simulations indicate that these hydrogen-bonding
networks serve as conduits that facilitate proton transfer from the
solution phase to the surface through the center of the defect site
and into the solution on the opposite side of the membrane via a
Grotthuss mechanism53 involving proton shuttling. Discussion
T
l
d Snapshots (c,d) and energetics (e,f) from the nudged elastic
band calculations for proton transfer through the oxygen- and OH-
terminated defect sites marking (region I) release of proton from H3O þ to
oxygen and OH groups, respectively; (region II) relay of proton between
oxygen and OH groups, respectively; (region III) release of proton from
oxygen and OH groups to H3O þ, respectively. Denotations of spheres:
grey ¼ carbon; red ¼ oxygen; white ¼ hydrogen atoms. Figure 3 | Reactive force field calculations. Proton channel formation fro
ReaxFF calculations of water-mediated proton transfer through atomic
defects terminated in six OH groups (a), four OH groups and one oxyge
atom (b), two OH groups and two oxygen atoms (c), and three oxygen
atoms (d). Denotations of spheres: grey ¼ carbon; red ¼ oxygen;
white ¼ hydrogen atoms. Figure 3 | Reactive force field calculations. Proton channel formation from
ReaxFF calculations of water-mediated proton transfer through atomic
defects terminated in six OH groups (a), four OH groups and one oxygen
atom (b), two OH groups and two oxygen atoms (c), and three oxygen
atoms (d). Denotations of spheres: grey ¼ carbon; red ¼ oxygen;
white ¼ hydrogen atoms. Figure 3 | Reactive force field calculations. Proton channel formation from
ReaxFF calculations of water-mediated proton transfer through atomic
defects terminated in six OH groups (a), four OH groups and one oxygen
atom (b), two OH groups and two oxygen atoms (c), and three oxygen
atoms (d). Denotations of spheres: grey ¼ carbon; red ¼ oxygen;
white ¼ hydrogen atoms. 2.0
1.5
1.0
0.5
0.0
0
2
4
6
8
10 12
Reaction coordinate (Å)
(I)
(III)
(II)
Energy (eV) 0
2
4
6
8
Reaction coordinate (Å)
0.75
(I)
(III)
(II)
0.50
0.25
Energy (eV)
0.00 bottom three defect hydroxyl groups on the other side, and finally
release into the aqueous phase. While solution-phase proton
shuttling occurs with activation barriers o0.2 eV, the barrier for
transferring the proton through the defect sites in graphene via
the proton relay mechanism is just 0.68 eV (DFT, Fig. 2b, well
reproduced by ReaxFF (0.61 eV)), indicating proton transfer will
occur at room temperature. Reaction coordinate (Å) Reaction coordinate (Å) Figure 2 | Density functional theory calculations. Side and top views of Figure 2 | Density functional theory calculations. Side and top views of
oxygen- (a) and OH- (b) terminated defect models used in the DFT
calculations. Discussion
T
l
d To elucidate the mechanisms for proton transfer, we discuss
findings from density functional theory (DFT) calculations
(Fig. 2) and ReaxFF reactive force field molecular dynamics
(Fig. 3)50,51 simulations (Methods). DFT simulations track
the detailed changes in the electronic structure and quantify the
corresponding activation barriers as protons transfer from the
water layer above the surface through the graphene interface and
exit into solution on the opposite side of the surface. The ReaxFF
simulations provide a larger scale representation of the interfaces
and explicitly include dynamics. All of the defect terminations considered are energetically
favourable as compared with the bare quad-vacancy system
(Supplementary Note 6 and Supplementary Fig. 12). The removal
of four carbon atoms in a central aromatic ring in the graphene
layer leads to the formation of the quad-vacancy site as shown in
Supplementary Fig. 13. This site is comprised of six coordina-
tively unsaturated carbon atoms that are either terminated with 3 & 2015 Macmillan Publishers Limited. All rights reserved ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7539 bottom three defect hydroxyl groups on the other side, and final
release into the aqueous phase. While solution-phase proto
shuttling occurs with activation barriers o0.2 eV, the barrier fo
transferring the proton through the defect sites in graphene v
the proton relay mechanism is just 0.68 eV (DFT, Fig. 2b, we
reproduced by ReaxFF (0.61 eV)), indicating proton transfer w
occur at room temperature. Additional ReaxFF simulations show that a proton transf
channel, consisting o water molecules that transfer the proton
through Grotthuss-type reactions, thins and finally vanishes whe
the pairs of OH groups terminating the defect site are successive
replaced with oxygen atoms (Fig. 3a–d). These transfer paths a
selective to aqueous protons as helium and H2 transfer requir
barriers exceeding 1 9 eV (Supplementary Note 7 and Supplemen
2.0
1.5
1.0
0.5
0.0
0
2
4
6
8
0
2
4
6
8
10 12
Reaction coordinate (Å)
Reaction coordinate (Å)
0.75
(I)
(III)
(II)
(I)
(III)
(II)
0.50
0.25
Energy (eV)
Energy (eV)
0.00
Figure 2 | Density functional theory calculations. Side and top views of
oxygen- (a) and OH- (b) terminated defect models used in the DFT
calculations. NATURE COMMUNICATIONS | 6:6539 | DOI: 10.1038/ncomms7539 | www.nature.com/naturecommunications
& 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | 6:6539 | DOI: 10.1038/ncomms7539 | www.nature.com/naturecommunications
2015 M
ill
P bli h
Li
it d All i ht
d ARTICLE Solution pH was
adjusted with minimum amounts of dilute solutions of B1 M NaOH (Sigma-
Aldrich, 99.99%) and HCl (EMD ACS grade). The pH-jump experiments were
carried out using a fused silica hemisphere (ISP Optics, 1’’ diameter, QU-HS-25)
pressed against either a fused silica window (ISP Optics, 1’’ diameter, QI-W-25-1)
or a CVD-prepared graphene film transferred onto a silica window in an
experimental setup previously reported13,56. The hemisphere and the fused silica
window were cleaned before experiments by first treating the surface of interest
with NoChromix (Godax Laboratories) for 1 h, rinsing with Millipore water and
then storing in Millipore water overnight for SHG experiments the next day. On
the day of the experiment, the bare fused silica window and hemisphere were
sonicated in methanol for 6 min, dried in a 110 C oven for 30 min, oxygen plasma
cleaned (Harric Plasma) on high for 30 s, and then stored in Millipore water until
the experiment. The graphene samples were not cleaned with this procedure, but
were instead cleaned by flushing with B2 l of Millipore water before each
experiment. Supplementary Note 9 describes the graphene characterization and
analysis by Raman and ultraviolet–visible spectroscopy (Supplementary Fig. 16)
prior and after the pH-jump experiments. barriers are comparable to recent experimentally determined
activation energy barriers for proton transfer through graphene
subjected to an externally applied potential12. From the SHG
signal jump rates and the time required for 2D proton diffusion,
we estimate that the presence of as few as a handful of atomic
defects in a 1 mm2 area sample of single-layer graphene is
sufficient to allow for the apparent unimpeded protonation and
deprotonation of the interfacial silanol groups within 10 s
(Supplementary Note 8 and Supplementary Fig. 15). Yet, we
caution that given the limited accuracy with which the defect
density can be determined in large (mm)-scale graphene, aqueous
protons may transfer across single-layer graphene not only along
the path discussed here but also along others as well. The
identification of low barriers specifically for water-assisted
transfer of protons through OH-terminated atomic defects in
graphene, and high barriers for oxygen-terminated defects could
be an important step towards the preparation of zero-crossover
proton-selective membranes. Flow system and flow cell. As shown in Fig. ARTICLE ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7539 Table 1 | DFT and ReaxFF-calculated activation barriers for
proton transfer through different vacancy sites on graphene
in water. Graphene
surface
(number of
vacancies)
Bottom layer
Defect
termination
Activation
barrier DFT
(eV)*
Activation
barrier
ReaxFF*
None
Water
No
termination
3.9
Not
computed
1
Water
No
termination
42.0
3.54 eV
4
Water
No
termination
0.25
0.22 eV
4
Water
3O ether
capped
1.8
1.7 eV
4
Water
6 OH
hydroxyl
capped
0.68
0.61 eV
4
Water þ SiO2
3 O ether
capped
2.5
2.53 eV
4
Water þ SiO2
6 OH
hydroxyl
capped
0.7
0.75 eV
DFT, density functional theory. *The energy difference that is reported is due to the initial reference (or reactant) state. Table 1 | DFT and ReaxFF-calculated activation barriers for
proton transfer through different vacancy sites on graphene
in water. Aberration-corrected scanning transmission electron microscopy. To confirm
the single-layer nature of graphene synthesized using the CVD method13, atomic-
resolution STEM imaging was performed at room temperature with an aberration-
corrected Nion UltraSTEM-100 (ref. 58) equipped with a cold field-emission
electron source. The microscope was operated at 60 kV, which is below the knock-
on damage threshold for graphene. The CVD-prepared graphene specimens were
transferred to a SiN-supported silicon microchip transmission electron microscopy
grid. Before STEM imaging, the specimen was heated at 160 C in vacuum
(10 5 torr) for 8 h to remove surface contamination. Following heating in vacuum,
the specimen was immediately transferred to the UltraSTEM for ADF STEM
imaging. The surface of the graphene still retains residual PMMA that was used in
the transfer processes to the transmission electron microscopy grid as shown in
Supplementary Fig. 26; however, there are large areas that are devoid of the
PMMA, which made it feasible to directly image the lattice structure and confirm
the single-layer nature using atomic-resolution STEM imaging. The images were
filtered using a smoothing function in Digital Micrograph, and the contrast and
brightness were adjusted to enhance the contrast of the graphene. Aqueous solution and substrate preparation. The aqueous solutions were pre-
pared with Millipore water, prepared the day before an experiment and left open to
air overnight to equilibrate with atmospheric CO2 and NaCl (Alfa Aesar, 99 þ %). The concentration of NaCl was confirmed using a conductivity meter (Fisher
Traceable Conductivity and TDS meter, Fisher Scientific). ARTICLE 1a, the graphene-on-fused silica
sample or the silica window were clamped face down against a Viton O-ring on the
Teflon flow cell13,56 so that the surface of interest was in contact with the aqueous
phase. The fused silica hemisphere was then clamped on top of the window with a
Millipore water layer in between in order to minimize the change of refractive
index between the phases and to avoid the use of an index-matching fluid. Throughout the duration of the experiment, it was also necessary to maintain a
ring of Millipore water around the bottom of the hemisphere in order to avoid
evaporation of the sandwiched water layer. All of the experiments were completed
with a 0.9 ml s 1 flow using variable flow peristaltic pumps as previously
reported13,56. Using the flow system depicted in Fig. 1a, the pumps were switched
to pull solutions from two different reservoirs. For the experiments reported here
(excluding pKa experiments, see Supplementary Note 2), the two reservoirs
contained 1 mM NaCl Millipore solutions adjusted to either pH 3 or 10. At the
start and end of each pH-jump experiment, a 1 mM NaCl aqueous solution
adjusted to pH 7 was pumped through the system, and the SHG signal was
collected until it reached a steady state. It is assumed that the steady-state
conditions were reached once the SHG signal remained at a stable intensity for a
minimum of 300 s. After the system reached the steady state at pH 7, the flow was
switched back and forth between the pH 3 and 10 aqueous solutions, each time
allowing the SHG signal to reach steady state before switching to the next pH. After
several pH 3 to 10 and pH 10 to 3 jumps were completed, the pH was adjusted back
to pH 7, and the SHG signal was collected until the steady state was reached one
last time. None of the liquid flow effects, reported for fused silica/water interfaces
subjected to high shear rates59, were observed under the creeping flow conditions
used here. Supplementary Note 10 and Supplementary Table 2 assess the flow
dynamics in the cell. Methods
CVD
h CVD graphene synthesis. We used graphene having a grain size of B100 mm
(ref. 54) grown on copper foils by atmospheric pressure CVD54,55. The graphene
was transferred using spin coating of poly(methyl methacrylate) (PMMA) followed
by copper etching in a FeCl3 solution and PMMA removal in acetone. The transfer
was made onto clean fused silica substrates (ISP Optics, 1’’ diameter, QI-W-25-1,
flatness 1 wave per inch at 633 nm) to fill B1 cm2 with a single layer. Following
annealing in a flow of 4% H2 in Ar for 30 min at 300 C, vibrational sum-frequency
generation spectra showed no evidence for CH stretches56. Similar to the finding of
water layers between graphene and mica by atomic force microscopy57, there is
probably water located between the graphene samples and the fused silica
substrates used here. Laser and detection system. A detailed description of our SHG setup has been
described previously60–63. Briefly, we use a regeneratively amplified Ti:sapphire
system (Hurricane, Spectra Physics) that operates at a kHz repetition rate to
produce 120 fs pulses to pump an optical parametric amplifier (OPA-CF, Spectra
Physics) tuned to produce 600 nm light. After exiting the OPA, the beam is then
directed through a variable density filter to attenuate the pulse energy to either
0.3±0.05 mJ per pulse for bare silica studies or 0.15±0.05 mJ per pulse for graphene
studies. The pulse energy used for the graphene films equates to a power density of
2.1(7) 104 mJ cm 2 per pulse with a 30 mm focal spot, which is well below the
damage threshold of graphene as previously reported13,56. At an angle just below Scanning electron microscopy. SEM images were collected from the center of the
graphene film. Graphene on an optical window was imaged using a Hitachi S-4800
scanning electron microscope (SEM) operating at 2 kV with a probing current
of 10 mA and an Everhart–Thornley detector. Copper tape was used to reduce
charging effects. Individual images were taken at 1,200 magnification with
1,280 960 resolution. An array of 5 5 images (529 397 mm, pixel size 176 nm)
was stitched together using Adobe Illustrator. Automatic brightness and contrast
adjustment on each frame was carried out using the ‘auto adjust’ feature in Preview
(Apple, Inc.). No other postedit feature or change was applied. Discussion
T
l
d This proton
transfer mechanism identified here involves relaying the proton
from one of the top three defect hydroxyl groups to the next
hydroxyl group and the next, subsequent transfer to one of the three oxygen atoms in epoxide-like arrangements reminiscent
of pyrylium cations (different from the crown ethers recently
reported by Guo et al.)52, or with six hydroxide groups. Proton
transfer through the pyrylium-terminated quad-vacancy site
requires 1.7 eV (Fig. 2a), attributed to the protophobicity of
pyrylium cations and their in-plane localization, which leaves a
3.4 Å gap between water and the graphene substrate that prevents
proton transfer. The hydroxyl-terminated site (Fig. 2b), however,
provides hydrogen-bonding networks (Fig. 2d) that interconnect
the graphene surface to the water layers above and below it. DFT
and ReaxFF simulations indicate that these hydrogen-bonding
networks serve as conduits that facilitate proton transfer from the
solution phase to the surface through the center of the defect site
and into the solution on the opposite side of the membrane via a
Grotthuss mechanism53 involving proton shuttling. This proton
transfer mechanism identified here involves relaying the proton
from one of the top three defect hydroxyl groups to the next
hydroxyl group and the next, subsequent transfer to one of the We conclude that aqueous protons transfer through single-
layer graphene via rare, OH-terminated atomic defects at room
temperature. While the rarity of the atomic defect sites would
make it challenging to follow proton exchange across graphene
using the pH-sensitive electrodes, the close proximity of the
graphene layer and the charged fused silica surface, where the
experimental observation of surface protonation and deprotona-
tion is made by SHG, allows for the experimental observation of
proton exchange across these rare defects. The associated energy NATURE COMMUNICATIONS | 6:6539 | DOI: 10.1038/ncomms7539 | www.nature.com/naturecommunications
& 2015 Macmillan Publishers Limited. All rights reserved. 4 ARTICLE Am. Chem. Soc. 130, 15444–15447
(2008). 16. Konek, C. T. et al. Interfacial acidities, charge densities, potentials, and energies
of carboxylic acid-functionalized silica/water interfaces determined by second
harmonic generation. J. Am. Chem. Soc. 126, 11754–11755 (2004). pp
y
g
All of the calculations were carried out within the generalized gradient
approximation using Perdew–Burke–Ernzerhof functional66 to treat exchange and
correlation gradient corrections and projector augmented wave pseudopotentials67
to describe the electron–ion interactions. Plane wave basis sets with a cutoff energy
of 400 eV were used to solve the Kohn–Sham equations for calculations for systems
without water. Calculations for systems that include water solvation were carried
out with cutoff energies for C and O of 283 eV. The surface Brillouin zone was
sampled using a Monkhorst–Pack mesh of 3 3 1. All electronic energies were
converged to within a tolerance of 1 10 5 eV. All of the atoms were allowed to
relax in the geometry optimizations until the forces on each atom were
o0.03 eV Å 1. Spin polarization was examined for all of the systems explored and
applied when needed. Transition states were isolated using the nudged elastic band
method44,45 together with the dimer method68. The nudged elastic band method
was used to provide an initial transition state structure that was used in the
subsequent dimer simulations to isolate the transition state. The reaction barrier
was defined as the energy difference between the transition state and the reaction
state minimum. The intrinsic barrier is defined as the energy gap between a
transition state and its immediate reaction state. Given the importance of surface
relaxation in atomically defected graphene layers69, all of our calculations on
the single, di and quad carbon vacancy sites and the oxygen-terminated sites
explicitly modeled surface relaxation (Supplementary Note 11 and Supplementary
Figs 17–19). g
17. Stumm, W. & Morgan, J. J. Aquatic Chemistry, Chemical Equilibria and Rates
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The ReaxFF simulations were performed using the stand-alone ReaxFF
implementation to study proton transfer through pristine graphene and graphene
with di and quad vacancies. We then compared with results from long ab initio
MD to validate predictions of force field in describing water/graphene systems
(Supplementary Note 12 and Supplementary Fig. 20). In our simulations, we used a
(6 6) periodic graphene sheet with water molecules placed in random
configurations on either side of the graphene sheet. The dimensions of the
simulation cell are 15.01 17.83 Å parallel to the sheet and 30 Å in the direction
perpendicular to the sheet. All MD simulations have been performed in the
canonical (NVT, constant number of atoms (N), constant volume (V) and constant
temperature (T)) ensemble, with a time step of 0.25 fs using the Berendsen
thermostat with a coupling time constant of 100 fs to control temperature of the
entire system. To obtain the density plots in Fig. 3, we first divided the simulation
cell into a mesh of cubic boxes with dimensions (0.30 0.30 0.30 Å). We then
counted the number of times a particular atom type (for example, oxygen) was
located in each of the grids through the entire length of simulation and normalized
these numbers by the highest count recorded in any of the grids. We used these
normalized values to obtain the resulting density plots in Fig. 3. The ReaxFF results
reproduce the DFT results, described well in further detail in Supplementary
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the hydrated proton in the perfluorosulfonic acid membrane nafion. J. Phys. Chem. B 110, 18594–18600 (2006). 69. Buettner, M., Choudhury, P., Johnson, J. K. & Yates, J. T. Vacancy clusters as
entry ports for cesium intercalation in graphite. Carbon 49, 3937–3952 (2011). 44. Hashimoto, A., Suenaga, K., Gloter, A., Urita, K. & Iljima, S. Direct evidence for
atomic defects in graphene layers. Nature 430, 870–873 (2004). References 1. Joshi, R. K. et al. Precise and ultrafast molecular sieving through graphene
oxide membranes. Science 343, 752–754 (2014). 31. Atkins, P. & de Pablo, J. Physical Chemistry 8th edn (W.H. Freeman and
Company, 2006). 2. Mi, B. Graphene oxide membranes for ionic and molecular sieving. Science 343,
740–742 (2014). 32. Heberle, J. R. J., Thiedemann, G., Oesterhelt, D. & Dencher, N. A. Proton
migration along the membrane surface and retarded surface to bulk transfer. Nature 370, 370–382 (1994). 3. Gao, W. et al. Ozonated graphene oxide film as a proton-exchange membrane. Angew. Chem. Int. Ed. 53, 1–6 (2014). 3. Gao, W. et al. Ozonated graphene oxide film as a proton-exchange membrane. Angew. Chem. Int. Ed. 53, 1–6 (2014). NATURE COMMUNICATIONS | 6:6539 | DOI: 10.1038/ncomms7539 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | 6:6539 | DOI: 10.1038/ncomms7539 | www.nature.co 6 & 2015 Macmillan Publishers Limited. All rights reserved. Additional information Supplementary Information accompanies this paper at http://www.nature.com/
naturecommunications 51. van Duin, A. C., Dasgupta, S., Lorant, F. & Goddard, W. A. ReaxFF: a reactive
force field for hydrocarbons. J. Phys. Chem. A 105, 9396–9409 (2001). 52. Guo, J. et al. Crown ethers in graphene. Nat. Commun. 5, 5389 (2014). Competing financial interests: The authors declare no competing financial interests. 53. de Grotthuss, C. J. T. Sur la de´composition de l’eau et des corps qu’elle tient en
dissolution a` l’aide de l’e´lectricite´ galvanique. Ann. Chim. 58, 54–73 (1806). Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions. g
q
54. Vlassiouk, I. et al. Graphene nucleation density on copper: fundamental role of
background pressure. J. Phys. Chem. C 117, 18919–18926 (2013). How to cite this article: Achtyl, J.L. et al. Aqueous proton transfer across single-layer
graphene. Nat. Commun. 6:6539 doi: 10.1038/ncomms7539 (2015). 55. Huang, P. et al. Grains and grain boundaries in single-layer graphene atomic
patchwork quilts. Nature 469, 389–392 (2010). 56. Achtyl, J. L. et al. Interaction of magnesium ions with pristine single-layer and
defected graphene/water interfaces studied by second harmonic generation. J. Phys. Chem. B 118, 7739–7749 (2014). 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/ 57. Xu, K., Cao, P. & Heath, J. R. Graphene visualizes the first water adlayers on
mica at ambient conditions. Science 329, 1188–1191 (2010). 58. Krivanek, O. L. et al. An electron microscop for the abberation-corrected era. Ultramicroscopy 108, 179–195 (2008). 7 NATURE COMMUNICATIONS | 6:6539 | DOI: 10.1038/ncomms7539 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved.
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https://openalex.org/W2753902188
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https://www.scienceopen.com/document_file/c88897fc-480b-4467-a0a8-f921dd840c2c/ScienceOpen/420_Goldman.pdf
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English
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Poetry Inspired, Physics Driven, Experimental Animation
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Electronic workshops in computing
| 2,017
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cc-by
| 855
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DOI: http://dx.doi.org/10.14236/ewic/EVA2017.83 DOI: http://dx.doi.org/10.14236/ewic/EVA2017.83 1. INTRODUCTION work. For example, our visualisation “Sky Pacers”
was partially inspired by an ancient Chinese star
catalogue in the form of a poem specifying the
names of stars, “The Song of the Sky Pacers”. The
ancient Chinese stone carvings representing the
constellations were also inspirational in our
deliberations. Another line of poetry that influenced
the work came from William Blake’s poem “The
Tyger”. “When the serenity of the great void and the fury
of
the
cosmos
intersect,
when
quantum
mechanics and relativity come face to face,
tiptoe and waddle, skitter and scuttle, whirl and
whisk, behold: The Prophecy of the Sky Pacers
is now! There is no escaping our cosmic
destiny.” “Glints of light, passing of shadows, the
choreography of perpetual existence sets the
stage for this Delphian ballet. The impenetrable
flow of life’s rhythms, their Sisyphean inceptions
and cessations are punctuated with eternity’s
ephemeral modulations. The transmigration has
begun, the beginning of the end, the end of the
beginning.” “When the stars thew down their spears and
watered heaven with their tears.” “When the stars thew down their spears and
watered heaven with their tears.” “When the stars thew down their spears and
watered heaven with their tears.” Emily Dickinson’s “The First Day's Night Had
Come” creates an inspiring poetic image for
another section of the piece: “She said her Strings were snapt—
Her Bow—to Atoms blown—
And so to mend her—gave me work
Until another Morn—” Above are two descriptions of some of the works
we will look examine. The essence of the
presentation is to demonstrate how two seemingly
opposite
approaches,
a
humanistic,
poetic
approach and that of digital imaging technology
can be integrated in the creation of new works of
art. Robert Frost’s epigram "Pertinax," contains the
following illuminating lines: Let chaos storm! Let cloud shapes swarm! I wait for form. Let chaos storm! Let cloud shapes swarm! I wait for form. Poetry Inspired, Physics Driven,
Experimental Animation Harvey Goldman
University of Massachusetts, Dartmouth
340 Barney’s Joy Road
Dartmouth, MA 02748, USA
hg_art@icloud.com 2. THE POETICS OF DIGITAL VISUALISATION. Once these poetic depictions are gathered and
internalised, our process is to let them percolate
while filtering the feelings they evoke through some
highly sophisticated software. Both 2D and 3D
animation techniques along with physics based
particle systems are employed with the hope of
capturing the organic nature of the poetic imagery. The ability to work with gravity, wind, air, viscosity
and turbulence as creative tools has opened up
new areas of exploration for us. For the last 5 years composer Jing Wang and I
have
been
collaborating
on
a
series
of
experimental electronic visualisations. The work
falls under the genres of “Visual Music” or
“Experimental Animation.” One of our less luminous
objectives has been to bring a more organic and
natural feel to our digital based creative work. The
field of electronic visualisation has a strong
inclination towards mathematics. Geometric forms
and movements are common place. In an attempt to move past these stereotypes, we
have attempted to incorporate the use of poetry as
inspiration for both the form and structure of our © Goldman. Published by
BCS Learning and Development Ltd. Proceedings of Proceedings of EVA London 2017, UK 420 Poetry Inspired, Physics Dri
Harvey
Figure 1: Still from: “Sky Pacers”. Figure 2: Still from: “Ouroboros”. Poetry Inspired, Physics Driven, Experimental Animation
Harvey Goldman
Figure 1: Still from: “Sky Pacers”. Figure 2: Still from: “Ouroboros”. We are currently worki
“Uriel” based on a poem
transcendentalist poet R
this work, we are not o
imagery created in the w
and form/structure of the
more poignant sections f
“With a look that solved
and stirred the devils e
Gave his sentiment div
Against the being of a l Poetry Inspired, Physics Driven, Experimental Animation
Harvey Goldman
Figure 1: Still from: “Sky Pacers”. Figure 2: Still from: “Ouroboros”. We are currently working on a new piece titled
“Uriel” based on a poem of the same name by the
transcendentalist poet Ralph Waldo Emerson. In
this work, we are not only inspired by the poetic
imagery created in the work but the “dramatic arc”
and form/structure of the poem as well. One of the
more poignant sections follows:
“With a look that solved the sphere
and stirred the devils everywhere,
Gave his sentiment divine
Against the being of a line.” Poetry Inspired, Physics Driven, Experimental Animation
Harvey Goldman Figure 1: Still from: “Sky Pacers”. 2. THE POETICS OF DIGITAL VISUALISATION. We are currently working on a new piece titled
“Uriel” based on a poem of the same name by the
transcendentalist poet Ralph Waldo Emerson. In
this work, we are not only inspired by the poetic
imagery created in the work but the “dramatic arc”
and form/structure of the poem as well. One of the
more poignant sections follows: “With a look that solved the sphere
and stirred the devils everywhere,
Gave his sentiment divine
Against the being of a line.” Figure 1: Still from: “Sky Pacers”. Figure 1: Still from: “Sky Pacers”. Figure 2: Still from: “Ouroboros”. Figure 2: Still from: “Ouroboros”. Figure 2: Still from: “Ouroboros”. 421
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https://openalex.org/W4391514876
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https://wjso.biomedcentral.com/counter/pdf/10.1186/s12957-024-03319-3
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English
| null |
The management of uterine tumor resembling an ovarian sex cord tumor (UTROSCT): case series and literature review
|
World journal of surgical oncology
| 2,024
|
cc-by
| 6,175
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Open Access Open Access Abstract Aims To present a case series of 11 rare uterine tumors resembling ovarian sex cord tumors (UTROSCTs), and review
the literature on this topic to offer up-to-date treatment management for UTROSCTs. Method Eight cases from Fujian Cancer Hospital between January 2017 and May 2023 and three patients from Fujian
Union Hospital between October 2012 and October 2020 were retrospectively reviewed. All cases were pathologically
confirmed as UTROSCTs by two senior and experienced pathologists. Clinical behaviors, medical data, histopathologi-
cal features, therapy approaches, and survival outcomes were discussed. Results The median age at initial diagnosis was 53 years (29–70 years). 3 (27.3%) patients were under 40. Seven
cases presented with abnormal vaginal bleeding, one with menstrual disorder, one with abnormal vaginal secre-
tion, and two patients were accidentally found by physical examination without any symptoms. Three patients were
initially misdiagnosed with endometrial cancer by MRI. Curettage was performed in all cases. Nine of them were well
diagnosed by routine curettage, except for two samples, which were identified after surgery. Immunohistochemical
biomarkers, such as CD99, Desmin, WT-1, CK, Vimentin, SMA, α-Inhibin, Ki67, CD56, ER, PR, and CR, tend to be positive
in UTRO SCs patients. Six patients underwent hysterectomy with bilateral salpingo-oophorectomy. Two cases received
a radical hysterectomy with bilateral salpingo-oophorectomy, retroperitoneal lymph node dissection, and omentum
dissection. Three UTROSCTs were under observation after mass resection. The median PFS was 24 months (range
1–125 months). Conclusion UTROSCT is a rare mesenchymal tumor with low malignant potential. Treatment modalities should be
carefully considered to balance the therapy outcomes and patient needs. Surgery conservative management might
be suitable for young women with fertility desires. Keywords UTROSCT, Prognosis, Hysterectomy, Conservative management †Jie Lin and Linying Liu contributed equally to this work. Lin et al. World Journal of Surgical Oncology (2024) 22:42
https://doi.org/10.1186/s12957-024-03319-3 Lin et al. World Journal of Surgical Oncology (2024) 22:42
https://doi.org/10.1186/s12957-024-03319-3 World Journal of
Surgical Oncology Lin et al. World Journal of Surgical Oncology
https://doi.org/10.1186/s12957-024-03319-3 Radiological imaging characteristics
d l
l
ld h Radiological imaging sometimes could have been deceiv-
ing due to the small population size and limited knowl-
edge about UTROSCT. The max SUV value in the
myometrium is 4.5 under PET-CT examination after
hysteroscopic curettage in case 3 (Fig. 1A) with con-
servative therapy. An increasing endometrial thickness
was observed in cases 4 and 10 (Fig. 1B, C). A mass with
myometrial invasion of case 11 which was misdiagnosed
as endometrial cancer was finally proved to be UTRO-
SCT (Fig. 1D). Case 6 was also suspected as endometrial
cancer by MRI examination (Fig. 1E), so as case 10. No
residual tumor was noted after curettage (Fig. 1F). Other
patients didn’t conduct a radiology examination. The
radiological imaging findings are presented in Fig. 1. †Jie Lin and Linying Liu contributed equally to this work. g
sunyang@fjzlhospital.com
Full list of author information is available at the end of the article © The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom-
mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Lin et al. World Journal of Surgical Oncology (2024) 22:42 Page 2 of 9 Lin et al. World Journal of Surgical Oncology (2024) 22:42 Pathological findings
Gl
d l
h l Glandular epithelium, basal-like, and myoepithelial cells
are the three main kinds of cells that could be found in
UTROSCT patients. Under the light microscope, we
could see specific common structures: myxedema, sex
cord-like structure, collagen degeneration, foam-like
cells, organ-like structure, and muscle layer infiltration,
which are often mixed and concurrence (Fig. 2). Materials and methods
Clinical data A total of 11 UTROSCT patients from two institutions
from October 2012 and October 2020 were included. Intrauterine tissue and surgical specimens were reviewed
and confirmed by two senior and experienced pathol-
ogists. The clinical and follow-up information was
obtained through medical files and telephone contact. We collected information such as menopausal status,
parity, symptoms, tumor positions, tumor biomarkers,
image data, immunohistochemical biomarkers, treat-
ment approaches, and survival status. May 31, 2023, was
the final censoring date for assessing the survival time. Results Uterine tumor resembling ovarian sex cord tumor
(UTROSCT) is a sporadic and controversial disease with
unclear origins, named for resembling the morphol-
ogy of ovarian sex cord tumors [1]. It mainly occurs in
women in their 50 s and occasionally in young women
with childbearing needs [2]. UTROSCT patients always
present with irregular vaginal bleeding or chronic pelvic
pain. However, some can be discovered by accidents [3]. Although UTROSCT is generally considered benign or
has a low-malignant potential, it could metastasize some-
times. There are no established treatment protocols for
UTROSCT so far. Surgical intervention is usually recom-
mended with a favorable prognosis. Clinical features and treatment outcomes The age ranged from 29 to 70 (median = 53) at the
initial diagnosis. Seven cases presented with vaginal
bleeding, one with menstrual disorder, and one with
vaginal secretion. While two showed no symptoms. No
elevation of serum tumor biomarkers such as CA125,
CA199, and CEA were observed in 11 cases. The mass
positions were in the endometrium (4 cases) and myo-
metrium (7 cases). None of the patients received fur-
ther postoperative treatment. The median follow-up
time was 24 months (1–125 months), and no recur-
rence or metastasis was noted. Details are presented in
Table 1. Although there has been modest literature on UTRO-
SCT in recent years, focusing mainly on clinicopatho-
logical characteristics and gene variations, relatively few
advances have been made in treatment strategies due
to the low incidence. Here we reported 11 UTROSCT
cases with different therapy approaches together with
an updated literature review to expand our knowledge of
UTROSCT management. Interventions Pathology is the golden standard for UTROSCT diag-
nosis. Patients with UTROSCT showed a diverse phe-
notype profile, according to immunohistochemistry
performed on UTROSCT patients (Fig. 3, Table 2). Bio-
markers such as inhibin, CD99, CD56, WT-1, and CK
tend to be positive in UTROSCTs. Most tumors had
exhibited at least one of the standard sex-cord mark-
ers, including CD99, WT-1, and α-Inhibin (Table 2). The
positive expression rates of α-inhibin (Fig. 3A), CD99
(Fig. 3B), Vimentin (Fig. 3C) and WT-1 (Fig. 3D) were
3/10, 7/9, 5/6, 8/8, respectively. Almost all patients (9/11)
expressed one or more smooth muscle markers, and the
positive expression rates of desmin (Fig. 3E) and SMA All patients underwent diagnostic curettage, three
of which were hysteroscopically assisted. For further
steps, six patients underwent hysterectomy with bilat-
eral salpingo-oophorectomy, two cases received radi-
cal hysterectomy with bilateral salpingo-oophorectomy,
retroperitoneal lymph node dissection, and omentum
dissection, and three were under observation after tumo-
rectomy due to their desires for children-bearing in the
near future. All surgeries were performed by experienced
gynecology surgeons. After treatment, patients were
advised to have follow-up visits every three months in
the first 2 years, every six months in the 3–5 years, and
once a year after 5 years. Lin et al. Abbreviations: NA not available, − negative Interventions World Journal of Surgical Oncology (2024) 22:42 Page 3 of 9 Table 1 Clinical features of UTROSCT patients
bb
l bl
Case
Age
Menopausal
status
Parity
Symptoms
Tumor position
Tumor biomarkers
Treatment approaches
Recurrence
CA199
U/ml
CEA
ng/ml
CA125
U/ml
1
39
No
2–0-0–2
Vaginal bleeding
Submucosa
(myometrium)
13.61
2.59
9.06
Mass resection
No (1 month)
2
34
No
1–0-0–1
Menstrual disorder
Submucosa
(myometrium)
−
NA
NA
Mass resection
No (2 months)
3
29
No
1–0-1–1
No symptoms
Submucosa
(myometrium)
−
NA
NA
Mass resection
No (54 months)
4
40
No
0–0-0–0
No symptoms
Endometrium
9.39
NA
23
Hysterectomy
No (24 months)
5
67
Yes
2–0-0–2
Vaginal bleeding
Submucosa
(myometrium)
17.72
2.5
6.64
Hysterectomy
No (5 months)
6
53
No
2–0-0–2
Vaginal bleeding
Endometrium
6.64
1.49
12.44
Hysterectomy
No (6 months)
7
55
Yes
2–0-1–2
Vaginal bleeding
Submucosa
(myometrium)
NA
NA
NA
Hysterectomy
No (66 months)
8
70
Yes
5–0-1–5
Vaginal bleeding
Submucosa
(myometrium)
NA
NA
NA
Hysterectomy
No (125 months)
9
52
Yes
2–0-0–2
Vaginal bleeding
Endometrium
14.64
2
8
Hysterectomy
No (29 months)
10
57
Yes
3–0-0–3
Vaginal secretion
Endometrium
NA
NA
NA
Radical surgery
No (31 months)
11
59
Yes
2–0-0–2
Vaginal bleeding
Submucosa
(myometrium)
-
1.54
NA
Radical surgery
No (13 months) Fig. 1 UTROSCT (arrow) in different images of PET-CT and MRI. A A max SUV was 4.5 in the myometrium. B–D UTROSCT is mildly hypointense
compared to endometrium signals on sagittal T2 sequence. E UTROSCT is similar to uterus signal and well-delineated on dynamic
contrast-enhanced sequence. F No mass observed after curettage Fig. 1 UTROSCT (arrow) in different images of PET-CT and MRI. A A max SUV was 4.5 in the myometrium. B–D UTROSCT is mildly hypointense
compared to endometrium signals on sagittal T2 sequence. E UTROSCT is similar to uterus signal and well-delineated on dynamic
contrast-enhanced sequence. F No mass observed after curettage PR (Fig. 3I) expressed variably among UTROSCTs. A
low Ki67(Fig. 3J) index was observed among the patients
(median 5%, range 2–30%). While CD56 (Fig. 3K) and (Fig. 3F) were 8/10 and 6/10, respectively. CK (Fig. 3G)
as an epithelial biomarker was positive in most cases
(10/11), and hormone receptors like ER (Fig. 3H) and Lin et al. World Journal of Surgical Oncology (2024) 22:42 Page 4 of 9 Page 4 of 9 Fig. 2 Morpholohistopathology of UTROSCT. Interventions UTROSCT is characterized by various architectures including A myxedema, B sex cord-like structure, C
collagen degeneration, D foam-like cell, E organ-like structure. F muscle layer infiltration (H&E × 10) Fig. 2 Morpholohistopathology of UTROSCT. UTROSCT is characterized by various architectures including A myxedema, B sex cord-like structure, C
collagen degeneration, D foam-like cell, E organ-like structure. F muscle layer infiltration (H&E × 10) Fig. 3 Immunohistochemical biomarkers of UTROSCT. A α-inhibin. B CD99. C Vimentin. D WT-1. E desmin. F SMA. G CK. H ER. I PR. J Ki67. K CD56. L
Calretinin (IHC × 10) Fig. 3 Immunohistochemical biomarkers of UTROSCT. A α-inhibin. B CD99. C Vimentin. D WT-1. E desmin. F SMA. G CK. H ER. I PR. J Ki67. K CD56. L
Calretinin (IHC×10) Fig. 3 Immunohistochemical biomarkers of UTROSCT. A α-inhibin. B CD99. C Vimentin. D WT-1. E desmin. F SMA. G CK. H ER. I PR. J Ki67. K CD56. L
Calretinin (IHC × 10) Lin et al. World Journal of Surgical Oncology (2024) 22:42 Page 5 of 9 Table 2 Immunohistochemical biomarkers in the 11-patient cohort
Abbreviations: + positive, − negative, NA not available
Case
CD99
Desmin
WT-1
CK
Vimentin
SMA
α-Inhibin
Ki67
CD56
ER
PR
CR
1
NA
+
+
+
NA
+
−
1%
+
+
+
+
2
+
+
+
+
+
+
−
2% +
+
+
+
NA
3
+
+
+
+
+
+
+
1% +
NA
NA
NA
+
4
−
−
NA
−
+
−
−
10% +
+
−
−
+
5
−
NA
+
+
NA
+
+
10% +
+
+
+
+
6
+
+
+
+
−
−
+
10% +
NA
+
+
NA
7
+
+
NA
+
NA
+
−
2%
NA
+
+
+
8
+
−
NA
+
+
−
−
NA
NA
NA
NA
NA
9
+
+
+
+
NA
+
−
30% +
+
+
NA
+
10
NA
+
+
+
NA
−
−
40%
+
+
NA
NA
11
+
+
+
+
+
NA
NA
10% +
NA
+
+
− Table 2 Immunohistochemical biomarkers in the 11-patient cohort Abbreviations: + positive, − negative, NA not available recorded that hysterectomy is the prior surgery approach
in 71 out of 75 UTROSCT patients [40]. Discussion In 1976, Clement and Scully first reported certain kinds
of rare uterine tumors with histologic resemblances to
ovarian sex-cord tumors [4]. Those types of tumors could
be divided into two groups. The first group is endometrial
stromal tumors with sex cord-like elements (ESTSCLE)
with a particular propensity to recur or metastasize, and
UTROSCT is the other group [5]. UTROSCT is rare and
usually recognized as a benign tumor. Clinical observa-
tions and literature data indicated a better prognosis [6]. However, sporadic reports showed it could metastasize
and recur sometimes. In 2017, Moore documented 34
UTROSCT cases, of which about 23.5% exhibited malig-
nant behavior [7]. Due to the potential malignancy and
limited experience, treatment recommendations have not
been well established. Interventions Besides, most
patients had good survival outcomes simply by remov-
ing the uterine [21, 22, 40]. Moreover, some scholars
claimed recurrence cases previously treated by hyster-
ectomy [11, 12, 20] could still get prolonged survival by
completely removing the recurrent mass. In our study, 6
out of 11 patients treated by hysterectomy did not experi-
ence recurrence and metastasis after a median follow-up
time of 26.5 months (range 5–125 months). However, we
still need a longer follow-up time and more cases to make
survival outcomes more convincing. Calretinin (Fig. 3L) are often used for differentiated
diagnosis. Radical surgery for UTROSCTs with high risk of recurrence
or metastasis UTROSCT has uncertain malignant potential due to
its low recurrence rate [7]. Distant metastasis and local
recurrence have been occasionally reported [11, 22, 28,
29]. Our review found that the recurrence rate was low
(19/117) during the median follow-up time between
20 days and 32 years. Currently, predictive features of
aggressive UTROSCTs are poorly understood. Risk fac-
tors, such as myometrial invasion, serosal involvement,
lymph-vascular space invasion (LVSI), GREB1-NCOA2
fusion, NCOA2 or NCOA3 [23, 41, 42], and high mitotic
activity [40, 43]have been proven to be associated with
tumor recurrence. Baris Boyraz concluded that high-
risk UTROSCT showed more than three of the following
five features: at least moderate cytologic atypia, tumor
size > 5 cm, above 3 mitoses/10 at high power fields
(HPFs), marginal infiltration, and necrosis [40].i Surgery is recommended as the preferred treatment for
UTROSCT patients [8]. Hysterectomy or mass resection
alone are possible options for its management [9]. A sys-
tematic review of the literature on UTROSCTs’ treatment
approaches is detailed in Table 3. With advancements in
medicine and humanities, individualized-oriented tumor
treatments have been deeply rooted in people’s hearts. We should pay more attention to patients with special
needs or in different conditions, and then make person-
alized plans to balance the therapy outcomes and their
needs. i
High-risk UTROSCT patients could benefit from radical
surgery. Miho believed hysterectomy alone was associated
with a higher rate of recurrence. Extended radical surgery,
including bilateral salpingo-oophorectomy, lymphadenec-
tomy, and omentectomy, may reduce the recurrence rate Hysterectomy for UTROSCTs in the middle and old age i
High-risk UTROSCT patients could benefit from radical
surgery. Miho believed hysterectomy alone was associated
with a higher rate of recurrence. Extended radical surgery,
including bilateral salpingo-oophorectomy, lymphadenec-
tomy, and omentectomy, may reduce the recurrence rate Hysterectomy for UTROSCTs in the middle and old age
Hysterectomy resection with or without salpingo-oopho-
rectomy should be a primary option for middle-aged
or older women with UTROSCT, especially in patients
whose follow-up cannot be guaranteed [9]. Baris Boyraz Lin et al. World Journal of Surgical Oncology (2024) 22:42 Page 6 of 9 of UTROSCT with sarcomatous features [34]. Based on
the review in Table 3, no recurrence or distant metastasis
was observed among 8 UTROSCT patients treated with
radical surgery, indicating radical surgery seemed to be the
optimal choice for high-risk patients. However, it should
be attention that high-risk UTROSCT is often detected
Table 3 Different therapies and outcomes of UTROSCT patients reported in the literature (2014 ~ 2023)
Abbreviations: Ned no evidence of disease. Conservative treatment: tumorectomy, myomectomy, and electrical resection, etc. Present cases
No
Year
Age
Outcome (follow-up)
Hysterectomy ± bilateral salpingo-oophorectomy
Gutierrez-Pecharroman, et al. [10]
1
2014
49
Ned (18 months)
Bakula-Zalewska, et al. [6]
4
2014
50–63
Ned (3–14.5 years)
Moore, et al. [7]
31
2017
26–86
7/31 recurrence (6–135 months)
Kondo, et al. [11]
1
2018
63
1/1 recurrence (6 years)
Croce, et al. [12]
1
2019
72
1/1 recurrence (17 months)
Dubruc, et al. [13]
1
2019
56
Ned (4 months)
Zhang, et al. [14]
2
2019
33, 64
Ned (12 days–1 year)
Lee, et al. [15]
2
2019
34, 47, 59
Ned (116 months–20 years)
Vilos, et al. [16]
2
2019
47, 52
Ned (1–3 years)
Dimitriadis, et al. [17]
1
2020
46
1/1 recurrence (2 years)
Grither, et al. [18]
1
2020
69
Ned (8 months)
Chang, et al. [19]
1
2020
57
1/1 recurrence (30 months)
Bennett, et al. [20]
3
2020
32, 37, 54
3/3 recurrence (7–32 years)
Kaur, et al. [21]
5
2020
42–50
1/5 recurrence (1 month–2 years)
Goebel, et al. [22]
10
2020
34–74
1/10 recurrence (1–319 months)
Zhou, et al. [8]
1
2021
51
Ned (58 months)
Devereaux, et al. [23]
1
2021
42
1/1 recurrence (6 months)
Shibahara, et al. [24]
1
2022
77
Ned (12 months)
Wang, et al. [3]
1
2022
42
Ned (2 months)
Pang, et al. Hysterectomy for UTROSCTs in the middle and old age Oriana Marrucci reported a rare vagi-
nal vault recurrence case 5 years after total hysterectomy
with bilateral salpingo-oophorectomy. She had a favorable
prognosis with only a second surgery [44]. Shigeaki Umeda
also represented a case with epiploic appendix metasta-
sis, who was treated with surgery and has been followed
up for 8 years without evidence of recurrence [28]. How-
ever, Michelle presented an unusual case of an aggressive
UTROSCT who underwent neoadjuvant chemotherapy
followed by optimal cytoreductive surgery and adjuvant
chemotherapy and still died 15 months after her initial
diagnosis [31]. Therefore, the selection of auxiliary treat-
ments needs to consider the specific situations of UTRO-
SCT patients. resection. After a comprehensive examination without
signs of residue tumor, case 2 opted for surveillance. The
menstruation returned to normal after conservative ther-
apy. She has been followed up for two months with dis-
ease-free. In case 3, a PET/CT showed a max SUV value
of 4.5 in the myometrium, which cannot be confirmed as
postoperative changes or residue tumors. After exhaus-
tive discussions with patients, she still chose to observe
due to her eager solid of fertility preservation. Surpris-
ingly, there are no signs of recurrence or metastasis after
a follow-up of 54 months. Case 1 had a myomectomy and
showed no sign of recurrence. She strongly desired to
preserve her organs and decided to be closely observed. A conservative, uterus-preserving treatment appears jus-
tified in those whose close follow-up can be guaranteed,
even in high-risk ones. Once recurrence, patients could
still get a favorable prognosis after salvage surgery [7]. Further investigations are needed to prove the safety of
organ-preserving strategy in UTROSCT. UTROSCT is somehow inert and quickly got the atten-
tion of irregular uterine bleeding. All patients in our
research were diagnosed in the early stages. A hysterec-
tomy seems enough. However, a 40-year-old female still
firmly chose to have extra salpingo-oophorectomy due to
psychological fear of tumor recurrence, even after being
fully educated on UTROSCT. Moreover, pre-surgery
examinations, such as MRI images and curettage pathol-
ogy, could sometimes be confusing, leading to over-
treatment for oncological safety. Clinicians should focus
more on aggressive UTROSCT features patients shared,
such as tumor size, myometrial invasion, LVSI, and gene
fusion, to determine the surgical approaches. Working
with an experienced pathologist is necessary. Still, the role of hormone therapy is also controversial. Sabrina M. Hysterectomy for UTROSCTs in the middle and old age [25]
2
2022
42, 46
Ned (4–35 months)
Ye, et al. [2]
4
2022
40–70
Ned (uncertain)
Zhou, et al. [26]
1
2023
49
Ned (1 months)
Lu, et al. [27]
11
2023
37–60
Ned (11–141 months)
Radical surgery
Umeda, et al. [28]
2
2014
38, 57
Ned (11 months–8 years)
Mačák, et al. [29]
1
2015
53
Ned (10 months)
Cetinkaya, et al. [30]
1
2016
52
Ned (17 months)
Kuznicki, et al. [31]
1
2017
49
Death (15 months)
Fan, et al. [32]
1
2018
62
Ned (20 days)
Segala, et al. [33]
1
2019
62
Ned (5 years)
Sato, et al. [34]
1
2020
57
Ned (3 years)
Conservative treatment
Bakula-Zalewska, et al. [6]
2
2014
24,25
Ned (4.5–7 years)
Watrowski, et al. [35]
1
2015
22
Ned (28 months)
Jeong, et al. [36]
1
2015
32
Ned (47 months)
De Franciscis, et al. [37]
1
2016
38
Ned (60 months)
Moore, et al. [7]
2
2017
12, 36
Ned (27–63 months)
Schraag SM, et al. [38]
2
2017
24, 28, 72
2/2 recurrence (6–39 months)
Lee, et al. [15]
1
2019
34
Ned (49 months)
Goebel, et al. [22]
1
2020
38
Ned (50 months)
Carbone, et al. [39]
2
2021
25, 30
Ned (96 months,16 months)
Ye, et al. [2]
1
2022
40
Ned (uncertain)
Lu, et al. [27]
6
2023
27–50
Ned (17–55 months) Table 3 Different therapies and outcomes of UTROSCT patients reported in the literature (2014 ~ 2023) radical surgery, indicating radical surgery seemed to be the
optimal choice for high-risk patients. However, it should
be attention that high-risk UTROSCT is often detected of UTROSCT with sarcomatous features [34]. Based on
the review in Table 3, no recurrence or distant metastasis
was observed among 8 UTROSCT patients treated with of UTROSCT with sarcomatous features [34]. Based on
the review in Table 3, no recurrence or distant metastasis
was observed among 8 UTROSCT patients treated with Lin et al. World Journal of Surgical Oncology (2024) 22:42 Lin et al. World Journal of Surgical Oncology Page 7 of 9 incidentally after a hysterectomy surgery, which makes the
initial treatment insufficient. For this reason, the roles of
adjuvant therapies should be carefully considered. Surpris-
ingly, the literature showed adjuvant treatments such as
chemotherapy, hormone therapy, or radiotherapy were still
in debate for the UTROSCTs with high risks of recurrence
or metastasis [35]. Conservative therapy for UTROSCTs with fertility desire Conservative therapy for UTROSCTs with fertility desire
With the development of minimally invasive surgery,
tumorectomy by hysteroscopy or laparoscopy could be
expected in young UTROSCTs who desire to preserve
their organs and fertility. Many scholars reported suc-
cessful pregnancies in young UTROSCT women treated
with fertility-preserving surgery and had no sign of
recurrence [38, 39], even in a myometrial invasion case
[36]. Only one relapsed case after 20 months of con-
servative surgery [38]. In our review, all UTROSCTs
with conservative therapy had favorable prognoses. Most
researchers believed UTROSCT patients could main-
tain fertility without affecting the survival rate. In our
study, three cases (Nos. 1, 2, and 3) were done with mass Hysterectomy for UTROSCTs in the middle and old age Schraag recorded that surgery and endo-
crine treatment were applied for a young UTROSCT
with recurrence. She was disease-free 34 months after
the last surgery and was still on a monitor [38]. High-
dose progesterone therapy was also applied after surgery
in a UTROSCT with pelvic lymph node metastasis, and
no recurrence was observed [28]. While Daisuke Endo
pointed out that hormone therapy may be ineffective for
recurrent tumors [45]. Some scholars even believed hor-
mone therapy such as tamoxifen progressed this disease
[33]. In our study, none patients received additional treat-
ments, and no recurrences were observed. To be pointed
out, two cases received radical surgery due to malignancy
features shown in MRI images and pathology ambigu-
ity. Based on the above, we believe the choice of surgi-
cal approaches should be carefully considered based on
whether she is a high-risk UTROSCT. As far as we know, few articles have summarized and
compared the different therapy approaches to UTROSCT. Our study gave a comprehensive knowledge of UTRO-
SCT therapy methods. However, the main limitation of
our study is that it was designed retrospectively. Specific
information about related cases is lacking, the follow-up
time for the young UTROSCT needs longer, and genetic
alterations and molecular detection remain to be explored. However, we still contributed 11 UTROSCTs to the medi-
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Medical University, China (Grant number: 2021QH1141), Joint Funds for
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script. Jie Lin checked and revised the manuscript. Linghua Wang helped to
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Risk factors associated with Dengue incidence in Bandung, Indonesia: a household based case-control study
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Risk factors associated with Dengue incidence in Bandung, Indonesia Risk factors associated with Dengue incidence in Bandung, Indonesia 45 Vol. 11, No. 1, June 2020 Unit of Health Research and Development of Pangandaran District, West Java Province, Ministry of Health of
Republic of Indonesia Unit of Health Research and Development of Pangandaran District, West Java Province, Ministry of Health of
Republic of Indonesia Corresponding author: Hubullah Fuadzy
Email: hubullah_fy@yahoo.com Corresponding author: Hubullah Fuadzy
Email: hubullah_fy@yahoo.com Received: Desember 9, 2019; Revised: Januari 20, 2020; Accepted: May Received: Desember 9, 2019; Revised: Januari 20, 2020; Accepted: May 19, 2020. Abstrak Latar belakang: Bandung memiliki daerah perkotaan dengan kualitas bangunan rumah yang memadai,
namun masih memiliki kasus endemik Demam Berdarah Dengue (DBD) yang tinggi. Penelitian ini
bertujuan untuk mengetahui karakteristik kejadian demam berdarah di tingkat rumah tangga. Metode: Data dianalisis dari 781 rumah tangga yang terdiri dari 261 kasus dan 522 kontrol. Pemilihan
sampel menggunakan metode kasus kontrol berpasangan dengan rasio 1:2. Tahapan penelitian terdiri
dari pengamatan status rumah menggunakan form ceklis pemeriksaan dan wawancara menggunakan
kuesioner tertutup. Analisis data korelasi pairwise spearmen, kemudian regresi logistik biner digunakan
untuk prediksi faktor risiko. Hasil: Faktor risiko usia produktif dan rendahnya tingkat pendidikan kepala keluarga, toilet yang kotor,
dan status rumah tidak sehat memiliki pengaruh yang signifikan terhadap peningkatan kasus demam
berdarah di Kota Bandung ((p<0.05). Faktor determinan adalah usia produktif kepala keluarga (31 - 60
tahun) dimana memiliki kemungkinan 2,53 (95%CI 1.34-4.78;p<0.05) kali lebih besar untuk memiliki
anggota rumah tangga yang menderita DBD di Bandung. Kesimpulan: Usia dan pendidikan kepala rumah tangga, kebersihan toilet dan status rumah sehat memiliki
peran penting dalam mempengaruhi kejadian demam berdarah. Dengan demikian, petugas kesehatan
perlu melakukan promosi kesehatan mengenai DBD secara intensif kepada kepala rumah tangga. (Health
Science Journal of Indonesia 2020;11(1):45-51) Kata kunci: Demam Berdarah Dengue (DBD), kepala rumah tangga, usia, sanitasi rumah, Kota Bandung Keywords: Dengue, head of household, ages, house sanitation, Bandung City DOI: dx.doi.org/10.22435/hsji.v11i1.3150 DOI: dx.doi.org/10.22435/hsji.v11i1.3150 Hubullah Fuadzy, Mutiara Widawati, Endang P. Astuti, Heni Prasetyowati, Joni Hendri, Rohmansyah W. Nurindra, Dewi N. Hodijah. Abstract Background: Bandung have urban areas with adequate housebuilding quality, yet still has high Dengue
endemic cases. This study aims to investigate the characteristics of dengue incidence at the household level. Methods: Data analyzed from 781 households consisted of 261 cases and 522 controls. We applied matched
case-control samples with a ratio of 1:2 (case: controls). The stages of the research consisted of a house status
observation using a form inspection checklist and interviews using a closed-ended questionnaire. The data
analyzed by pairwise spearmen correlation and binary logistic regression for risk factor prediction. Results: Risk factors for productive age and low level of education of family heads, dirty toilets, and
unhealthy house status have a significant effect on the increase of dengue cases in Bandung (p<0.05). The
determinant factor is the productive age of the head of the family (31 - 60 years), it means that the family
who has a family head in a productive age is 2.53 (95% CI 1.34-4.78; p <0.05) times more likely to have a
household member suffering from DHF in Bandung. Conclusion: The age and the level of education of the household heads, toilet hygiene, and healthy home
status have an essential role in influencing dengue fever. Therefore, health workers need to perform an
intensive health promotion regarding DHF to the household heads. (Health Science Journal of Indonesia
2020;11(1):45-51) Keywords: Dengue, head of household, ages, house sanitation, Bandung City Health Science Journal of Indonesia Fuadzy et al. 46 Dengue haemorrhagic fever (DHF) is a mosquito-borne
disease caused by Dengue virus and transmitted by Aedes
aegypti and Ae. albopictus. Dengue infection has become
a major public health problem worldwide, especially in
Asia. In recent decades, the disease outbreak has been
reported in 128 countries with more than 3.79 billion
people are at risk, mostly in urban and semi-urban areas.1
In Southeast Asia, Dengue is endemic in 12 countries. An
estimated 2.9 million dengue infections occur annually
with 5,906 among them died, thus impacting the annual
economic burden of US $ 1.65 (the US $ 1.06 - the US $
2.41) per capita.2 efforts were also closely related to the knowledge
and attitudes of the community. Several studies have
suggested that proper education and beliefs about
dengue are related to the prevention of dengue.6–8
These determinant factors significantly affect the
ongoing transmission of dengue in several regions. Abstract The region with the highest risk of increasing DHF
cases is the area in the center of Bandung, with
peak cases from January to August. Many people
are at risk of catching the dengue fever in the center
of Bandung due to socio-economic conditions,
especially slum housing conditions, population
density, relatively high mobility, and low education
levels.9 Respati’s research (2018) states that the most
decisive risk factors in the prevention and control
of dengue in Bandung are community participation,
housing, environmental factors, and climate.10 The government of Indonesia has conducted various
efforts to prevent dengue transmission by physical,
biology, chemical, empowering of community, and
integrated management control, but dengue morbidity
keeps increasing. Indonesia has reported that the
morbidity rate of dengue still increases to 700 times in
the last 45 years, whereas the mortality rate decreases to
56.16 times.3dengue vector-density and human mobility
cause rapid spread of dengue virus in Indonesia. We
investigated the changes in dengue haemorrhagic
fever (DHF However, Indonesia has not succeeded in
preventing and controlling Ae. Aegypti from dengue
virus transmission in both rural and urban settlements. In Bandung, most of the citizens use containers for
collecting and storing water for daily needs. These
conditions will trigger an increase in the population of
Aedes and the potential for the Dengue transmission. In 2010, at least of 3,430 Dengue cases were reported
in this city. However, in the following two years
(2013), the dengue cases were increased to 5,736
cases.11 The free larvae index was also increased
and close to the national target at 93.38%.12 In this
study, we have investigated the characteristic of the
dengue incidence at the household level in Bandung,
Indonesia Household activities may play a role in the transmission
of Dengue Fever. Various factors, such as house
sanitation, room construction, occupants’ behavior,
management of water containers4sanitation and
hand hygiene in low- and middle-income settings
and provide an overview of the impact on other
diseases.\\n\\nMETHODS:
For
estimating
the
impact of water, sanitation and hygiene on diarrhea,
we selected exposure levels with both sufficient
global exposure data and a matching exposure-risk
relationship. Global exposure data were estimated
for the year 2012, and risk estimates were taken from
the most recent systematic analyses. We estimated
attributable deaths and disability-adjusted life years
(DALYs, socio-economic conditions and knowledge
about dengue fever have the potential to influence
the incidence of dengue fever. Abstract Communities with
less stable socio-economics condition tend to have
inadequate or unhygienic houses. The previous study
about risk factor analysis showed that residents in
rented houses are more at risk of dengue than those
who live in their own houses (AOR = 2.2, 95%
CI= 1.1 - 4.6; p < 0.05). In addition, residents of
unhygienic dwellings were more likely to get dengue
compared to residents in hygienic houses (AOR =
3.4, 95% CI = 1.0 – 11.7; p < 0.05).5 The behavior
of increasing house hygiene and vector control Study Subjects Bandung is the capital of the West Java Province,
located at coordinates of 107o E and 6o55’ S. Bandung has 75 PHCs (Primary Health Care/Pusat
Kesehatan Masyarakat), as a unit in sub-district
level that responsible for the community health
efforts, consisting of public health activities for the
population within their working area. This study has
been carried out in 4 PHCs areas, i.e. Dago, Sekejati,
Kopo, and Cipamokolan. The location of this study
was determined based on the highest dengue case. This study also refers to research on Determining
Risk Factors for Residential Sanitation in Dengue
Hemorrhagic Fever (DHF) in Bandung in 2016.13
This study aims to explain what factors cause the
number of healthy home coverage continues to show
an increasing trend every year, despite the increasing
trend of dengue fever as well. Risk factors associated with Dengue incidence in Bandung, Indonesia Vol. 11, No. 1, June 2020 47 variables were analyzed through binary logistic
regression using Minitab version 19. This research is observation research with a matched
case-control design with a ratio of 1:2 so that the
confounding factor in both groups can be controlled. The case group population is a house that has household
members who have had DHF in 2015 in Bandung,
while the control population is a house where household
members have never had DHF in Bandung. The sample
size was calculated using the odds ratio estimation
formula, and a total sample of 783 households was
obtained, consisting of 261 case groups and 522 control
groups. The criteria for selecting a research location
were if the puskesmas has a high number of dengue
cases in 2015 in the city of Bandung. The sample
selection of case group households uses a systematic
random sampling technique, while the control group
uses purposive sampling techniques with the criteria
of a control group household being at a radius of 100
meters from the sample cases. Data Collection Data were collected through observation and inter
views. The observation using the Ministry of Health’s
standard questionnaire viz healthy housing inspec
tion checklist to determine the house status based
on the regulation of the Ministry of Health, Republic
of Indonesia No. 829/Menkes/VII/1999. There were
four observed places, such as living/family room, bed
room, bathroom, and kitchen. This study evaluated all
room construction (ceilings, walls, floors, windows,
vents, chimney, and lighting), and sanitation facili
ties (water facilities, toilets, wastewater disposal, and
garbage cans). After observation on four rooms, KAP
interviews using structural questionnaire were con
ducted with some question to respondents about vec
tor, transmission, and control of Dengue Result of the head of household characteristic
component for case and control groups showed that the
majority of households considered as a settled family
with the indicator of age included into the productive
period (95.02% and 88.70%), level of education ranged
from high school to higher education (65.52% and
56.90%), and income (70.11% and 65.33%) was more
than the regional minimum wage of Bandung (2,310,000
IDR), respectively. There are different age period and
education level of head of household between case and
control group (p<0.05) (Table 1). Overall, the value of
the house component consisting of the construction of
houses, and sanitation facilities, both in case and control
group was relatively similar. For a few different house
components, namely the ventilation, and chimney,
(p<0.05) (Table 1). Characteristics of Dengue Incidences The data samples of 783 respondents were successfully
collected, which distributed to four Public Health
Centers. As for the distribution consisted of 67 cases and
134 controls in Dago PHCs, 60 cases and 120 controls in
Sekejati PHCs, 72 cases and 144 controls in Kopo PHCs,
also 62 cases and 124 controls in Cipamokolan PHCs. Ethics Approval As this research involving humans as subjects, the
research ethics approval was required. Therefore,
research ethics approval letter has been published
by the National Institute of Health Research and
Development of Indonesia with Registration Number
LB.02.01/5.2/KE.056/2016. The respondents gave a
statement written in informed consent. Data Analysis Status of a healthy house determined by calculating
the weight of each component. The result of the
calculation compared with the average standard
value. The value for room construction is 44, and
the value for sanitation facilities is 31. Based on
the Arikunto criteria (2003), a 75% cut-off14 can be
used to determine the status of healthy and unhealthy
houses. Then the house is characterized as a healthy
house if the total amount achieved is 641 - 855 and
an unhealthy house if the total amount obtained is
less than 641. Then, the data analyzed by bivariate
analysis using pairwise Spearman correlation. After that, multivariate analyses were performed
to determine the dominant risk factor in affecting
the incidence of Dengue in Bandung. Later, those House status showed that the majority of the Bandung
citizen still included in the category of an unhealthy
house, both in case and control group (83.14% and
89.08%). Moreover, the significantly different result
was related to the house status to the incidence of
Dengue in Bandung (p<0.05) (Table 2).The majority of
Bandung citizen had inadequate knowledge (75.48%
and 72.99%) regarding the transmission and eradication
of Dengue, yet they had a right attitude (93.87% and
91.76%), and action (60.15% and 66.48%) concerning
the prevention of Dengue. No significant different
results related to knowledge, attitudes, and practices to
the incidence of Dengue (p>0.05) (Table 3). Health Science Journal of Indonesia Fuadzy et al. 48 Fu
48 48 Table 2. Association of house status with Dengue incidences
No
House Status
Houses
p-value
Case,
n=261
Control,
n=522
1
Houses
Healthy
44(16.86)
57(10.92)
0.019*
Unhealthy
217(83.14)
465(89.08)
> Houses status are revealed healthy when whole of the house
components assessment has value more than 641.25 based on
Decree of the Minister of Health of the Republic of Indonesia No.829/
Menkes/VII/1999 concerning the requirements of healthy housing
Table 3. Association of knowledges, attitudes, and practices
with Dengue incidences
No
Variables
Houses
p-value
Case,
n=261
Control,
n=522
1
Knowledge
0.456
Good
64(24.52)
141(27.01)
Poor
197(75.48)
381(72.99)
2
Attitudes
0.293
Positive
245(93.87)
479(91.76)
Negative
16(6.13)
43(8.24)
3
Practices
0.082*
Good
157(60.15)
347(66.48)
Poor
104(39.85)
175(33.52)
* Candidates for Multivariate Analysis Table 2. Association of house status with Dengue incidences Table 1. Assosiation between house components with
Dengue incidences Table 1. Determinants of Dengue The goodness of fit on logistic regression analysis
showed that p-value of Hosmer Lemeshow was
0.499, indicated that the model is fit to be postulated. The value of concordant is 65.1, which means that
probably there is only 65.1% risk factor variable
correlated with the incidence of Dengue in Bandung. Risk factors that influence the increase in DHF are
the age and the education of the household head,
toilet, and the status of the healthy house (p<0.05). As for the most dominant risk factor of the spread
of Dengue in Bandung is the age of household head
which is still productive which leads to a tendency of
dengue infection risk with OR 2.53 (95% CI, 1.34-
4.78). Data Analysis Assosiation between house components with
Dengue incidences
No
House Components
Houses
p-value
Case,
n=261
Control,
n=522
1
Household head
characteristics
Age of household head
0.004*
Productive ( 16-60
Year)
248(95.02) 463(88.70)
Not Productive (>60
Year)
13(4.98)
59(11.30)
Education of household
head
0.020*
≥ Senior High School
171(65.52) 297(56.90)
< Senior High School
90(34.48) 225(43.10)
Income of household
head`
0.180*
≥ 3,000,000 IDR
183(70.11) 341(65.33)
< 3,000,000 IDR
78(29.89) 181(34.67)
2
House constructions
Ceiling
0.477
Healthy
203(77.78) 394(75.48)
Unhealthy
58(22.22) 128(24.52)
Wall
0.635
Healthy
233(89.27) 460(88.12)
Unhealthy
28(10.73)
62(11.88)
Floor
0.789
Healthy
237(90.80) 477(91.38)
Unhealthy
24(9.20)
45(8.62)
Bedroom window
0.956
Healthy
184(70.50) 367(70.31)
Unhealthy
77(29.50) 155(29.69)
Living room window
0.206*
Healthy
230(88.12) 475(91.00)
Unhealthy
31(11.88)
47(9.00)
Ventilation
0.045*
Healthy
140(53.64) 319(61.11)
Unhealthy
121(46.36) 203(38.89)
Chimney
0.011*
Healthy
161(61.68) 369(70.69)
Unhealthy
100(38.32) 153(29.31)
Sunlight
0.076*
Healthy
155(59.39) 275(52.68)
Unhealthy
106(40.61) 247(47.32)
3
Sanitation facilities
Clean water
0.858
Healthy
201(77.01) 399(76.44)
Unhealthy
60(22.99) 123(23.56)
Toilet
0.234*
Healthy
152(58.24) 327(62.64)
Unhealthy
109(41.76) 195(37.36)
Waste disposal
0.946
Healthy
218(83.52) 437(83.72)
Unhealthy
43(16.48)
85(16.28)
Garbage disposal
0.559
Healthy
193(73.95) 396(75.86)
Unhealthy
68(26.05) 126(24.14)
> House components is Part of Physical building inside of house
and householder behaviours, and was revealed of healthy when it’s
still functioning well and could prevent the disease’s transmission; DISCUSSIONS In this study, the majority of cases of dengue infection
in Bandung in 2016 lived in urban areas with good
socioeconomic status, but they lived in houses with
poor sanitation and lack of practice and behavior in
vector control. This study also revealed that dirty
toilets, bathtubs that are rarely drained, frequent use
of insecticides, and lack of vector control efforts will Risk factors associated with Dengue incidence in Bandung, Indonesia Vol. 11, No. 1, June 2020 49 Table 4. Binary Logistic Regression Analysis on
determinants of dengue incidence characteristics
No
Variables
Odds Ratio 95% CI,
p-value
1
Age of household head
2.53 (1.34-4.78)
0.004**
2
Education of
household head
1.39 (1.00-1.93)
0.048*
3
Living room window
0.65 (0.39-1.77)
0.095
4
Chimney
0.73 (0.52-1.03)
0.078
5
Toilet
0.68 (0.48-0.96)
0.027*
6
Practices
0.73 (0.53-1.00)
0.055
7
Healthy house
1.72 (1,05-2.83)
0.031* increase the risk of family members infected with
dengue fever / DHF. In Kediri-Indonesia it was found
that the activity to prevent Dengue through mosquito
breeding sites eradication in the form of water
container management has a significant correlation
with the increase in Dengue cases.15 Previous studies
also reported that the condition of toilets in parts
of Kuala Lumpur16 and Thailand17 related to the
existence of Aedes spp; as well as in Venezuela. It stated that the practice of vector prevention was
negatively associated with dengue cases.18 Table 4. Binary Logistic Regression Analysis on
determinants of dengue incidence characteristics The attitudes and actions of the people of Bandung
City have shown functional categories in efforts to
control the vector of DHF, but it is not supported
by good knowledge as well. The impact is the
community makes efforts to clean the house
environment as daily tasks, but the city has not
made an effort to eradicate mosquitoes routinely
and precisely. Some people of the City of Bandung
admit that attempts to drain water are often but not
routine and not accompanied by brushing actions, so
the possibility of mosquito eggs attached to the walls
of water containers can grow into adult mosquitoes. Therefore, comprehensive knowledge about Dengue
should also be taken into consideration since
inadequate knowledge will not have an impact on
the behaviors of mosquito breeding sites eradication
in total.19–21 Important information in this study is that the
majority of DHF patients and their household heads
are in the phase where they are productive. DISCUSSIONS They
occupy a sturdy house but pay less attention to
aspects of vector control of DHF. This phenomenon
is probably due to the lack of complete community
knowledge about DHF. This phenomenon can
result in the spread of DHF from the workplace to
the residential environment, and transmit DHF to
household members who live at home, to reduce
family productivity. The mean number of workdays
lost for adult DHF patients was 7.6 (SD 3.1) days;27
as well as the head of the family who must treat
patients while in hospital. Therefore, serious efforts
are needed from the Bandung local government
to provide systematic education on the control of
DHF vectors at the household level to the head of
the household, so that they are motivated to make
efforts to eradicate mosquito nests regularly and
appropriately. The hope is that the head of the house
can invite all members of his family to start a clean
and healthy life, especially in preventing dengue
transmission. The existence of water containers in the toilet
can become a potential habitat for Aedes aegypti
breeding such as bathtubs and buckets.22,23 Stagnant
water in containers that are not drained and brushed
for more than nine days can be a habitat that is
loved by Aedes aegypti to complete the life cycle
of mosquitoes ranging from eggs, larvae, pupae, to
adults.24 Aedes aegypti adults infected with dengue
virus can become dengue vectors during their lifetime
between 15-65 days 25; as long as its blood needs are
met, Aedes aegypti will always be active inside the
house. Therefore, in dengue-endemic areas, it was
reported that household members who live at house
with dengue patient have the opportunity to become
infected with dengue 3.2 times (95% CI 1.8-5.7).26 The limitation of this study was that this study did
not do in-depth interviews related to the explanation
of why the people of Bandung City were low in
knowledge but had good vector control attitudes
and actions, though the means to access media
information about DHF is very easy to obtain. Besides, no more digging about the mobility of
household heads in maintaining the socioeconomic
status of the community in the city of Bandung. The results of the binary logistic regression analysis
showed that the productive age of the head of
the household had a dominant role in dengue
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https://openalex.org/W3092063058
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https://annals-csis.org/proceedings/2020/drp/pdf/197.pdf
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English
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Feasibility of computerized adaptive testing evaluated by Monte-Carlo and post-hoc simulations
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Annals of Computer Science and Information Systems
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Proceedings of the Federated Conference on Proceedings of the Federated Conference on
Computer Science and Information Systems pp. 359–367
DOI: 10.15439/2020F197
ISSN 2300-5963 ACSIS, Vol. 21 Feasibility of computerized adaptive testing
evaluated by Monte-Carlo and post-hoc simulations Patrícia Martinková
Institute of Computer Science of the Czech Academy of Sciences
Pod Vodárenskou vˇeží 2, Praha 8
Faculty of Education, Charles University, Myslíkova 7, Praha 1
martinkova@cs.cas.cz Lubomír Štˇepánek
Institute of Biophysics and Informatics
First Faculty of Medicine, Charles University
Salmovská 1, Praha 2
lubomir.stepanek@lf1.cuni.cz Lubomír Štˇepánek
Institute of Biophysics and Informatics
First Faculty of Medicine, Charles University
Salmovská 1, Praha 2
lubomir.stepanek@lf1.cuni.cz tests [3] on the other hand involve assigning blocks of items
adaptively depending on the ability estimate from the previous
test section. Abstract—Computerized adaptive testing (CAT) is a modern
alternative to classical paper and pencil testing. CAT is based
on an automated selection of optimal item corresponding to
current estimate of test-taker’s ability, which is in contrast to
fixed predefined items assigned in linear test. Advantages of CAT
include lowered test anxiety and shortened test length, increased
precision of estimates of test-takers’ abilities, and lowered level of
item exposure thus better security. Challenges are high technical
demands on the whole test work-flow and need of large item
banks. Basic principles of computerized adaptive test are presented
in Figure 1. select and show
the first item
test-taker’s response
their ability estimation
select the next item
termination criteria
met? exit and output the
final ability estimate
yes
no
Fig. 1. Computerized adaptive testing flowchart In this study, we analyze feasibility and advantages of comput-
erized adaptive testing using a Monte-Carlo simulation and post-
hoc analysis based on a real linear admission test administrated
at a medical college. We compare various settings of the adaptive
test in terms of precision of ability estimates and test length. We find out that with adaptive item selection, the test length
can be reduced to 40 out of 100 items while keeping the precision
of ability estimates within the prescribed range and obtaining
ability estimates highly correlated to estimates based on complete
linear test (Pearson’s ρ .= 0.96). We also demonstrate positive
effect of content balancing and item exposure rate control on
item composition. termination criteria
met? I. INTRODUCTION M M
ULTI-ITEM assessment instruments find their use in
number of areas including admission or other educa-
tional tests, psychological measurement, health-related ques-
tionnaires, and other behavioral measurements. A usual way
to perform achievement testing is by assigning a fixed set of
items which are supposed to measure construct of interest,
such as knowledge of biology, level of depression, fatigue, or
respondent’s quality of life. Fig. 1. Computerized adaptive testing flowchart An adaptive test is initialized by the selection and adminis-
tration of the first item. The first item can be selected randomly
or based on prior ability estimate of the respondent. Average
ability can be used as an uninformed estimate, alternatively,
initial estimate may be based on respondent’s answers to one
or a small number of pre-test items. Given that the abilities may greatly differ across test-takers,
the respondents with higher levels of ability may be bored by
easier items, while those with lower levels of ability might
experience inconvenient stress. An effective and appropriate
selection of items which suit the best the test-takers of a given
ability can thus be more convenient for respondents, may save
time and moreover provide estimates of better precision than
fixed tests of the same length. Depending on the answer to the first item, the test-taker
ability estimate is updated. If the termination criterion (such
as number of administered items or precision of the estimate)
is not met, the updated ability estimate is used to select the
next optimal item. This cycle is repeated until the a priori
specified termination criterion is met; then, eventually, the test
is stopped and final estimate of the test-taker ability is provided
as an output. Adaptive tests [1], [2] have been an alternative to linear
tests for decades. The most complex version of adaptive
tests is the one in which the item selection is done after
each item administration depending on the current estimate
of test-taker’s ability which is iteratively updated. Multistage PROCEEDINGS OF THE FEDCSIS. SOFIA, 2020 PROCEEDINGS OF THE FEDCSIS. SOFIA, 2020 360 (IRT) model was fitted to describe the probability of a correct
answer given applicant’s ability [12], tests have been demonstrated in areas of educational testing
[4], testing of psychological distress [5], [6], as well as
health-related measurements such as in mobility surveys [7],
and testing general disability [8]. While the adaptive tests
are usually shorter in terms of number of items and overall
time needed to complete the test, they also enable to estimate
test-taker’s ability with better precision than linear tests of sim-
ilar length. The lower level of item exposure usually implies
also better security when items are administered adaptively. pi (θp) = Pr (Upi = 1|θp, ξi) = Ψ [ai (θp −bi)]
=
exp [ai (θp −bi)]
1 + exp [ai (θp −bi)],
(1 (1) where θp is the ability of subject p ∈{1, 2, . . . , N}, vector
ξi = (ai, bi)T stands for set of item parameters (discrimination
and difficulty, respectively) for item i ∈{1, 2, . . . , I}, and
Ψ(•) is the logistic function. However, since the adaptive testing is more complex it re-
quires higher technical facility and support of trained experts. The initial setting of the adaptive test may provide number
of options which may have crucial impact on functioning of
the adaptive test. Therefore, feasibility and optimal setting of
CAT with respect to the given item bank and population of
test takers need to be analyzed in order to apply the adaptive
test effectively and profitably. In the item calibration phase, the item parameters (ai, bi)T
were estimated. To estimate the item parameters, we used
marginal maximum likelihood (MML) as follows [13]. Let
us assume local independence, i. e. independence of item
responses for the same subject given their ability θp (within
subject). Then the probability Pr (up|θp, ξ) of response pattern
up of subject p follows the form In this work, we use Monte Carlo simulations and post-hoc
analysis based on real data of admission test administrated at
a medical college with the aim to derive the optimal setting
of adaptive test. We also compare the precision of different
settings and estimate the correlation between the adaptively
estimated ability and estimates based on answers to complete
set of 100 items. We discuss results for different levels
of precision, and various test termination criteria. PROCEEDINGS OF THE FEDCSIS. SOFIA, 2020 We also
implement content balancing and item exposure rate control to
see how it affects performance and properties of the adaptive
test. We discuss the findings in context of the admission testing
and other educational testing at medical faculties. Pr (up|θp, ξ) =
IY
i=1
Pr (Upi = upi|θp, ξi) . (2) (2) Supposing there is no cooperation between subjects, we
can also assume independence between subjects (in-between). Let’s further denote ξ = (ξ1, . . . , ξI) the matrix of parameters
for all items i. Then the marginal likelihood function takes
form L (ξ, µ, σ; U) =
N
Y
p=1
Pr (up|ξ, µ, σ)
(3) (3) with The paper proceeds as follows. We firstly describe the
data and introduce all necessary background theory, including
underlying models, settings of adaptive tests and design of
the simulation studies in the Research Methodology section. We then present results of the post-hoc analysis and Monte
Carlo simulation in Section Results. Finally, discussion and
final remarks are provided in Conclusion section. Pr (up|ξ, µ, σ) =
=
Z
. . . Z
Pr (up|θp, ξ) g (θp|µ, σ) dθp, where µ and σ are the expected value and the variance
of respondent ability θp. With this approach, abilities θp
are treated as stochastic variables with normal distribution,
θp ∼N (µ, σ) and are integrated out [14]. The first-order derivatives with respect to ability parameters
θp result into the likelihood equations [15] that could be
numerically estimated using Expectation-Maximization (EM)
algorithm [16], producing the desired estimates of item pa-
rameters ˆξ =
ˆξ1, . . . , ˆξI
. A. Data and item calibration We used data from a real fixed admission test administrated
to 2372 test-takers (applicants) at First Faculty of Medicine,
Charles University, Prague in 2015 [9], also see [10]. Interac-
tive presentation of psychometric properties of the admission
test is available in R package ShinyItemAnalysis [11]. A. Comparison of linear and adaptive testing Both the linear and adaptive test scenario have their advan-
tages and disadvantages, respectively. Advantages of adaptive IEEE Catalog Number: CFP2085N-ART ©2020, PTI 359 LUBOM´IR ˇSTˇEP ´ANEK, PATRICIA MARTINKOVA: FEASIBILITY OF COMPUTERIZED ADAPTIVE TESTING LUBOM´IR ˇSTˇEP ´ANEK, PATRICIA MARTINKOVA: FEASIBILITY OF COMPUTERIZED ADAPTIVE TESTING 361 the maximum likelihood estimates (MLE) and their standard
errors [18]. Content balancing. Balancing of an adaptive test content
is usually treated as a combinatorial constrained optimization
problem [22]. Alternatively, it is based on a shadow-test
approach by projection of rest of the test at the current moment
(after k −1 items are administrated), which is a nonlinear
program using maximum-information rule and constrained by
domain attributes and other conditions [22]. While we used MLE to estimate ability in most results
shown here, other methods are available. In case the ability is
only unidimensional, a popular approach is the weighted like-
lihood estimator (WLE) [19]; which maximizes equation (4)
weighted by a function w(θ), thus In the post-hoc analysis described in this paper, we used one
of the combinatorial designs, where we initially set desired
proportions of expected administration rate to each of the
three domains (genetics, taxonomy, human biology). The items
were selected in a way to minimize differences between the
currently observed and initially set proportions. L (θ; up) = w(θ)L (θ|up) . (5) (5) Finally, Bayesian ability estimation [20] specifies prior
ability distribution p (θp|µ, σ) and maximizes posterior dis-
tribution of θp given up of the following form: p (θp|up, ξ, µ, σ) =
Pr (up|θp, ξ) p (θp|µ, σ)
R
. . . R
Pr (up|θp, ξ) p (θp|µ, σ) dθp
. Item exposure rate control. The rates of how many times
each item is administrated to one or more of test-takers
throughout one adaptive test session may be controlled to
minimize their unwanted leakage outside the tested population. Hetter-Sympson experiment is commonly applied to face this
problem and was also used in our simulation study [23]. The algorithm was run before the optimally selected item
was administrated, output of which was a decision either to
administer the item, or to pass and select the next best item
at the current estimate of ability ˆθp,k. The administered items
were removed from the item pool. Hetter-Symspon experiment
is based on evaluation of joint conditional probabilities of
item administration; thus cumbersome and usually must be
numerically simulated. Item selection. We used likelihood-based item selec-
tion [17], i. e. LUBOM´IR ˇSTˇEP ´ANEK, PATRICIA MARTINKOVA: FEASIBILITY OF COMPUTERIZED ADAPTIVE TESTING in each step, the next (k-th) item was selected
to maximize the observed Fisher information ik ≡arg max
j
n
IUk−1,Uj
ˆθk−1
o (6) at θp = ˆθp,k−1 given a subject p, where Uk−1 is an answer
pattern up to the (k −1)-th item [17]. This rule is also known
as the maximum-information rule in adaptive testing. Other item selection procedures include naive approach
such as Urry’s criterion picking always an item with diffi-
culty closest to the current ability estimate [21]. In Bayesian
framework, the posterior distribution of θp after the preceding
item serves as the prior distribution for the selection of the
next item. If the posterior distribution after k −1 items has
density p (θ|uk−1), then the k-th item is selected such that the
posterior distribution There are also some alternatives – an experiment determin-
ing which items are eligible for subjects and which not [24]. If an item is eligible, it remains in the pool for the subject
p; otherwise it is removed. This works as a principle of
"self-adjustment"; when an item was highly exposed within
previous p −1 subjects, it is likely not to be eligible for the
p-th subject. p (θ|uk−1, Uik) ∝p (θ|uk−1) p (Uik = uik|θ) is optimized in some sense [20]. the maximum
errors [18].
While we
shown here, o
only unidimen
lihood estima
weighted by
Finally, Ba
ability distrib
tribution of θ
p (θp|up, ξ
Item selec
tion [17], i. e
to maximize
at θp = ˆθp,k−
pattern up to
as the maxim
Other item
such as Urry
culty closest
framework, th
item serves a
next item. If
density p (θ|u
posterior distr
p (θ|u
is optimized i
Termination
ability estima
IUp,k−1(ˆθp,k−
where Uk−1
(inclusively)
ˆθp,k−1 is
S
LUBOM´IR ˇSTˇE LUBOM´IR ˇSTˇEP ´ANEK, PATRICIA MARTINKOVA: FEASIBILITY OF COMPUTERIZED ADAPTIVE TESTING B. Settings of adaptive tests Initialization. Initial item was selected as the one maximiz-
ing observed Fisher information at ability θ0 = 0, see [17]. Ability estimation. Test-taker’s ability is iteratively updated
whenever the respondent answers to a given item and the
answer is collected. Beginning with the equation (2), the
likelihood is as follows The test consisted of 100 dichotomously scored items
covering different Biology topics. For the purpose of this
analysis, items were classified into three general domains
– genetics, taxonomy, and human biology, respectively. The
mutual proportions of these three domains were of nearly equal
size. L (θ; up) =
IY
i=1
P (Upi = upi|θ, ξi)
(4) (4) and is maximized with respect to θ. Then, the first-order
and second-order partial derivatives are needed to compute To evaluate psychometric properties of the items, unidi-
mensional two-parameter logistic (2PL) item-response theory C. Post-hoc analysis {E} ;
Algorithm 2: Investigation of average adaptive test
length depending on the z-score from the original
linear test
Data: data of the real test
Result: boxplots of average test lengths for groups
based on z-scores from the original linear test
1 δ = 0.05
// neighbourhood around z∗;
2 {A} = ∅
// list of vectors of lengths;
3
// for different z-scores;
4 {Z}
// list of original z-scores;
5 {Z∗} = ∅// list of z∗-scores;
6 for j = 1 : 17 do
7
z∗= −2.00 + 0.25 · j;
8
{Z∗} = {Z∗} ∪{z∗};
9
{D} = ∅;
10
for all subjects with z ∈Z such that |z −z∗| ≤δ
do
11
run an adaptive test for the subject with
stopping criterion SE(θ)max = 0.30 and save
its length as d;
12
{D} = {D} ∪{d};
13
end
14
{A} = {A} ∪{D};
15 end
16
make a boxplot of {A} vs. {Z∗} ; Algorithm 1: Investigation of adaptive test length
depending on the precision of ability estimate Data: data of the real test
Result: boxplots of test lengths for CAT with different
standard errors of ability estimates Result: boxplots of average test lengths for groups
based on z-scores from the original linear test 1 δ = 0.05
// neighbourhood around z∗;
2 {A} = ∅
// list of vectors of lengths;
3
// for different z-scores;
4 {Z}
// list of original z-scores;
5 {Z∗} = ∅// list of z∗-scores; 1 δ = 0.05
// neighbourhood around z∗;
2 {A} = ∅
// list of vectors of lengths;
3
// for different z-scores;
4 {Z}
// list of original z-scores;
5 {Z∗} = ∅// list of z∗-scores; r j = 1 : 17 do
z∗= −2.00 + 0.25 · j;
{Z∗} = {Z∗} ∪{z∗};
{D} = ∅; {
}
for all subjects with z ∈Z such that |z −z∗| ≤δ
do {
}
for all subjects with z ∈Z such that |z −z∗| ≤δ
do 1
run an adaptive test for the subject with
stopping criterion SE(θ)max = 0.30 and save
its length as d; its length as d;
12
{D} = {D} ∪{d};
3
end
4
{A} = {A} ∪{D};
d {A} = {A} ∪{D};
d 17
make a boxplot of {A} vs. {E} ; 17
make a boxplot of {A} vs. {E} ; end
make a boxplot of {A} vs. C. Post-hoc analysis {Z∗} ; δ = 0.05 and z∗∈{−2.00, −1.75, −1.50, . . . , +1.75, +2.00}
were supposed to virtually take the adaptive test, keeping the
SE(θ)max = 0.30 for equation (7) constant. The z∗neighbour-
hood δ = 0.05 was chosen empirically, but consequently, one
can realize that δ = 0.125 would cover continuously the entire
range of all z-scores. For each z∗, a vector of all the adaptive
tests’ lengths was displayed in the final boxplot. The schema
of the simulation is provided in Algorithm 2. mismatch rate for adaptive tests with stopping criteria
SE(θ)max ∈{0.20, 0.30, 0.40, 0.50}. While the best fifth for
the linear test was calculated using the z-scores, the MML
ability estimates were used for the adaptive test. D. Monte Carlo simulation studies Whereas the post-hoc analysis requires real data from an
administrated test, the Monte-Carlo simulation study starts
from the scratch – it generates abilities of "virtual" test-takers
usually following normal distribution and responses based on
selected model (e. g. the 2PL IRT model) with given item
parameters. We first simulated the linear test, then, based on
the simulated answers, the adaptive scenario was simulated. We then correlated ability estimates from the adaptive test
with the true ability values. Finally, we displayed lengths
of adaptive test and we correlated the ability estimate with
the true ability. Other comparisons and analyses are possible
(length with respect to the true ability score, etc.), but not
presented here. The algorithm of the simulation is technically
described in Algorithm 3. Similarly, the effect of content balancing and item exposure
rate control was analyzed. When an adaptive test was admin-
istered to each test-taker from a randomly selected subset, we
counted how many times individual items occur in the tests. Absolute numbers of the items’ occurrences were then counted
up for different scenarios – besides the situation when neither
the content balancing nor the item exposure was applied,
the case of (only) the content balancing and (only) the item
exposure rate controlling was taken into account. Eventually,
using the fact, the items were classified into three domains
(Genetics, Taxonomy, Human Biology), their counts could be
clearly plotted using boxes in a boxplot. Finally, to study the impact of adaptive test with different
settings on the admission process, we enumerated the
admission mismatch rate between linear and adaptive tests. We assumed the best fifth of all the applicants would be
admitted and we calculated the mismatch rate as the ratio
of students who would be admitted based on their score
in the linear test but not based on the score in adaptive
test and vice versa. We then compared the admission Analyses were performed in R programming language and
environment [25] using the package mirtCAT [26]. C. Post-hoc analysis {E} ; h
ent
s;
s;
ing
h
Algorithm 2: Investigation of average adaptive test
length depending on the z-score from the original
linear test
Data: data of the real test
Result: boxplots of average test lengths for groups
based on z-scores from the original linear test
1 δ = 0.05
// neighbourhood around z∗;
2 {A} = ∅
// list of vectors of lengths;
3
// for different z-scores;
4 {Z}
// list of original z-scores;
5 {Z∗} = ∅// list of z∗-scores;
6 for j = 1 : 17 do
7
z∗= −2.00 + 0.25 · j;
8
{Z∗} = {Z∗} ∪{z∗};
9
{D} = ∅;
10
for all subjects with z ∈Z such that |z −z∗| ≤δ
do
11
run an adaptive test for the subject with
stopping criterion SE(θ)max = 0.30 and save
its length as d;
12
{D} = {D} ∪{d};
13
end
14
{A} = {A} ∪{D};
15 end
16
make a boxplot of {A} vs. {Z∗} ; Algorithm 1: Investigation of adaptive test length
depending on the precision of ability estimate
Data: data of the real test
Result: boxplots of test lengths for CAT with different
standard errors of ability estimates
1 {S}
// set or subset of respondents;
2
// of the real test;
3 {A} = ∅
// list of vectors of lengths;
4
// for different standard;
5
// errors;
6 {E} = ∅
// list of standard errors;
7 for j = 1 : 7 do
8
SE = 0.15 + 0.05 · j;
9
{E} = {E} ∪{SE};
10
{D} = ∅;
11
for p ∈S do
12
run an adaptive test for subject p with stopping
criterion SE(θ)max = SE and save its length
as d;
13
{D} = {D} ∪{d};
14
end
15
{A} = {A} ∪{D};
16 end
17
make a boxplot of {A} vs. C. Post-hoc analysis Termination criteria. In our simulation studies, we used
ability estimate precision as a stopping rule. Assuming the
IUp,k−1(ˆθp,k−1) is observed Fisher information [17] at ˆθp,k−1
where Uk−1 is an answer pattern until the (k −1)-th item
(inclusively) given a subject p, then standard error of ability
ˆθp,k−1 is In post-hoc analysis, the item parameters and the response
patterns of the respondents were used to rerun the test under
adaptive conditions. By doing this, the properties of the
adaptive test (such as the test length, precision of estimated
abilities etc.) were "post-hoc" evaluated and compared to the
original linear test. SE(ˆθp,k−1) =
1
r
IUp,k−1
ˆθp,k−1
. (7) (7) Considering the dataset of test-takers taking the real test,
we varied the maximal allowed standard error of the ability
estimates and ran the adaptive version of the test for each
of the test-takers investigating how many items were needed
to complete the test. The pseudocode of this simulation is
provided in Algorithm 1. For the adaptive test, we specified the maximal allowed
standard error SE(θ)max of the ability estimate based on the
distribution of standard errors of the ability estimates from the
full 100-item test. For subject p, the test was terminated just
after administration of the k-th item if SE(ˆθp,k) ≤SE(θ)max
and SE(ˆθp,k−1) > SE(θ)max. Otherwise, the test was stopped
if the length of 100 items was reached and all available items
were used. Similarly, we calculated the z-score for each subject using
the test scores from the real test, z-score = xp −¯x
sx
, Whenever the termination (stopping) criterion is met, the
adaptive test is ended and final estimate of test-taker’s ability
is provided. where xp is a test score of a subject p, ¯x is an average test score
and sx is a standard deviation of all test scores. All test-takers
having their z-scores in the interval of |z −z∗| ≤δ, where where xp is a test score of a subject p, ¯x is an average test score
and sx is a standard deviation of all test scores. All test-takers
having their z-scores in the interval of |z −z∗| ≤δ, where PROCEEDINGS OF THE FEDCSIS. C. Post-hoc analysis SOFIA, 2020 362 Algorithm 2: Investigation of average adaptive test
length depending on the z-score from the original
linear test
Data: data of the real test
Result: boxplots of average test lengths for groups
based on z-scores from the original linear test Algorithm 2: Investigation of average adaptive test
length depending on the z-score from the original
linear test
Data: data of the real test
Result: boxplots of average test lengths for groups
based on z-scores from the original linear test
1 δ = 0.05
// neighbourhood around z∗;
2 {A} = ∅
// list of vectors of lengths;
3
// for different z-scores;
4 {Z}
// list of original z-scores;
5 {Z∗} = ∅// list of z∗-scores;
6 for j = 1 : 17 do
7
z∗= −2.00 + 0.25 · j;
8
{Z∗} = {Z∗} ∪{z∗};
9
{D} = ∅;
10
for all subjects with z ∈Z such that |z −z∗| ≤δ
do
11
run an adaptive test for the subject with
stopping criterion SE(θ)max = 0.30 and save
its length as d;
12
{D} = {D} ∪{d};
13
end
14
{A} = {A} ∪{D};
15 end
16
make a boxplot of {A} vs. {Z∗} ; Algorithm 1: Investigation of adaptive test length
depending on the precision of ability estimate
Data: data of the real test
Result: boxplots of test lengths for CAT with different
standard errors of ability estimates
1 {S}
// set or subset of respondents;
2
// of the real test;
3 {A} = ∅
// list of vectors of lengths;
4
// for different standard;
5
// errors;
6 {E} = ∅
// list of standard errors;
7 for j = 1 : 7 do
8
SE = 0.15 + 0.05 · j;
9
{E} = {E} ∪{SE};
10
{D} = ∅;
11
for p ∈S do
12
run an adaptive test for subject p with stopping
criterion SE(θ)max = SE and save its length
as d;
13
{D} = {D} ∪{d};
14
end
15
{A} = {A} ∪{D};
16 end
17
make a boxplot of {A} vs. A. Post-hoc analysis Post-hoc analysis used the real test-takers data to simulate
the results under scenario of an adaptive test with selected
parameters. As an example, Fig. 5 demonstrates iteratively
estimated ability estimates and order of items in which they
would be administered to the 1-st subject under adaptive
scenario with terminating criterion SE(θ)max = 0.30. We can
see that the initial item would be item number 81, the last
item would be item number 70. The width of the grey belt
stands for precision of the ability estimate at each step k,
equal to two standard errors 2SE(ˆθ)p,k of the ability estimate
of person p. The belt becomes more narrow as the test-taker
answers more and more items. Note that the standard error
after 18 administered items is SE(ˆθ)1,18 ≤0.30 while after
17 administered items it is SE(ˆθ)1,17 > 0.30. ∀i ∈{1, 2, . . . , 100}, resulting in a spectrum of the item
information curves. q
P(q)
0.0
0.2
0.4
0.6
0.8
1.0
-6
-4
-2
0
2
4
6
Fig. 2. Item characteristic curves of the linear test estimated using 2PL IRT
model. q
P(q)
0.0
0.2
0.4
0.6
0.8
1.0
-6
-4
-2
0
2
4
6 ,
As a result of Algorithm 1, Fig. 6 presents how the number
of items needed to stop the adaptive test depends on the
termination criterion. We can see that the higher the maximal
standard error is applied as the termination criterion, the lower
the number of items is needed to terminate the adaptive test. As a result of simulation described with Algorithm 2,
Fig. 7 illustrates how the respondent ability (estimated with
a z-score) affects the number of items needed to stop the
adaptive test. The size of maximal allowed standard error
of the ability estimates as the stopping criterion was set to
SE(θ)max = 0.30 based on the distribution of the standard As a result of Algorithm 1, Fig. 6 presents how the number
of items needed to stop the adaptive test depends on the
termination criterion. We can see that the higher the maximal
standard error is applied as the termination criterion, the lower
the number of items is needed to terminate the adaptive test. Fig. 2. Item characteristic curves of the linear test estimated using 2PL IRT
model. III. RESULTS 10 for p = 1 : n do p
11
apply 2PL IRT model on p-th ability of {S} and
simulate an answer pattern ; p
13
{A} = {A} ∪ˆθp;
14
{D} = {D} ∪{d};
d Fig. 4. Histogram of standard errors of the ability estimates. 17
make a scatterplot of {A} vs. {S}, calculate
a correlation of {A} and {S} ; III. RESULTS All items of the linear test were calibrated using the 2PL
IRT model as described by equation (1). Item characteristic
curves and item information curves are plotted in Fig. 2 and
Fig. 3. All items have positive discrimination ai > 0 for LUBOM´IR ˇSTˇEP ´ANEK, PATRICIA MARTINKOVA: FEASIBILITY OF COMPUTERIZED ADAPTIVE TESTING 363 Algorithm 3: Investigation of ability estimates based
on adaptive tests using a Monte-Carlo simulation
Data: generated abilities following N(0, 12),
item parameters (estimated from real data), adaptive
test’s stopping criterion SE(θ)max = 0.30, item
selection using maximum-information rule
Result: a list of ability estimates based on adaptive test
1 n = 300
// number of generated;
2
// abilities;
3 {S}
// list of n generated;
4
// abilities;
5
// following N(0, 12);
6 {A} = ∅
// list of adaptive-based;
7
// ability estimates;
8 {D} = ∅// list of lengths;
9
// of adaptive tests;
10 for p = 1 : n do
11
apply 2PL IRT model on p-th ability of {S} and
simulate an answer pattern ;
12
use the answer pattern and run an adaptive test for
p-th ability and save its length as d and ability
estimate as ˆθp ;
13
{A} = {A} ∪ˆθp;
14
{D} = {D} ∪{d};
15 end
16
make a boxplot of {D} ;
17
make a scatterplot of {A} vs. {S}, calculate
a correlation of {A} and {S} ; Algorithm 3: Investigation of ability estimates based
on adaptive tests using a Monte-Carlo simulation q
I(q)
0.0
0.2
0.4
0.6
0.8
-6
-4
-2
0
2
4
6
Fig. 3. Item information curves of the linear test estimated using 2PL IRT
model. q
I(q)
0.0
0.2
0.4
0.6
0.8
-6
-4
-2
0
2
4
6 1 n = 300
// number of generated;
2
// abilities;
3 {S}
// list of n generated;
4
// abilities;
5
// following N(0, 12);
6 {A} = ∅
// list of adaptive-based;
7
// ability estimates;
8 {D} = ∅// list of lengths;
9
// of adaptive tests; Fig. 3. Item information curves of the linear test estimated using 2PL IRT
model. standard error of ability estimates
absolute frequency
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0
200
400
600
800
Fig. 4. Histogram of standard errors of the ability estimates. A. Post-hoc analysis errors, displayed in Fig. 4. We can see that the closer the
z-score is to zero, the lower number of items is needed
to complete the adaptive test while meeting the required
ability estimate precision defined by SE(θ)max = 0.30. This
corresponds to the fact that the information functions for
majority of items have the maxima for ability around zero as
demonstrated in Fig. 3. Contrary, for z-scores far from zero,
the observed Fisher information is small for most of the items,
thus a larger number of items is needed to meet the stopping
criterion, and often not even meeting it using all 100 items
available. Table I provides mismatch matrices for linear and adaptive
tests with different stopping criteria SE(θ)max. As expected,
the mismatch rate increases with increased allowed standard
error applied as a stopping criterion in the adaptive test. The
mismatch rate is 0.036, 0.083, 0.102 and 0.118 for adaptive
tests with stopping rules SE(θ)max = 0.20, 0.30, 0.40 and
0.50, respectively. A. Post-hoc analysis A plot of progress of 1-st subject in an adaptive test with the
terminating criterion set to maximal allowable standard error of the ability
estimates of SE(θ)max = 0.30. Fig. 7. Number of items needed to stop the adaptive test in respondents of
different ability levels. is employed, there is no visible change in comparison to no
application of the exposure control. 0.20
0.25
0.30
0.35
0.40
0.45
0.50
20
40
60
80
100
standard error of θ
# of items needed to stop the adaptive test 0.20
0.25
0.30
0.35
0.40
0.45
0.50
20
40
60
80
100
standard error of θ
# of items needed to stop the adaptive test
Fig. 6. Number of items needed to stop the adaptive test versus a size of
standard error of the ability estimate as the stopping criterion. 0
10
20
30
40
50
# of times item is administered
neither balancing nor control
content balancing
exposure control
genetics
taxonomy
human biology
Fig. 8. Number of items belonging to the domains genetics, taxonomy, human
biology, respectively, as were administrated with application of neither content
balancing nor item exposure control, with application of content balancing,
and with application of item exposure rate control only. 0
10
20
30
40
50
# of times item is administered
neither balancing nor control
content balancing
exposure control
genetics
taxonomy
human biology Fig. 6. Number of items needed to stop the adaptive test versus a size of
standard error of the ability estimate as the stopping criterion. Fig. 8. Number of items belonging to the domains genetics, taxonomy, human
biology, respectively, as were administrated with application of neither content
balancing nor item exposure control, with application of content balancing,
and with application of item exposure rate control only. errors, displayed in Fig. 4. We can see that the closer the
z-score is to zero, the lower number of items is needed
to complete the adaptive test while meeting the required
ability estimate precision defined by SE(θ)max = 0.30. This
corresponds to the fact that the information functions for
majority of items have the maxima for ability around zero as
demonstrated in Fig. 3. Contrary, for z-scores far from zero,
the observed Fisher information is small for most of the items,
thus a larger number of items is needed to meet the stopping
criterion, and often not even meeting it using all 100 items
available. A. Post-hoc analysis When applying the 2PL IRT model on the data from the
linear test, we get, besides other, also standard errors of
the ability estimates for each test-taker. Histogram of these
standard errors in in Fig. 4. Range of the standard errors of
the ability estimates is between 0.20 to 0.50, with majority of
values within the interval ⟨0.20, 0.30⟩. As a result of simulation described with Algorithm 2,
Fig. 7 illustrates how the respondent ability (estimated with
a z-score) affects the number of items needed to stop the
adaptive test. The size of maximal allowed standard error
of the ability estimates as the stopping criterion was set to
SE(θ)max = 0.30 based on the distribution of the standard PROCEEDINGS OF THE FEDCSIS. SOFIA, 2020 364 CAT Standard Errors
Item
θ
−1.0
−0.5
0.0
0.5
1.0
0
81
72
57
82
73
76
18
78
56
74
90
30
80
53
89
37
70
Fig. 5. A plot of progress of 1-st subject in an adaptive test with the
terminating criterion set to maximal allowable standard error of the ability
estimates of SE(θ)max = 0.30. CAT Standard Errors
Item
θ
−1.0
−0.5
0.0
0.5
1.0
0
81
72
57
82
73
76
18
78
56
74
90
30
80
53
89
37
70
Fig. 5. A plot of progress of 1-st subject in an adaptive test with the
terminating criterion set to maximal allowable standard error of the ability
estimates of SE(θ)max = 0.30. −2.00
−1.50
−1.00
−0.50
0.00
0.50
1.00
1.50
2.00
20
40
60
80
100
z−score
# of items needed to stop the adaptive test
standard error of θ = 0.3
Fig. 7. Number of items needed to stop the adaptive test in respondents of
different ability levels. CAT Standard Errors
Item
θ
−1.0
−0.5
0.0
0.5
1.0
0
81
72
57
82
73
76
18
78
56
74
90
30
80
53
89
37
70 CAT Standard Errors −2.00
−1.50
−1.00
−0.50
0.00
0.50
1.00
1.50
2.00
20
40
60
80
100
z−score
# of items needed to stop the adaptive test
standard error of θ = 0.3
Fig. 7. Number of items needed to stop the adaptive test in respondents of
different ability levels. −2.00
−1.50
−1.00
−0.50
0.00
0.50
1.00
1.50
2.00
20
40
60
80
100
z−score
# of items needed to stop the adaptive test
standard error of θ = 0.3 Fig. 5. TABLE I
MISMATCH MATRICES OF ADMITTED TEST-TAKERS BY LINEAR AND
ADAPTIVE TEST WITH STOPPING CRITERION
SE(θ)max ∈{0.20, 0.30, 0.40, 0.50}. TABLE I
MISMATCH MATRICES OF ADMITTED TEST-TAKERS BY LINEAR AND
ADAPTIVE TEST WITH STOPPING CRITERION
SE(θ)max ∈{0.20, 0.30, 0.40, 0.50}. MISMATCH MATRICES OF ADMITTED TEST-TAKERS BY LINEAR AND
ADAPTIVE TEST WITH STOPPING CRITERION
SE(θ)max ∈{0.20, 0.30, 0.40, 0.50}. SE(θ)max = 0.20
admitted by adaptive test
no
yes
admitted by linear test
no
1842
38
yes
48
435
SE(θ)max = 0.30
admitted by adaptive test
no
yes
admitted by linear test
no
1787
93
yes
103
380
SE(θ)max = 0.40
admitted by adaptive test
no
yes
admitted by linear test
no
1762
118
yes
123
360
SE(θ)max = 0.50
admitted by adaptive test
no
yes
admitted by linear test
no
1737
143
yes
136
347 Fig. 10. A scatterplot of the generated abilities and their estimates based on
the adaptive tests. The blue line stands for an axis of the first quadrant of the
plot. 75% shortening as compared to the linear test, while keeping
the same precision of ability estimates for most respondents. When even larger standard errors of the ability estimates
are tolerated, the length of the test could be reduced even
more, e. g. to only 10 items per one test, as was shown in
the post-hoc analysis of the average adaptive test length with
varying stopping criterion. 20
40
60
80
100
adaptive test
# of items needed to stop the adaptive test
# of items needed
to complete the linear test The post-hoc simulation also demonstrated that an average
length for adaptive tests is shorter for average ability levels. While the content balancing with the combinatorial ap-
proach showed a significant improvement in test domain
equalizing, an effect of the item exposure rate did not seem
to be so eminent under our setting. The lower the tolerated standard error as a stopping criterion
of the adaptive test is, the lower is the mismatch error rate
when using an adaptive test instead of the linear one. The
mismatch rate was less than 10% for adaptive test with
stopping criterion of SE(ˆθ)max = 0.30. The Monte-Carlo simulation study also indicated that abil-
ity estimates provided by the adaptive tests can be tightly
correlated with their true (generated) values; thus, although
the shortened length, the adaptive test can provide precise
estimates of the respondent abilities. adaptive test Fig. 9. TABLE I
MISMATCH MATRICES OF ADMITTED TEST-TAKERS BY LINEAR AND
ADAPTIVE TEST WITH STOPPING CRITERION
SE(θ)max ∈{0.20, 0.30, 0.40, 0.50}. A boxplot of number of items needed to be answered to complete the
adaptive test based on Monte-Carlo simulated test-takers’ abilities. The blue
dashed line shows a length of the linear test (100 items). To conclude, usage of adaptive testing seems to be a promis-
ing alternative to classic linear tests and offers many advan-
tages as showed by the simulations. Pearson’s correlation between the generated abilities and
their estimates based on the adaptive tests is about ρ .= 0.960,
which is depicted also in Fig. 10. IV. CONCLUSION Research was supported by Charles University grant
PRIMUS/17/HUM/11. Research was supported by Charles University grant
PRIMUS/17/HUM/11. Both the post-hoc analysis and Monte-Carlo simulation
study showed that average test lengths can be shortened with
adaptive tests, while keeping the standard error of the ability
estimates at the same level for most of the respondents. The
shortening of the test within the adaptive test with SE(ˆθ)max =
0.30 was by about 75 % percent, i. e. while the original linear
test had 100 items, the adaptive one was ended on average
after answering 25 items only. B. Monte-Carlo simulation study In Fig. 8, we plot numbers of occurrences of items in all
individual adaptive tests for randomly selected 50 test-takers,
considering that each item belongs to one of the following
three domains – either to genetics, taxonomy, or human biol-
ogy, respectively. While the proportions of the three domains
of items as they were administrated vary a lot in Fig. 8 where
neither the content balancing nor the item exposure rate control
is applied, these numbers are near equal when the content
balancing is applied. When the item exposure rate control As a result of the Monte-Carlo simulation study described
by Algorithm 3, Fig. 9 provides a boxplot illustrating the
mean length of the adaptive test for the set of test-takers with
the generated abilities. While each test-taker has to answer
to all (100) items within the linear fashion, they would only
have to answer about 25 % of items to finish the simulated
adaptive test with the termination criterion SE(θ)max = 0.30. The length of the test using this adaptive scenario provides LUBOM´IR ˇSTˇEP ´ANEK, PATRICIA MARTINKOVA: FEASIBILITY OF COMPUTERIZED ADAPTIVE TESTING LUBOM´IR ˇSTˇEP ´ANEK, PATRICIA MARTINKOVA: FEASIBILITY OF COMPUTERIZED ADAPTIVE TESTING 365 -2
-1
0
1
2
-2
-1
0
1
2
generated abilities
abilities estimated based on the adaptive tests
Fig. 10. A scatterplot of the generated abilities and their estimates based on
the adaptive tests. The blue line stands for an axis of the first quadrant of the
plot. -2
-1
0
1
2
-2
-1
0
1
2
generated abilities
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http://www.nomos-elibrary.de/10.5771/1611-5821-2012-5-52.pdf
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German
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Analyse der Mitgliederentwicklung sowie weiterer Kennzahlen der bundesunmittelbaren Krankenkassen ohne und mit Zusatzbeitrag bzw. Prämie
|
Gesundheits- und Sozialpolitik
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cc-by
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THEMA THEMA 1 Dieser Beitrag gibt die persönlichen Auffas-
sungen der Verfasser wieder. Maximilian GaSSner,
Frank Otto,
Eva Ludwig Dr. Maximilian Gaßner ist
Präsident des Bundesversiche-
rungsamts (BVA) in Bonn Dr. Maximilian Gaßner ist
Präsident des Bundesversiche-
rungsamts (BVA) in Bonn Frank Otto ist Referatsleiter
im Referat Finanzaufsicht in
der Krankenversicherung im
BVA Eva Ludwig ist Referentin im
Referat Finanzaufsicht in der
Krankenversicherung im BVA Die nachfolgende Untersuchung be-
schäftigt sich mit der Frage der Mit-
gliederentwicklung sowie weiterer
Kennzahlen der bundesunmittelbaren
Krankenkassen ohne und mit Zusatz-
beitrag bzw. Prämie. Die Ergebnisse
müssen als statistisch nicht signifikante
Zwischenergebnisse interpretiert wer-
den. Endgültige Feststellungen verbie-
ten sich in Folge der geringen Zahl der
Kassen und der besonderen Situation im
betrachteten Zeitraum (01.01.2009 bis
heute). Einfluss auf die Zahlen dürften
zudem politische Interpretationen der
gesetzlichen Regelungen, aber auch
sonstige exogene Ereignisse, wie z. B. die
Intervention des Bundeskartellamtes ge-
gen die gemeinsame Pressekonferenz der
betroffenen Kassen zur Erhebung des
Zusatzbeitrages gehabt haben, sodass
die Zahlen nicht nur auf die Wirkungen
des Preissignals zurückzuführen sind. Analyse der Mitgliederentwicklung
sowie weiterer Kennzahlen
der bundesunmittelbaren
Krankenkassen ohne und mit
Zusatzbeitrag bzw. Prämie1 Mit Einführung des Gesundheitsfonds im Jahr
2009 wurden die individuellen Beitragssätze der
gesetzlichen Krankenkassen durch ein System
mit einheitlichem Beitragssatz, Zusatzbeiträgen
und Prämien ersetzt. Einige Krankenkassen haben
seitdem Zusatzbeiträge eingeführt und wieder
abgeschafft. Andere Krankenkassen waren in der
Lage, Prämien an ihre Mitglieder auszuzahlen. Welche
Erfahrungen haben diese Krankenkassen mit den
neuen Instrumenten gemacht? Eine erster Überblick. Maximilian GaSSner,
Frank Otto,
Eva Ludwig
Dr. Maximilian Gaßner ist
Präsident des Bundesversiche-
rungsamts (BVA) in Bonn
Frank Otto ist Referatsleiter
im Referat Finanzaufsicht in
der Krankenversicherung im
BVA
Eva Ludwig ist Referentin im
Referat Finanzaufsicht in der
Krankenversicherung im BVA https://doi.org/10.5771/1611-5821-2012-5-52
Generiert durch IP '195.242.1.103', am 24.10.2024, 05:55:08.
Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig Einleitung Soweit der Finanzbedarf einer Kranken-
kasse durch die Zuweisungen aus dem
Gesundheitsfonds nicht gedeckt ist, hat
sie gem. § 242 Abs. 1 SGB V einen »ein-
kommensunabhängigen Zusatzbeitrag«
zu erheben. Der Zusatzbeitrag setzt ein
klares Preissignal, das von vielen Ge-
sundheitsökonomen und den Vertretern
von Prämienmodellen zur Verschärfung
des Wettbewerbs immer gefordert wur-
de. Dieses Preissignal ist von den Mit-
gliedern der gesetzlichen Krankenversi-
cherung auch »gut« verstanden worden. Viele haben die Kassen gewechselt. Zu-
mal dieser Kassenwechsel durch ein Son-
derkündigungsrecht gem. § 175 Abs. 4
S. 5 SGB V vom Gesetzgeber auch noch
erleichtert wird. Die Transparenz wird
noch verstärkt durch die Verpflichtung
der Krankenkasse gem. § 175 Abs. 4
S. 6 SGB V, ihre Mitglieder auf das
Sonderkündigungsrecht ausdrücklich
hinzuweisen. G+S
5/2012 G+S
5/2012 52 THEMA Tabelle 1: Mitgliederentwicklung bundesunmittelbarer Krankenkassen mit
Zusatzbeitrag
Bundesunmittelbare Kranken
kassen mit ZB
Höhe*
Von
Bis
Mitglieder
veränderung
im ZB-Zeit
raum
Unter 200.000
Mitglieder
Ungewichteter
Durchschnitt
8,83 €
Feb 2010
Okt 2011
-20,45%
Gewichteter
Durchschnitt
9,23 €
Mrz 2010
Sep 2011
-29,48%
Über 200.000
Mitglieder
Ungewichteter
Durchschnitt
8,00 €
Jun 10
Feb 12
-12,08%
Gewichteter
Durchschnitt
8,00 €
Nov 10
Jun 12
-7,75%
Quelle: KM1. Als Gewicht wurde die Anzahl der Mitglieder verwendet. * Ohne prozentuale Zusatzbeiträge Tabelle 1: Mitgliederentwicklung bundesunmittelbarer Krankenkassen mit
Zusatzbeitrag Im Zeitraum vom 01.01.2009 bis
heute (Juli 2012) gab es insgesamt 14
(9,6 %) bundesunmittelbare Kranken-
kassen mit Zusatzbeitrag und 7 (4,8 %)
mit einer Prämie. Hinzu kommen
weitere 3 (2,1 %) landesunmittelbare
Krankenkassen mit Zusatzbeitrag und
3 (2,1 %) landesunmittelbare Kranken-
kassen mit Prämie. 2 Vgl. Eibich, Schmitz und Ziebarth im DIW
Wochenbericht Nr. 51+52, 2011, S. 8. 1. Entwicklung und Kennzahlen der
Krankenkassen mit Zusatzbeitrag
Übersicht und Mitglieder
entwicklung Der Zusatzbeitrag wurde in Höhe von
6,50 Euro bis 15 Euro erhoben sowie in
Höhe von 1 % des beitragspflichtigen
Einkommens. Die folgende Tabelle zeigt
eine Übersicht der Durchschnittswerte
für bundesunmittelbare Krankenkassen
mit Zusatzbeitrag. Quelle: KM1. Als Gewicht wurde die Anzahl der Mitglieder verwendet. * Ohne prozentuale Zusatzbeiträge Quelle: KM1. Als Gewicht wurde die Anzahl der Mitglieder verwendet. * Ohne prozentuale Zusatzbeiträge nige bis gar keine Mitgliederverluste. 2 Vgl. Eibich, Schmitz und Ziebarth im DIW
Wochenbericht Nr. 51+52, 2011, S. 8.
3 Vgl. ebd., S. 9. nige bis gar keine Mitgliederverluste. se übereinstimmt, sind die Zahlen nicht
vergleichbar. buchen. Dieses Konto wird nach dem
Soll-Prinzip der zeitlichen Rechnungs-
abgrenzung geführt, dieses sieht die Ver-
buchung in jener Rechnungsperiode vor,
für die die Rechnungsstellung erfolgt
ist. Die Differenz dieser beiden Posten
lässt sich damit als Unterschiedsbetrag
zwischen dem maximal zu erreichenden
Zusatzbeitragseinnahmen und den tat-
sächlichen Rechnungsstellungen für den
Zusatzbeitrag interpretieren. nige bis gar keine Mitgliederverluste. Thema Zusatzbeitrag vor. Die Autoren
berechneten, dass die Einführung eines
Zusatzbeitrags im Durchschnitt zu Ver-
sichertenverlusten von –7,61 % führt.2
Die individuelle Wahrscheinlichkeit für
einen Krankenkassenwechsel verdop-
pelt sich durch den Zusatzbeitrag, so
die Autoren.3 Da die Art und Weise der
Berechnung nicht mit der dieser Analy- Die Mitgliederentwicklung der Kran-
kenkassen über 200.000 Mitglieder mit
Zusatzbeitrag zeigt ein homogeneres
Bild, als das der kleineren Krankenkas-
sen. Es wird deutlich, dass sich der Zu-
satzbeitrag je nach Krankenkassegröße
unterschiedlich stark auf die Mitglieder-
entwicklung auswirkt. Der Anstieg im
Januar 2012 ergibt sich aus einer Fusion. Die Erhebung des Zusatzbeitrags
wirkte sich auf die Mitgliederzahl un-
terschiedlich aus. Die folgende Abbil-
dung zeigt die Mitgliederentwicklung
der bundesunmittelbaren Krankenkas-
sen mit Zusatzbeitrag. Im Wochenbericht Nr. 51+52/2011
vom 21. Dezember 2011 legte das
Deutsche Institut für Wirtschaftsfor-
schung (DIW) eigene Analysen zum
Diagramm1 Die Krankenkassen mit dem stärksten
Mitgliederverlust finden sich im Bereich
der mittleren Mitgliederstärke. Kleine
geschlossene Krankenkassen hatten we- 53
5/2012
G+S
Abbildung 1: Mitgliederentwicklung bundesunmittelbarer Krankenkassen mit Zusatzbeitrag
Page 1
Abbildung 1: Mitgliederentwicklung bundesunmittelbarer Krankenkassen mit Zusatzbeitrag
0
200.000
400.000
600.000
800.000
1.000.000
1.200.000
1.400.000
1.600.000
1.800.000
2.000.000
2010.01
2010.02
2010.03
2010.04
2010.05
2010.06
2010.07
2010.08
2010.09
2010.10
2010.11
2010.12
2011.01
2011.02
2011.03
2011.04
2011.05
2011.06
2011.07
2011.08
2011.09
2011.10
2011.11
2011.12
2012.01
2012.02
2012.03
2012.04
2012.05
2012.06
0
20.000
40.000
60.000
80.000
100.000
120.000
140.000
160.000
180.000
200.000
Durchschnitt; über 200.000 Mitglieder
Durchschnitt; unter 200.000 Mitglieder
https://doi.org/10.5771/1611-5821-2012-5-52
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https://doi.org/10.5771/1611-5821-2012-5-52
Generiert durch IP '195.242.1.103', am 24.10.2024, 05:55:08. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig. 53 THEMA Tabelle 2: Rückstandsquoten des Zusatzbeitrags Tabelle 2: Rückstandsquoten des Zusatzbeitrags
Theoretische Soll-
Einnahmen ZB 2010
Soll-Einnahmen ZB
gemäß
KJ1 2010
Forderungen auf ZB
2010
Differenz zu den
Soll-Einnahmen
KJ1 in % 2010
Quelle:
KM1 2010
Kt. 2500, KJ1 2010
Kt. 0210, KJ1 2010
Eig. Berechnung
Unter 200.000 Mitglieder
Ungewichteter Durchschnitt
3.019.165 €
2.949.699 €
664.386 €
22,52%
Gewichteter Durchschnitt
7.943.505 €
7.875.940 €
2.022.810 €
25,68%
Über 200.000 Mitglieder
Ungewichteter Durchschnitt
136.196.266 €
126.138.097 €
16.937.853 €
13,43%
Gewichteter Durchschnitt
282.397.428 €
265.393.676 €
30.024.052 €
11,31%
Summe für alle bundesunmittelbare
Krankenkassen mit Zusatzbeitrag
699.096.320 €
648.388.682 €
88.675.582 €
13,68%
Als Gewicht wurde die Anzahl der Mitglieder verwendet. Ohne Krankenkassen mit prozentualem Zusatzbeitrag. Verpflichtungen auf Zusatzbeitrag
wurden hier nicht berücksichtigt. 3. Vergleich der
Mitgliederveränderungen bei
Krankenkassen ohne bzw. mit
Zusatzbeitrag oder Prämie Bei den Einnahmen gemäß KJ1 wur-
de die Schlüsselnummer 2500 herange-
zogen. Sie entspricht dem Konto 2500,
auf welchem die Krankenkassen ihre
Einnahmen aus dem Zusatzbeitrag ver- Im Vergleich der Mitgliederverände-
rung (Abbildung 3) wird deutlich, dass
Krankenkassen mit Zusatzbeitrag im
Durchschnitt Mitglieder verloren haben,
während Krankenkassen mit Prämie
im Durchschnitt Mitglieder gewonnen
haben. Betrachtet man die prozentua-
le Mitgliederänderung bezogen auf die
Ausgangswerte im Januar 2009, sind
nur für die Krankenkassen mit Zusatz-
beitrag extrem negative Veränderungs-
raten zu beobachten. Tabelle 3: Mitgliederentwicklung bundesunmittelbarer Krankenkassen mit Prämie
Höhe
Von
Bis
Mitgliederzuwachs im
Prämien-Zeitraum
Bundesunmittelbare Krankenkassen mit Prämie
Ungewichteter
Durchschnitt
5,38 €
Mai. 10
Fast alle
bis heute
12,06%
Gewichteter
Durchschnitt
4,70 €
Apr. 10
10,48%
Quelle: KM1. Als Gewicht wurde die Anzahl der Mitglieder verwendet Tabelle 3: Mitgliederentwicklung bundesunmittelbarer Krankenkassen mit Prämie
Höhe
Von
Bis
Mitgliederzuwachs im
Prämien-Zeitraum
Bundesunmittelbare Krankenkassen mit Prämie
Ungewichteter
Durchschnitt
5,38 €
Mai. 10
Fast alle
bis heute
12,06%
Gewichteter
Durchschnitt
4,70 €
Apr. 10
10,48%
Quelle: KM1. Als Gewicht wurde die Anzahl der Mitglieder verwendet Tabelle 3: Mitgliederentwicklung bundesunmittelbarer Krankenkassen mit Prämie Die Daten wurden für den Boxplot
auf den aktuellen Fusionsstand (Juli
2012) gebracht. Diese Vorgehensweise Quelle: KM1. Als Gewicht wurde die Anzahl der Mitglieder verwendet https://doi.org/10.5771/1611-5821-2012-5-52
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Realisierungsquoten
des Zusatzbeitrags Die ersten Prämien gab es bereits ab dem
01.01.2009. Von den 7 bundesunmittel-
baren Krankenkassen, die seither eine
Prämie auszahlen, sind 3 geschlossene
BKKen, die ausgezahlten Prämien bewe-
gen sich in einer Spanne von 2,50 Euro
bis 10 Euro im Monat. Tabelle 3 zeigt
die Durchschnittswerte für die Auszah-
lung von Prämien. Tabelle 2 zeigt eigene Berechnungen des
BVA auf Basis der amtlichen Statistik
für das Jahr 2010. Die Soll-Einnahmen wurden für je-
den Monat mit Zusatzbeitrag berechnet,
indem der erhobene Zusatzbeitrag mit
der Mitgliederzahl multipliziert wurde. Dieses Vorgehen führt allerdings nur zu
einer näherungsweisen Lösung, da die
Mitglieder, die vom Zusatzbeitrag be-
freit sind, nicht berücksichtigt werden. Hierbei ist insbesondere zu beachten,
dass Mitglieder, die bis zum Zeitpunkt
der erstmaligen Fälligkeit kündigen, den
Zusatzbeitrag nicht zahlen. In der folgenden Spalte ist die Schlüs-
selnummer 0210 abgebildet. Unter dem
korrespondierenden Konto verbuchen
die Krankenkassen die Forderungen auf
den Zusatzbeitrag, d. h. die in Rechnung
gestellten, aber nicht eingegangenen
Zahlbeträge. Da laut Kontenrahmen
Forderungen jedoch gemäß ihres Aus-
fallrisikos bewertet werden müssen, ist
es möglich, dass hier bereits mit dem
Ausfallrisiko wertberichtigte Beträge
verbucht wurden. Die Differenz zwi-
schen den Soll-Einnahmen gemäß KJ1
und den Forderungen auf Zusatzbeitrag
kann somit mit Einschränkungen als
Rückstandsquote interpretiert werden. In Abbildung 2 sind die Mitglieder-
entwicklungen der Krankenkassen mit
Prämie über den gesamten Zeitraum von
01.01.2009 bis heute abzulesen. G+S
5/2012 54 THEMA Abbildung 2: Mitgliederentwicklung bundesunmittelbarer Krankenkassen mit Prämie
Abbildung 2: Mitgliederentwicklung bundesunmittelbarer Krankenkassen mit Prämie
72.000
74.000
76.000
78.000
80.000
82.000
84.000
86.000
88.000
90.000
92.000
2009.01
2009.02
2009.03
2009.04
2009.05
2009.06
2009.07
2009.08
2009.09
2009.10
2009.11
2009.12
2010.01
2010.02
2010.03
2010.04
2010.05
2010.06
2010.07
2010.08
2010.09
2010.10
2010.11
2010.12
2011.01
2011.02
2011.03
2011.04
2011.05
2011.06
2011.07
2011.08
2011.09
2011.10
2011.11
2011.12
2012.01
2012.02
2012.03
2012.04
2012.05
2012.06
Mitglieder
Durchschnitt Abbildung 2: Mitgliederentwicklung bundesunmittelbarer Krankenkassen mit Prämie
Abbildung 2: Mitgliederentwicklung bundesunmittelbarer Krankenka haben bzw. hatten. Das System »Zusatz-
beiträge und Prämien« ist damit aktu-
ell nicht vollständig verwirklicht. Ein
Zusatzbeitrag wird von den Versicher-
ten und den Krankenkassen noch nicht
als reguläres Finanzierungsinstrument
der gesetzlichen Krankenversicherung
wahrgenommen und eine tiefere (Preis-)
Differenzierung hat nicht stattgefunden. Bei den Krankenkassen, die einen
Zusatzbeitrag erhoben haben, hat dies
teilweise zu starken Mitgliederverlusten
geführt, die weit über den bekannten
Mitgliederschwankungen bei Beitrags-
satzanpassungen lagen. Dieses Verhal-
ten deutet auf die politisch gewünschte
höhere Transparenz des Preissignals
hin. Insbesondere ab einer bestimmten kann zu Verzerrungen führen, wenn
Krankenkassen mit und ohne Zusatz-
beitrag fusioniert haben, da die Daten
dieser Krankenkassen vermischt wer-
den. Verzerrungen der Steigerungsraten
aufgrund von Fusionen werden durch
diese Vorgehensweise jedoch vermieden. Die Boxen entsprechen dem Bereich, in
dem 50 % der Daten, d. h. der Verände-
rungsraten liegen. Die Antennen stellen
die restlichen Werte (ca. 45 %) außer-
halb der Box dar, die übrigen (ca. 5 %)
werden als Extremwerte bzw. Ausrei-
ßer bezeichnet und sind im Boxplot aus
Datenschutzgründen nicht dargestellt. Die Linie innerhalb der Boxen stellt den
Median der Veränderungsraten mit dem
jeweiligen Kriterium (ohne Prämie/Zu-
satzbeitrag, mit Prämie und mit Zusatz-
beitrag) dar. Abbildung 3: Veränderung der Mitgliederzahlen in Prozent
Anmerkung: Bei geschlossenen Krankenkassen wurde die Mitgliederzahl zum Schlie-
ßungszeitpunkt herangezogen. Abbildung 3: Veränderung der Mitgliederzahlen in Prozent Besonders extreme Werte kommen
durch den langen Betrachtungszeitraum
zustande, der im Januar 2009 beginnt
und im Juni 2012 bzw. mit Schließungs-
zeitpunkt endet. 4. Fazit Zusammenfassend lässt sich festhal-
ten, dass relativ wenige Krankenkassen
einen Zusatzbeitrag oder eine Prämie Anmerkung: Bei geschlossenen Krankenkassen wurde die Mitgliederzahl zum Schlie-
ßungszeitpunkt herangezogen. 55
5/2012
G+S
https://doi.org/10.5771/1611-5821-2012-5-52
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Generiert durch IP '195.242.1.103', am 24.10.2024, 05:55:08. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig. 55 THEMA H
di
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de
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Patientenversorgung im
internationalen Vergleich
Comparing Chronic Disease Patterns
between Germany and the US
International Comparisons of Treatment Patterns,
Disease Interactions and Healthcare Use and Costs
Herausgegeben von Prof. Dr. Norbert Klusen, Dr. Frank Verheyen
2012, 179 S., brosch., 24,– €
ISBN 978-3-8329-7477-0
(Beiträge zum Gesundheitsmanagement, Bd. 35)
Comparing Chronic Disease Patterns
between Germany and the US
Norbert Klusen/Frank Verheyen (eds.)
International Comparisons of Treatment Patterns,
Disease Interactions, and Healthcare Use and Costs
Beiträge zum Gesundheitsmanagement
35
Nomos
Nomos Höhe des Zusatzbeitrags überraschten
die extremen Mitgliederbewegungen, da
sie weit über den Erfahrungswerten des
bisherigen »Beitragssatz-Wettbewerbs«
lagen. Comparing Chronic Disease Patterns
between Germany and the US International Comparisons of Treatment Patterns,
Disease Interactions and Healthcare Use and Costs
Herausgegeben von Prof. Dr. Norbert Klusen, Dr. Frank Verheyen
2012, 179 S., brosch., 24,– €
ISBN 978-3-8329-7477-0
(Beiträge zum Gesundheitsmanagement, Bd. 35) International Comparisons of Treatment Patterns,
Disease Interactions and Healthcare Use and Costs
Herausgegeben von Prof. Dr. Norbert Klusen, Dr. Frank Verheyen
2012, 179 S., brosch., 24,– €
ISBN 978-3-8329-7477-0
(Beiträge zum Gesundheitsmanagement, Bd. 35) Ländervergleich USA-Deutschland: Welche Unterschiede existieren
in der Versorgung chronischer Krankheiten? Worin unterscheiden
sich Qualität und Kosten? Was können wir voneinander lernen? Ausgehend von Hunderttausenden an Abrechnungsdaten diesseits
und jenseits des Atlantiks werden erstmals Behandlungsmuster
und -ergebnisse miteinander verglichen. Weitere Informationen: www.nomos-shop.de/14664 Weitere Informationen: www.nomos-shop.de/14664 Patientenversorgung im
internationalen Vergleich Schließlich konnte in der vorliegen-
den Analyse gezeigt werden, dass die
Auszahlung einer Prämie weniger starke
Mitgliederbewegungen nach sich zieht. Comparing Chronic Disease Patterns
between Germany and the US
Norbert Klusen/Frank Verheyen (eds.)
International Comparisons of Treatment Patterns,
Disease Interactions, and Healthcare Use and Costs
Beiträge zum Gesundheitsmanagement
35
Nomos
Nomos Die Analyse hat
gezeigt, dass die
Auszahlung einer
Prämie weniger starke
Mitgliederbewegungen
als die Erhebung eines
Zusatzbeitrages nach
sich zieht. Dies kam den vorwiegend kleinen Kran-
kenkassen mit Prämie zugute, die einen
zu großen Mitgliederansturm verwal-
tungstechnisch womöglich nicht hätten
bewältigen können. g
Infolge der Einführung des Systems
»Zusatzbeiträge und Prämien« ist eine
Verschärfung des Krankenkassenwett-
bewerbs dergestalt zu beobachten, dass
die Krankenkassen sich stark bemühen,
keinen Zusatzbeitrag zu erheben bzw. diesen abzuschaffen. Die Anreize, Ein-
sparungen zu realisieren und die Durch-
führung der Krankenversicherung mög-
lichst wirtschaftlich zu organisieren,
sind deshalb sehr stark. Allerdings gibt
es ebenso starke Anreize, die Entschei-
dung über die Erhebung eines Zusatzbei-
trags möglichst lange hinauszuzögern,
auch wenn dadurch eine prekäre finan-
zielle Situation entsteht, wenngleich die-
se Gefahr aktuell durch die generell gute
Finanzausstattung der Kassen durch den
Gesundheitsfonds nicht so brisant ist.n Comparing Chronic Disease Patterns
between Germany and the US
International Comparisons of Treatment Patterns,
Disease Interactions and Healthcare Use and Costs
Herausgegeben von Prof. Dr. Norbert Klusen, Dr. Frank Verheyen
2012, 179 S., brosch., 24,– €
ISBN 978-3-8329-7477-0
(Beiträge zum Gesundheitsmanagement, Bd. 35) Nomos https://doi.org/10.5771/1611-5821-2012-5-52
Generiert durch IP '195.242.1.103', am 24.10.2024, 05:55:08. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig. G+S
5/2012 G+S
5/2012 56
|
https://openalex.org/W2159055716
|
https://europepmc.org/articles/pmc3125199?pdf=render
|
English
| null |
A novel method to prepare L-Arabinose from xylose mother liquor by yeast-mediated biopurification
|
Microbial cell factories
| 2,011
|
cc-by
| 9,213
|
* Correspondence: mzxyjiang@163.com; zxdeng@sjtu.edu.cn
1Laboratory of Microbial Metabolism and School of Life Sciences and
Biotechnology, Shanghai Jiao Tong University, 800# Dongchuan Road,
Shanghai, China
2School of Chemistry and Ecology Engineering, Guangxi University for
Nationalities, Nanning, China
Full list of author information is available at the end of the article © 2011 Cheng 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. Cheng et al. Microbial Cell Factories 2011, 10:43
http://www microbialcellfactories com/content/10/1/ Cheng et al. Microbial Cell Factories 2011, 10:43
http://www microbialcellfactories com/content/10/1/ Cheng et al. Microbial Cell Factories 2011, 10:43 Cheng et al. Microbial Cell Factories 2011, 10:43
http://www.microbialcellfactories.com/content/10/1/43 A novel method to prepare L-Arabinose from
xylose mother liquor by yeast-mediated
biopurification airong Cheng1, Hengwei Wang1, Jiyang Lv1, Mingguo Jiang2*, Shuangjun Lin1 and Zixin Deng1 Abstract Background: L-arabinose is an important intermediate for anti-virus drug synthesis and has also been used in food
additives for diets-controlling in recent years. Commercial production of L-arabinose is a complex progress
consisting of acid hydrolysis of gum arabic, followed by multiple procedures of purification, thus making high
production cost. Therefore, there is a biotechnological and commercial interest in the development of new cost-
effective and high-performance methods for obtaining high purity grade L-arabinose. Results: An alternative, economical method for purifying L-arabinose from xylose mother liquor was developed in
this study. After screening 306 yeast strains, a strain of Pichia anomala Y161 was selected as it could effectively
metabolize other sugars but not L-arabinose. Fermentation in a medium containing xylose mother liquor permitted
enrichment of L-arabinose by a significant depletion of other sugars. Biochemical analysis of this yeast strain
confirmed that its poor capacity for utilizing L-arabinose was due to low activities of the enzymes required for the
metabolism of this sugar. Response surface methodology was employed for optimization the fermentation
conditions in shake flask cultures. The optimum conditions were: 75 h fermentation time, at 32.5°C, in a medium
containing 21% (v/v) xylose mother liquor. Under these conditions, the highest purity of L-arabinose reached was
86.1% of total sugar, facilitating recovery of white crystalline L-arabinose from the fermentation medium by simple
methods. Conclusion: Yeast-mediated biopurification provides a dynamic method to prepare high purity of L-arabinose from
the feedstock solution xylose mother liqour, with cost-effective and high-performance properties. inhibits the sucrase activity of intestinal mucosa [3-5]. It
also suppresses increase of blood glucose after sucrose
loading in a dose-dependent manner, but shows no
effect after starch loading in mice [5]. These observa-
tions have suggested the possibility of application of
L-arabinose, mixed with small quantities of sucrose, in
controlled diets such as those for weight-loss or for dia-
betics [6]. Media and screening of yeast strains To obtain a yeast strain that could utilize xylose, xylitol,
galactose but not L-arabinose from xylose mother
liquor, 306 yeast strains from our laboratory stock were
screened by inoculating each culture in solid 1% (w/v)
yeast nitrogen base medium (YNB, Difco, Detroit, MI,
USA) containing 2% (w/v) agar, supplemented with
either 1% (w/v) L-arabinose (YNA), 1% (w/v) xylose
(YNX), 1% (w/v) xylitol (YNXL) or 1% (w/v) galactose
(YNGL) and cultured at 28°C for 5 days. Cultures which
could grow on other media but not on YNA were
inoculated into solid YNB medium supplemented with
30% (v/v) xylose mother liquor (YNXM) and cultured at
28°C for 7 days. The culture that showed the fastest
growth rate based on the colony size was selected. This
strain was adapted to a medium containing 1% (w/v)
yeast extract, 1% (w/v) tryptone, 30% (v/v) xylose
mother liquor, 2% (w/v) agar (YPX) by repeated cultur-
ing for 30 cycles to further improve its adaptability in
xylose mother liquor. Xylose mother liquor is an acid hydrolysate by-pro-
duct in the preparation of xylose from corncob or sugar-
cane bagasse and is rather abundant in China. It
contains more than 350-400 g L-1 xylose and 150-180 g
L-1 L-arabinose, as well as galactose and glucose. L-ara-
binose is difficult to separate and crystallize by general
procedures due to the chemical complexity. Simulated
moving bed chromatography has been practiced to sepa-
rate L-arabinose from the mixture [9], but it demands
expensive equipment and high running costs and has
poor separation efficacy, limiting its large-scale use in
industry. Therefore, development of a simpler method
to obtain L-arabinose from xylose mother liquor is
crucial. For taxonomic identification of this yeast strain, geno-
mic DNA was extracted according to the simple phenol
lysis method [13] to amplify partial sequences of its 18S
rDNA and cytochrome c oxidase subunit 2 genes. The
primers used for amplification of 18 S rDNA were: 5’-
ATC CTG CCA GTA GTC ATA TGC TTG TCT C-3’
and 5’-GAG GCC TCA CTA AGC CAT TCA ATC
GGT A-3’; while those for the cytochrome c oxidase
subunit 2 gene were: 5’-AATATAATGTTTTATTTAG-
TATTAATA-3’ and 5’-TTTGATAGGCATCGCAC-
TATGAGC-3’. PCR conditions were as follows: 95°C for
3 min, 30 cycles of denaturation at 94°C for 35 s,
annealing at 61°C for 40 s, extension at 72°C for 90 s,
and a final extension at 72°C for 10 min. Media and screening of yeast strains Each PCR pro-
duct was independently ligated into T-vector (pMD18,
Takara, Dalian, China) and sequenced. Homology search
was performed using the Basic local alignment tool
(BLAST) available from National Center for Biotechnol-
ogy Information (NCBI, Bethesda, MD, USA) [14]. In recent times, biological removal and bio-purifica-
tion have become increasingly attractive approaches for
producing high-value compounds from crude sugar
feedstocks [10,11]. Most recently, Cheng et al designed
a strategy to produce xylitol from xylose mother liquor
through biological removal of growth inhibitors, glucose
and L-arabinose, and enrich xylose which was then
reduced to xylitol by a yeast [12]. Thus, a possible
approach for the efficient separation of L-arabinose
from xylose mother liquor would be to biologically
deplete other sugars such as xylose, glucose and galac-
tose from the xylose mother liquor, thus enriching L-
arabinose in this liquor and facilitating its crystallization
by simple methods. In the present study, we identified Pichia anomala
Y161 (China General Microbiological Culture Collection
Center, accession no. 2480) that could grow well in a
medium containing xylose, xylitol and galactose, but not
L-arabinose, as a carbon source, and could also grow
well in a medium containing 10-30% (v/v) xylose mother
liquor. Therefore, this strain was used to biologically
enrich L-arabinose from xylose mother liquor. Response Background The sugar, L-arabinose is named after gum arabic from
which it was first isolated. It is a five-carbon sugar and
is widely found in nature as a component of biopoly-
mers such as hemicellulose and pectin. L-arabinose is
traditionally used in the flavour industry in Maillard
reaction and in culture media. Recently, L-arabinose has
been used in food additives and as an intermediate in
drug synthesis [1,2]. The effects of L-arabinose on
intestinal absorption of sucrose have been investigated. Physiological experiments have revealed that L-arabinose Commercial production of L-arabinose consists of an
initial step of acid hydrolysis of gum arabic, followed by
its purification through multiple procedures such as neu-
tralization reaction, ion exchange and other chromato-
graphic separations. Recently, Lim et al. (2011) reported
a new preparation method for L-arabinose from arabinan
by the combination of endo-and exo-arabinanases, which
yielded 16 g L-1 L-arabinose from 20 g l-1 arabinan (80%
yield) [7]. Earlier, Ahmed et al. (1999) reported a novel Page 2 of 11 Cheng et al. Microbial Cell Factories 2011, 10:43
http://www.microbialcellfactories.com/content/10/1/43 Cheng et al. Microbial Cell Factories 2011, 10:43
http://www.microbialcellfactories.com/content/10/1/43 L-arabinose production method using Mycobacterium
smegmatis to transform ribitol, first by oxidization to L-
ribulose that is then isomerized into L-arabinose [8]. However, these methods need expensive raw materials
and complex separation processes for purification of L-
arabinose, which render these methods impractical on a
large-scale. surface methodology and Box-Wilson central composite
design (CCD) were used to optimize the fermentation
conditions by P. anomala Y161. Optimal fermentation
conditions were deduced and were applied in 3 L fer-
mentors to enrich L-arabinose by depleting xylose and
glucose and some of the galactose present. Crystallized
L-arabinose was obtained from this enriched fermenta-
tion medium by simple purification procedures. In 2010, China produced about 500 tons of L-arabi-
nose at the cost of more than 70 USD per kg. This pro-
hibitive cost for obtaining high grade L-arabinose limits
its use in applications such as food additives and drug
synthesis. Therefore, there is a biotechnological and
commercial interest in the development of new cost-
effective and high-performance methods for obtaining
high purity grade L-arabinose, which has biotechnologi-
cal applications as a valuable starting material for manu-
facture
of
various
life-saving
drugs
and
other
commercially important high-value products. Yeast seed culture Yeast pre-culture was prepared by transferring a loop of
culture obtained as above to test tubes containing 10 mL
seed culture medium (15% xylose mother liquor (v/v), 1% Page 3 of 11 Cheng et al. Microbial Cell Factories 2011, 10:43
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http://www.microbialcellfactories.com/content/10/1/43 Page 3 of 11 Cheng et al. Microbial Cell Factories 2011, 10:43
http://www.microbialcellfactories.com/content/10/1/43 Experimental design and statistical analysis tryptone (w/v), 1% yeast extract (w/v)). The pre-culture
was grown at 32°C on a rotary shaker at 200 rpm for
24 h and used to inoculate Erlenmeyer flasks (500 mL
cap.) containing 90 mL of fermentation media with vary-
ing concentrations of xylose mother liquor (containing
153 g L-1 L-arabinose, purchased from Jiahe Sugar Co. Ltd., Changyi City, Shandong Province, China). The
cultural conditions were modified according to the
experimental design. The speed of rotary shaker incuba-
tor was set to 250 rpm. All tests were conducted in dupli-
cate and results presented are the mean values of two
independent experiments. p
g
y
Based on the results of our preliminary experiments, the
selected yeast strain was grown under various cultural
conditions to obtain optimized purity of L-arabinose. In
these trials, three parameters were kept constant: med-
ium contained 2.5% (w/v) yeast extract, pH was 5.5 and
aeration was by shaking at 250 rpm. Yeast extract has
been shown to be a good nitrogen source, increasing
aldose (xylose) reductase activity, thus enhancing the
metabolism rate of xylose [15]. An aeration rate of 250
rpm improved significantly the assimilation and metabo-
lism rate of xylitol presumably by providing more cofac-
tor NAD, thus shortening the fermentation time to
enrich L-arabinose. Three parameters, namely, fermenta-
tion time (X1), temperature (X2) and concentration of
xylose mother liquor in the medium (X3), were varied
to obtain the optimized purity of L-arabinose according
to the response surface methodology (RSM) (Design-
Expert 7). A central composite design (CCD) was
employed for fast multifactor screening to determine the
most important independent factor [16]. A CCD at five
levels was conducted with three independent variables
of X1, X2 and X3 (Table 1). The complete design con-
sisted of 20 experimental points (8 factorial points, 6
axial points and 6 center points) as shown in Table 2,
and the experiments were carried out in a random
order. Enzymatic assays Y161 culture was grown aerobically in 250 ml shaken
flasks with 50 ml of medium containing 0.67% (w/v) YNB
supplemented with 1% xylose (w/v), 1% xylitol (w/v), or
1% galactose (w/v), and incubated at 28°C on a rotary
shaker at 250 rpm. 2 ml of cell suspension from each
late-exponential phase culture (OD600 6-7) were har-
vested by centrifugation at 5000 g for 5 min, washed
three times with extraction buffer (50 mM Tris-Cl
[pH7.5], 0.1 mM EDTA and 2 mM dithiothreitol) and
centrifuged at 5000 g for 5 min. The pelleted cells (about
35 mg dry weight cells per tube) were re-suspended in
400 μl of the above extraction buffer and were sonicated
using an ultrasonic homogenizer in an ice water bath for
fifty, 5 s bursts, interrupted by 5 s cooling intervals at
600 Watt. Crude cell-free extracts were obtained by reco-
vering the supernatant after spinning down cell debris. This preparation was used to determine enzyme activ-
ities with L-arabinose, L-arabitol and L-xylulose as
substrates and identify the enzymatic products by
HPLC. For L-arabinose reductase (LAR, EC 1.1.1.21)
and L-xylulose reductase (LXR, EC 1.1.1.10), activities
were determined in Tris-HCl buffer (100 mM, pH 7.0),
NADPH or NADH (0.2 mM) containing 1% (w/v, 66
mM) L-arabinose or 1% (v/v) L-xylulose. NAD- or
NADP-dehydrogenase (EC 1.1.1.12) activities were
assayed in glycine buffer (100 mM, pH 8.5), MgCl2
(50 mM), 1% (w/v, 66 mM) L-arabitol or 1% (w/v,
66 mM) xylitol containing either NAD or NADP
(3.0 mM) respectively. All assays were carried out at 30°C
for 5 min. One unit of enzyme activity was defined as the
amount of activity necessary to convert 1 μmol of sub-
strate to product (NADPH to NADP; or NADH to NAD;
or NAD to NADH; or NADP to NADPH) per min. The
protein concentration in crude extract was determined
according to the Bradford method with bovine serum
albumen (BSA) as a standard. The enzymatic reaction
mixes (1 hour reaction) were extracted with phenol to
remove proteins and 40 μL aliquots were applied to HPLC
to identify the products Isolation and purification of L-arabinose
h
d
l
d
d Isolation and purification of L arabinose
The yeast seed culture prepared as described above was
inoculated at 5% inoculum size into four 3 L capacity fer-
mentors (Bioflo 110, New Brunswick Scientific, Edison,
NJ, USA) each containing 2 L of the optimum fermenta-
tion medium with 2.5% (w/v) yeast extract as nitrogen
source at pH 5.5 and cultured under the optimum fer-
mentation conditions obtained from response surface
methodology. After fermentation, yeast cells were
removed by centrifugation, and the supernatant was
rotary decolorized with activated carbon at 80°C. Then
the colorless fermentation broth thus obtained was clari-
fied by removal of activated carbon via filtration, and was
applied to ion-exchange resin columns for removal of
metal ions, which was verified by a conductivity less than Table 1 Values of parameters used in the response
surface design
Factors
Factor level
-2
-1
0
1
2
Fermentation time (h)
50
60
70
80
90
Fermentation temperature (°C)
30
31
32
33
34
Xylose mother liquor (%, v/v)
15
20
25
30
35 Table 1 Values of parameters used in the response
surface design
Factors
Factor level
-2
-1
0
1
2
Fermentation time (h)
50
60
70
80
90
Fermentation temperature (°C)
30
31
32
33
34
Xylose mother liquor (%, v/v)
15
20
25
30
35 Table 1 Values of parameters used in the response
surface design Cheng et al. Microbial Cell Factories 2011, 10:43
http://www.microbialcellfactories.com/content/10/1/43 Cheng et al. Microbial Cell Factories 2011, 10:43
http://www.microbialcellfactories.com/content/10/1/43 Page 4 of 11 Table 2 Results of experimental trials (20 trials)
conducted as per the central composite design (CCD) for
optimization of yield in terms of purity of L-arabinose
obtained from xylose mother liquor
Run
No. Time
(h)
Temp. (°C)
Xylose mother
liquid (%, v/v)
Purity degree of
L-arabinose (%)
1
70
30
25
65.6
2
60
33
30
49.2
3
70
34
25
68.8
4
70
32
25
81.4
5
80
31
20
77.5
6
70
32
25
82.9
7
70
32
35
46.2
8
80
33
20
84.9
9
60
31
20
55.2
10
60
33
20
75.1
11
80
33
30
67.1
12
70
32
25
81.8
13
70
32
25
82.5
14
60
31
30
40.1
15
70
32
25
81.4
16
70
32
15
85.3
17
90
32
25
78.1
18
70
32
25
81.1
19
80
31
30
58.8
20
50
32
25
69.9 Materials and Methods. Isolation and purification of L-arabinose
h
d
l
d
d Based on the relative colony
sizes, one strain was found to grow faster than the other
three and was selected for further experiments. After 30
successive cycles of adaptation to YPX medium contain-
ing 30% (v/v) xylose mother liquor, the culture of this
strain was found to grow faster (colony size 1.0-1.2 mm)
than an unadapted culture (colony size 0.2-0.4 mm) on
YPX medium after 5 days at 28°C. This yeast strain
which utilizes well xylose, xylitol, galactose but not L-
arabinose, grew fastest on YNXM and was adapted to
YPX medium was designated as Y161. The growth char-
acteristics of yeast Y161 on YNA, YNX, YNXL, YNGL
and YPX solid medium are shown in Figure 1. The xylose mother liquor contains more than 350-400
g L-1 xylose and 150-200 g L-1 galactose plus glucose in
addition to 150-180 g L-1 L-arabinose. The approach fol-
lowed here for enriching L-arabinose was based on elim-
ination of other sugars from the xylose mother liquor. Hence the screen applied was aimed at selecting a yeast
strain with excellent properties for transport and rapid
metabolism of glucose, galactose and xylose, and capa-
city for adaptation to high concentration of xylose mother
liquor. The yeast strain Y161 met these requirements and
was adapted to a medium containing 30% (v/v) xylose
mother liquor. q
The taxonomic identity of Y161 was established based
on cloning and sequencing of its 18S rDNA and cyto-
chrome c oxidase subunit 2 genes (see Methods). The
two nucleotide sequences were analyzed for homology
by the BLAST search available from NCBI. The results
of this analysis showed that the 1.7 kb 18S rDNA fragment
from yeast Y161 (GenBank accession No. HQ901201) had
99% identity with that from Pichia anomala strain M34-1,
strain HS054 and strain IFO 10213T [17], as well as with
that from Pichia subpelliculosa NRRL Y-1683, Pichia sydo-
wiorum NRRL Y-7130, Pichia ciferrii NRRL Y-1031, Can-
dida silvicultrix NRRL Y-7789 [18]. Similarly, the 580 bp
fragment of cytochrome c oxidase subunit 2 gene from
yeast Y161 (GenBank accession No. HQ901202) showed
100% identity with a homologous sequence from Pichia
anomala isolate WM 825 [19] and 99% identity to that
from Wickerhamomyces anomalus NRRL Y-366 [20]. Both
the sequenced DNA fragments showed less than 90%
identity with corresponding sequences from other yeast
strains. Thus, the yeast Y161 was identified as Pichia
anomala. 20 μs/cm. Isolation and purification of L-arabinose
h
d
l
d
d The solution was adjusted to contain 70% (w/v)
of L-arabinose by concentrating, and cooled gradually
from 70°C to 4°C to allow L-arabinose crystallized. White
powdery crystals precipitated from solution, then were
centrifuged and washed with 95% ethanol, and dried. The
purity of L-arabinose was estimated by HPLC. Analytical methods L-arabinose, xylose, glucose, galactose and xylitol were
estimated by resolving samples on prepacked analytical
HPLC columns (Shodex SPO 810, 8 × 30 mm, Pb2+
cation exchange column), with distilled water as eluent,
at a flow rate of 1.0 mL/min, at 70°C. Sugars were iden-
tified using a refractive index detector (Shodex RI 101). Screening, selection and identification of yeast Pichia
anomala Y161 Among 306 strains of yeasts that were screened on
YNB-based media supplemented with different sugars,
all strains could utilize galactose as they were capable of
growth on solid YNGL medium. Of these, four strains
grew well on solid YNX, YNXL medium but grew
poorly on YNA. Cultures of these four yeast strains
were diluted appropriately with sterilized water and
spread onto solid YNXM medium as described in Growth pattern of Y161 in YPX liquid medium After 48 h, xylitol was exhausted as it
was almost undetectable, while trace amounts of L-arabi-
tol remained in the medium (less than 1.5 g L-1). At
this point (48 h), the concentration of L-arabinose was
13.5 g L-1. Comparing to the starting fermentation med-
ium which contains 15.3 g L-1 L-arabinose, 88.2% yield of
L-arabinose was achieved in the final fermentation
medium. Only 1.8 g L-1 L-arabinose was reduced to
L-arabitol, indicating that yeast Y161 could not utilize
well L-arabinose and showed a weak ability to reduce
L-arabinose to L-arabitol (Figure 2F). After 48 h of fer-
mentation, the degree of purity of L-arabinose increased
more than 3-fold, from the initial 26.2% to 85.6% in
terms of total sugars. YNA
YNX
YNGL
YNXL
YPX
YPX
Figure 1 Growth of yeast on solid medium containing different
sugars. Screening of 306 yeast strains was performed by observing
their growth on YNB medium supplemented with either L-arabinose
(YNA), xylose (YNX), xylitol (YNXL) or xylose mother liquor (YPX). Black arrows indicate the sector inoculated with the selected strain
that was capable of growth on all the above medium except YNA. After repeated culturing of this strain in YPX medium for 30 cycles,
it appeared to have adapted as seen from the comparison of its
growth (bottom left panel) with an unadapted culture (bottom right
panel). The adapted strain was then designated as strain Y161. On
YNA medium, Y161 showed poor growth in the first 3 days, and no
further increase in colony size or number was observed in the next
4 days. YPX YPX Xylose is a major constituent of plant material such as
corn cob and sugarcane bagasse. Xylose metabolic path-
way is therefore well developed in microorganisms that
live on decaying plant materials, including most strains
of yeasts such as Pichia stiptis [21], Candida tropicalis
[22], Candida shehatae [23] and H. anomala used in
this study. The metabolism of xylose in yeasts has been
described in detail in the literature [24-27]. Xylose
transport across the plasma membrane is the first step
in xylose metabolism pathway in yeasts. It is known that
xylose is taken up by active xylose/proton symporter
that mostly appeared to be associated with glucose
transport and showed a much higher affinity for glucose
than for xylose [28]. The uptake of xylose is highly
influenced by the presence of glucose. Growth pattern of Y161 in YPX liquid medium Growth pattern of Y161 in YPX liquid medium p
q
Y161 cells were grown in 500 ml capacity flasks contain-
ing 50 ml YPX liquid medium supplemented with 10%
(v/v) xylose mother liquor and incubated at 32°C on an
orbital shaker at 250 rpm. Samples were withdrawn per-
iodically to estimate the content change of glucose,
xylose, L-arabinose, galactose, L-arabitol and xylitol Page 5 of 11 Cheng et al. Microbial Cell Factories 2011, 10:43 Cheng et al. Microbial Cell Factories 2011, 10:43
http://www.microbialcellfactories.com/content/10/1/43 http://www.microbialcellfactories.com/content/10/1/43 the external medium (Figure 2C). After 30 h of fermenta-
tion, the amount of xylose and galactose decreased to
3.2 g L-1 and 2.5 g L-1, and the amount of xylitol
increased to 12.3 g L-1. Presumably, xylose was metabo-
lized and transformed to biomass and energy, while
L-arabitol was still unutilized (Figure 2D). After 36 h,
xylose from the medium had reached undetectable levels
and the amount of xylitol started to decrease. Trace
amounts of galactose and L-arabitol were found in med-
ium (Figure 2E). After 48 h, xylitol was exhausted as it
was almost undetectable, while trace amounts of L-arabi-
tol remained in the medium (less than 1.5 g L-1). At
this point (48 h), the concentration of L-arabinose was
13.5 g L-1. Comparing to the starting fermentation med-
ium which contains 15.3 g L-1 L-arabinose, 88.2% yield of
L-arabinose was achieved in the final fermentation
medium. Only 1.8 g L-1 L-arabinose was reduced to
L-arabitol, indicating that yeast Y161 could not utilize
well L-arabinose and showed a weak ability to reduce
L-arabinose to L-arabitol (Figure 2F). After 48 h of fer-
mentation, the degree of purity of L-arabinose increased
more than 3-fold, from the initial 26.2% to 85.6% in
terms of total sugars. the external medium (Figure 2C). After 30 h of fermenta-
tion, the amount of xylose and galactose decreased to
3.2 g L-1 and 2.5 g L-1, and the amount of xylitol
increased to 12.3 g L-1. Presumably, xylose was metabo-
lized and transformed to biomass and energy, while
L-arabitol was still unutilized (Figure 2D). After 36 h,
xylose from the medium had reached undetectable levels
and the amount of xylitol started to decrease. Trace
amounts of galactose and L-arabitol were found in med-
ium (Figure 2E). Growth pattern of Y161 in YPX liquid medium During the fer-
mentation of xylose mother liquor by the yeast Y161 in
this study, xylose uptake was not observed until glucose
was exhausted and xylitol was detected in medium only
after 8 h (Figure 2B), indicating that glucose has a strong
inhibitory effect on xylose uptake. Moreover, glucose was
shown to be an inhibitor of the intracellular enzymes
responsible for the catabolism of xylose, for example
reduction to xylitol [29]. Once glucose has been con-
sumed, enzymes for xylose metabolism are activated and
xylitol accumulates intracellularly and is then secreted to
the medium (Figure 2C-E). Extracellular xylitol concentra-
tion reached a maximum value just before nearly all of the
xylose was consumed (Figure 2D). The xylitol appears to
have been utilized for generating biomass and energy dur-
ing the later phases of fermentation, as seen from the sub-
sequent decline in concentration of extracellular xylitol YPX YPX YPX Figure 1 Growth of yeast on solid medium containing different
sugars. Screening of 306 yeast strains was performed by observing
their growth on YNB medium supplemented with either L-arabinose
(YNA), xylose (YNX), xylitol (YNXL) or xylose mother liquor (YPX). Black arrows indicate the sector inoculated with the selected strain
that was capable of growth on all the above medium except YNA. After repeated culturing of this strain in YPX medium for 30 cycles,
it appeared to have adapted as seen from the comparison of its
growth (bottom left panel) with an unadapted culture (bottom right
panel). The adapted strain was then designated as strain Y161. On
YNA medium, Y161 showed poor growth in the first 3 days, and no
further increase in colony size or number was observed in the next
4 days. during cultivation (Figure 2). The xylose mother liquor
used in this study contained glucose, xylose, galactose,
L-arabinose and an unknown substance (Figure 2A). The
amount of xylose was approximately 2.5 fold higher than
that of L-arabinose. Glucose was the first sugar to be
metabolized by Y161, while trace amounts of L-arabitol
were produced after 8 h indicating a low level reduction
of arabinose (Figure 2B). After 20 h fermentation, the
amount of xylose decreased significantly and was almost
equal to that of L-arabinose. The xylose appeared to be
first reduced to xylitol, of which 50% was secreted into Cheng et al. Microbial Cell Factories 2011, 10:43
http://www microbialcellfactories com/content/10/1/43 Cheng et al. Growth pattern of Y161 in YPX liquid medium However, Y161 grew poorly, if at all, on YNA
agar/liquid medium, indicating that despite a detectable
level of transport of this sugar, further metabolism may
not be efficient due to low activities of enzymes involved
in L-arabinose metabolism, such as L-arabinose specific
reductase (LAR), LAD or L-xylulose reductase (LXR). and NAD-dependent activity, with a higher preference
for NAD. An XDH activity of ~0.86 U mg-1 was found
in galactose-grown cells, which increased to 5.0 and
5.2 U mg-1 when the cells were grown on xylose or xyli-
tol, respectively. This suggests that XDH activity is more
effectively induced by xylose or xylitol compared with
the other three enzymes tested, which might explain
why yeast Y161 utilizes xylose or xylitol efficiently
(Figure 1). In contrast, the very low LAR, LAD and LXR
activities inY161 might explain its poor ability to utilize
L-arabinose (Figure 1). Through HPLC analysis, we also identified the enzy-
matic reaction products with L-arabinose, L-arabitol and
L-xylulose as substrates and NADPH or NAD as cofactors. These reactions were performed with crude cell free
extracts of Y161 grown on medium containing galactose,
xylose or xylitol as a carbon source. L-arabinose was
reduced to L-arabitol by the above crude extracts of Y161
in the presence of NADPH as a cofactor (Figure 3 A2-A4). L-xylulose was formed with L-arabitol as substrate
and NAD as cofactor, using the crude extracts of Y161
(Figure 3 B2-B4) while L-xylulose was the reduced to xyli-
tol (Figure 3 C2-C4). It should be noted that the amount
of L-xylulose oxidized from L-arabitol was significantly
less than the amount of L-arabitol reduced from L-arabi-
nose or xylitol reduced from L-xylulose (Figure 3), indicat-
ing that the activity of dehydrogenase harboring LAD was
very low, consistent with the results shown in Table 3. Y161 did not grow in a medium with L-arabitol as a car-
bon source (data not shown), although the cells exhibited
low LAD activity. There are a number of instances of a
polyol oxidized by the cell-free crude extracts of a yeast,
but not utilized for growth. Candida utilis could not uti-
lize D-glucitol but it could be oxidized in crude extract,
due to exogenous D-glucitol, which does not enter intact
cells of this strain [32]. Growth pattern of Y161 in YPX liquid medium Only a small amount of L-ara-
bitol (less than 1.2 g/L) was produced when Y161 was
cultured in YPX medium containing 10% xylose mother
liquor (Figure 2B-E). Subsequently, in the later stages of
fermentation, like in the case of xylitol, the amount of
extracellular L-arabitol decreased (Figure 2F). This indi-
cates that a certain amount of L-arabinose could be
transported across the plasma membrane and was meta-
bolized. However, Y161 grew poorly, if at all, on YNA
agar/liquid medium, indicating that despite a detectable
level of transport of this sugar, further metabolism may
not be efficient due to low activities of enzymes involved
in L-arabinose metabolism, such as L-arabinose specific
reductase (LAR), LAD or L-xylulose reductase (LXR). xylose metabolism pathway. Among these, Candida ara-
binofermentans PYCC 5603T and Pichia guilliermondii
PYCC 3012 received most attentions [28,30,31]. L-arabi-
nose transport across the yeast outer membrane is the
first step in its metabolism. In yeast C. arabinofermen-
tans PYCC 5603T and P. guilliermondii PYCC 3012
which have a high L-arabinose uptake rate, there are
two L-arabinose transport systems, one is the L-arabi-
nose/proton symporter with high-affinity transport com-
ponents, and the other is a facilitated diffusion transport
system with a relative low-affinity component. Once
inside the cytoplasm, L-arabinose is reduced to L-arabi-
tol by an unspecific NADPH-linked aldose reductase
which could also reduce xylose to xylitol [10,28]. L-ara-
bitol is then oxidized to L-xylulose by an NAD-depen-
dent L-arabitol 4-dehydrogenase (LAD), and L-xylulose
was reduced to xylitol, a common intermediate to the
xylose metabolism pathway. Y161 could efficiently meta-
bolize xylose and xylitol but showed a poor ability to
metabolize L-arabinose, probably because of the low
activities of the enzymes, L-arabinose reductase or L-
arabitol 4-dehydrogenase. Only a small amount of L-ara-
bitol (less than 1.2 g/L) was produced when Y161 was
cultured in YPX medium containing 10% xylose mother
liquor (Figure 2B-E). Subsequently, in the later stages of
fermentation, like in the case of xylitol, the amount of
extracellular L-arabitol decreased (Figure 2F). This indi-
cates that a certain amount of L-arabinose could be
transported across the plasma membrane and was meta-
bolized. Growth pattern of Y161 in YPX liquid medium Microbial Cell Factories 2011, 10:43
http://www.microbialcellfactories.com/content/10/1/43 Page 6 of 11 g
http://www.microbialcellfactories.com/content/10/1/43 until it was undetectable (Figure 2E-F) Ultimately L-ara-
commonly found in hemicellulose such as L-arabinnans
Figure 2 Sugar assimilation patterns during growth of Y161 in YPX medium containing 10% (v/v) xylose mother liquor. Aliquots were
removed at different times during fermentation (A, 0 h; B, 8 h; C, 20 h; D, 30 h; E, 36 h; F, 48 h) and analyzed by HPLC to identify the residual
sugars as indicated. Figure 2 Sugar assimilation patterns during growth of Y161 in YPX medium containing 10% (v/v) xylose mother liquor. Aliquots were
removed at different times during fermentation (A, 0 h; B, 8 h; C, 20 h; D, 30 h; E, 36 h; F, 48 h) and analyzed by HPLC to identify the residual
sugars as indicated. commonly found in hemicellulose such as L-arabinnans,
L-arabino-xylans in corn fiber which is associated to
xylose. Approximately a third of L-arabino-xylans in
corn fiber is L-arabinose. Many types of yeast harbor
the L-arabinose metabolism pathway as well as the until it was undetectable (Figure 2E-F). Ultimately, L-ara-
binose was the predominant sugar left in the fermentation
medium, thus reaching a high degree of purity. Similar to xylose, L-arabinose is also a natural sugar
and a component of plant carbohydrates which is Cheng et al. Microbial Cell Factories 2011, 10:43
http://www.microbialcellfactories.com/content/10/1/43 Page 7 of 11 xylose metabolism pathway. Among these, Candida ara-
binofermentans PYCC 5603T and Pichia guilliermondii
PYCC 3012 received most attentions [28,30,31]. L-arabi-
nose transport across the yeast outer membrane is the
first step in its metabolism. In yeast C. arabinofermen-
tans PYCC 5603T and P. guilliermondii PYCC 3012
which have a high L-arabinose uptake rate, there are
two L-arabinose transport systems, one is the L-arabi-
nose/proton symporter with high-affinity transport com-
ponents, and the other is a facilitated diffusion transport
system with a relative low-affinity component. Once
inside the cytoplasm, L-arabinose is reduced to L-arabi-
tol by an unspecific NADPH-linked aldose reductase
which could also reduce xylose to xylitol [10,28]. L-ara-
bitol is then oxidized to L-xylulose by an NAD-depen-
dent L-arabitol 4-dehydrogenase (LAD), and L-xylulose
was reduced to xylitol, a common intermediate to the
xylose metabolism pathway. Y161 could efficiently meta-
bolize xylose and xylitol but showed a poor ability to
metabolize L-arabinose, probably because of the low
activities of the enzymes, L-arabinose reductase or L-
arabitol 4-dehydrogenase. Values presented are means of duplicates. Enzymatic activities and products Table 3 Specific activities of L-arabinose reductase (LAR),
L-arabitol 4-dehydrogenase (LAD), L-xylulose reductase
(LXR) and xylitol dehydrogenase (XDH) in crude cell free
extracts of Y161 grown on different carbon sources
Specific activity (U, mg of protein-1)
Enzyme Cofactors Grown on
galactose
Grown on
xylose
Grown on
xylitol
LAR
NADPH
0.032
0.16
0.12
NADH
0.002
0.014
0.011
LAD
NAD
0.003
0.012
0.018
NADP
ND
ND
ND
LXR
NADPH
0.004
0.021
0.024
NADH
ND
ND
ND
XDH
NAD
0.86
5.0
5.2
NADP
0.007
0.18
0.26
Values presented are means of duplicates. ND not detectable Table 3 Specific activities of L-arabinose reductase (LAR),
L-arabitol 4-dehydrogenase (LAD), L-xylulose reductase
(LXR) and xylitol dehydrogenase (XDH) in crude cell free
extracts of Y161 grown on different carbon sources In order to investigate the possible reasons for poor uti-
lization of L-arabinose by Y161, activities of four known
enzymes, namely, LAR, LAD LXR and XDH, involved in
L-arabinose metabolism were assayed, in crude cell-free
extracts, in the presence of appropriate cofactors (Table
3). In extracts of Y161 cultures grown on galactose as a
carbon source the activity of LAR was 0.032 U mg-1
using NADPH as cofactor, and decreased to a tenth
with NADH as cofactor, indicating a preference for
NADPH. LXR activity was strictly NADPH-dependent. Weak LAD activity of ~0.003 U mg-1 was found in
these galactose-grown cells, which increased to around
0.012 and 0.018 U mg-1 when the cells were grown on
xylose and xylitol, respectively, suggesting LAD activity
is less effectively induced by xylose or xylitol. Similarly,
LAR and LXR were also less effectively induced by
xylose or xylitol. In contrast, XDH showed both, NADP- Table 3 Specific activities of L-arabinose reductase (LAR),
L-arabitol 4-dehydrogenase (LAD), L-xylulose reductase
(LXR) and xylitol dehydrogenase (XDH) in crude cell free
extracts of Y161 grown on different carbon sources Specific activity (U, mg of protein-1) Cheng et al. Microbial Cell Factories 2011, 10:43 Cheng et al. Microbial Cell Factories 2011, 10:43
http://www.microbialcellfactories.com/content/10/1/43 Page 8 of 11 Figure 3 HPLC analysis of enzymatic reaction products. Crude cell free extracts were used for analysis of various enzyme activities. The
reactions were allowed to run overnight and the solutions were extracted with phenol to remove proteins. Y = 80.27 + 5.32 × (X1) + 3.19 × (X2) −9.73 × (X3) −1.66 × (X1) × (X2)
+0.56 × (X1) × (X3) −1.24 × (X2) × (X3) −2.75 × (X1)2 −4.45 × (X2)2 −4.82 × (X3)2 Enzymatic activities and products Aliquots (40 μl) from the various
reactions were applied to HPLC column: A1, 1% (w/v) L-arabinose standard, no enzymatic reaction; A2-A4, 1% L-arabinose plus crude extracts of
Y161 grown with galactose, xylose or xylitol as a carbon source, respectively. B1, 1% (w/v) L-arabitol standard, no enzyme; B2-B4, L-arabitol plus
crude extracts of Y161 grown with galactose, xylose or xylitol as a carbon source, respectively. C1, 1% (v/v) L-xylulose standard; C2-C4, L-xylulose
plus crude extracts of Y161 grown with galactose, xylose or xylitol as a carbon source, respectively. Figure 3 HPLC analysis of enzymatic reaction products. Crude cell free extracts were used for analysis of various enzyme activities. The
reactions were allowed to run overnight and the solutions were extracted with phenol to remove proteins. Aliquots (40 μl) from the various
reactions were applied to HPLC column: A1, 1% (w/v) L-arabinose standard, no enzymatic reaction; A2-A4, 1% L-arabinose plus crude extracts of
Y161 grown with galactose, xylose or xylitol as a carbon source, respectively. B1, 1% (w/v) L-arabitol standard, no enzyme; B2-B4, L-arabitol plus
crude extracts of Y161 grown with galactose, xylose or xylitol as a carbon source, respectively. C1, 1% (v/v) L-xylulose standard; C2-C4, L-xylulose
plus crude extracts of Y161 grown with galactose, xylose or xylitol as a carbon source, respectively. Experimental design Microbial Cell Factories 2011, 10:43
http://www.microbialcellfactories.com/content/10/1/43 http://www.microbialcellfactories.com/content/10/1/43 Figure 4 Response surface for the enrichment of L-arabinose by Pichia anomala Y161. Impact on the purity of L-arabinose (%) of the
interactions between the different fermentation parameters was estimated: A: fermentation time (X1, h) and fermentation temperature (X2, °C);
B: fermentation time (X1, h) and concentration of xylose mother liquor in the medium (X3, %, v/v); C: fermentation temperature (X2, °C) and the
concentration of xylose mother liquor (X3, %, v/v). Figure 4 Response surface for the enrichment of L-arabinose by Pichia anomala Y161. Impact on the purity of L-arabinose (%) of the
interactions between the different fermentation parameters was estimated: A: fermentation time (X1, h) and fermentation temperature (X2, °C);
B: fermentation time (X1, h) and concentration of xylose mother liquor in the medium (X3, %, v/v); C: fermentation temperature (X2, °C) and the
concentration of xylose mother liquor (X3, %, v/v). Table 4 Analysis of variance of the degree of purity of L-
arabinose calculated with a quadratic equation obtained
from experimental results
Source
Sum of
Squares
df
Mean
Square
F-Value
Probability >
F
Model
3093.963
9
343.7737
5.603423 0.0063
X1
452.6256
1
452.6256
7.377682 0.0217
X2
163.2006
1
163.2006
2.660129 0.1339
X3
1515.156
1
1515.156
24.69665 0.0006
(X1)(X2)
22.11125
1
22.11125
0.360408 0.5616
(X1)(X3)
2.53125
1
2.53125
0.041259 0.8431
(X2)(X3)
12.25125
1
12.25125
0.199692 0.6645
(X1)2
190.7719
1
190.7719
3.109534 0.1083
(X2)2
498.9091
1
498.9091
8.132091 0.0172
(X3)2
583.413
1
583.413
9.509483 0.0116
Residual
613.5065
10 61.35065
Lack of
Fit
611.0115
5
122.2023
244.8944 < 0.0001
Pure
Error
2.495
5
0.499
Total
3707.47
19
X1, X2 and X3 correspond to fermentation time, temperature and
concentration of xylose mother liquor, respectively; df, degrees of freedom Table 4 Analysis of variance of the degree of purity of L-
arabinose calculated with a quadratic equation obtained
from experimental results In Figure 4B, the fermentation temperature was
fixed at the 0 level (32°C). The purity degree of L-ara-
binose increased when the concentration of the xylose
mother liquor was reduced from 35% to 15%. The
degree of purity of L-arabinose increased with the
prolonged fermentation and reached the maximum
value at 85 h. Figure 4C shows the 3-D response sur-
face diagram and the contour diagram with varying
ratios of xylose mother liquor and fermentation tem-
perature at a fixed fermentation time (0 level, 70 h). X1, X2 and X3 correspond to fermentation time, temperature and
concentration of xylose mother liquor, respectively; df, degrees of freedom Experimental design The statistical significance of the regression model was
checked by F-test. The fit of the polynomial model (R2)
was calculated to be 0.8345, indicating that 83.45% of
the variability in the response could be explained by the
model. Generally, a regression model having an R2 value
higher than 0.8 is considered to have a high correlation
[33]. Predicted R-squared was -0.3302, implying that the
overall mean is a better predictor of response than the
current model. The response equation represents a sui-
table model for L-arabinose enrichment from xylose
mother liquor. In general, the cost-effectiveness of any fermentation pro-
cess is governed by several parameters, including the cost
of the raw material and the efficacy of its conversion into
the desired product; the cost of energy consumption, influ-
enced by the time required to operate the fermentor, and
the optimum temperature to provide the maximum yield. Here, we modeled optimization of the fermentation pro-
cess based on three parameters, namely, time (X1), tem-
perature (X2) and concentration of xylose mother liquor
in the medium (X3). These three variables were tested in a
total of 20 trial runs in the current CCD as depicted in the
experimental design and the results were obtained from
experiments (Table 2). Maximum purity of L-arabinose
(85.3%) was achieved under the experimental conditions
of fermentation time 70 h, fermentation temperature 32°C
in a medium containing 15% (v/v) xylose mother liquor. By applying multiple regression analysis on the experimen-
tal data, the response variable, Y, for purity of L-arabinose
and the three test variables were related with the second-
order polynomial equation (using the above codes) as
given below: 3-D response surfaces plots were employed to determine
the interaction of the fermentation conditions and the
optimum levels of the parameters that have a significant
impact on the purity of L-arabinose. In Figure 4A, when
the 3-D response plot was developed for the enrichment
of L-arabinose with varying fermentation time (X1) and
fermentation temperature (X2) at a set concentration of
xylose mother liquor (X3, 25%), the degree of purity of L-
arabinose increased with increasing fermentation time,
and was enhanced with increasing fermentation tempera-
ture from 31 to 32.5°C, but then dropped at temperatures
between 32.5°C and 34°C, indicating that the fermentation
temperature has a crucial influence on the outcome. Cheng et al. Microbial Cell Factories 2011, 10:43 Page 9 of 11 Cheng et al. Authors’ contributions CHR and JMG designed research; CHR and WHH performed the yeast strain
screening and molecular identification; LJY performed HPLC analysis and
fermentation experiments; LSJ and DZX provided advice on organizing the
manuscript and on editorial quality; CHR performed the literature review and
drafted the manuscript. All authors have read and approved the final version
of the manuscript. Purification of L-arabinose from fermentation medium The purity of L-arabinose in the fermentation medium
using Y161 could reach around 86% of total sugars,
which exceeds the purity requirement for crystalliza-
tion of L-arabinose from a solution. From 8 L fermen-
tation broth of Y161containing 1.6 L xylose mother
liquor, 165 g white powdery crystals of L-arabinose
was obtained, an yield of 103.1 g L-arabinose per L of
xylose mother liquor. Theoretically, 240 g L-arabinose
should be obtained from the 8 L fermentation medium
containing 1.6 L xylose mother liquor (153 g L-arabi-
nose/L). About 30 g L-arabinose was lost during the
process of purification and there is a residual of 45 g
L-arabinose in the fermentation medium which could
not be recovered due to the increased conductivity
and impurities which interfere with the L-arabinose
crystallization. The final preparation of the purified
white powder was dissolved in water and applied for
HPLC analysis to confirm a purity of 99% (data not
shown). Author details
1 1Laboratory of Microbial Metabolism and School of Life Sciences and
Biotechnology, Shanghai Jiao Tong University, 800# Dongchuan Road,
Shanghai, China. 2School of Chemistry and Ecology Engineering, Guangxi
University for Nationalities, Nanning, China. Acknowledgements
Thi
k This work was supported by grants 973 and 863 from the Ministry of
Sciences and Technology, Shanghai Leading Academic Discipline Project
B203, and Guangxi Science Foundation Grant No.0832011. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Recently, yeast strains, especially S.cerevisiae, have
received intensive studies to act as cell factories in
green chemistry. Many natural products used in phar-
maceutical and nutraceutical industries such as isopre-
noids, flavonoids, and other fine chemicals could be
efficiently synthesized using S.cerevisiae as the green
synthesis factory [34,35]. On the other hand, yeast cells
could also be used as green decomposition factory to
biodegrade or metabolize unwanted compounds to
enrich the desired product [36,37]. Here, the yeast
strain P. anomala Y161 was employed as a green
decomposition factory to metabolize other low-valued
sugars and enrich high-valued L-arabinose from com-
positionally complex xylose mother liquor. To our
knowledge, this is the first attempt to apply this bio-
technological method for the preparation of L-arabi-
nose from xylose mother liquor. Latter is a viscous and
compositionally complex, low-value by-product of the
xylose production industry. Further studies on the fea-
sibility of this novel strategy for the scaled-up prepara-
tion of L-arabinose from xylose mother liquor are
currently underway. Received: 30 March 2011 Accepted: 7 June 2011 Published: 7 June 2011 Received: 30 March 2011 Accepted: 7 June 2011 Published: 7 June 20 Experimental design The concentration of xylose mother liquor had a
negative effect on the degree of purity of L-arabinose. The latter increased with decreasing concentration of
xylose mother liquor. Among the three fermentation parameters studied,
concentration of xylose mother liquor in the medium
had the highest impact on the degree of purity of L-ara-
binose, followed by fermentation time and fermentation
temperature, based on the regression coefficients in the
quadratic polynomial model (Table 4). To verify the predicted results of the model for opti-
mal purity of L-arabinose, experiments were performed Cheng et al. Microbial Cell Factories 2011, 10:43
http://www.microbialcellfactories.com/content/10/1/43 Cheng et al. Microbial Cell Factories 2011, 10:43
http://www.microbialcellfactories.com/content/10/1/43 Page 10 of 11 Cheng et al. Microbial Cell Factories 2011, 10:43
http://www.microbialcellfactories.com/content/10/1/43 Cheng et al. Microbial Cell Factories 2011, 10:43
http://www.microbialcellfactories.com/content/10/1/43 8.
Ahmed Z, Shimonishi T, Bhuiyan SH, Utamura M, Takada G, Izumori K:
Biochemical preparation of L-ribose and L-arabinose from ribitol: A new
approach. J Biosci Bioen 1999, 88:444-448. Conclusion in duplicate under the optimized fermentation condi-
tions: medium volume 100 mL, inoculation volume 5%
(v/v), fermentation time 75 h, fermentation tempera-
ture 32.5°C, 21% (v/v) xylose mother liquor, 2.5% yeast
extract and pH 5.5 in 500 mL Erlenmeyer flask. The
degree of purity of L-arabinose obtained from xylose
mother liquor reached 86.1% by HPLC analysis (data
not shown). The predicted value 87.05% is in good
agreement with experimental values, proving the accu-
racy of the model and the existence of optimum
points. in duplicate under the optimized fermentation condi-
tions: medium volume 100 mL, inoculation volume 5%
(v/v), fermentation time 75 h, fermentation tempera-
ture 32.5°C, 21% (v/v) xylose mother liquor, 2.5% yeast
extract and pH 5.5 in 500 mL Erlenmeyer flask. The
degree of purity of L-arabinose obtained from xylose
mother liquor reached 86.1% by HPLC analysis (data
not shown). The predicted value 87.05% is in good
agreement with experimental values, proving the accu-
racy of the model and the existence of optimum
points. In this study, we described a novel biological method to
obtain high purity L-arabinose from xylose mother
liquor. A strain of P. anomala Y161 was recovered in a
screen designed to identify a yeast strain with a poor
capacity of utilizing L-arabinose but a good capacity for
utilizing other sugars that are present in xylose mother
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and take full advantage of: Submit your next manuscript to BioMed Central
and take full advantage of: 30. Fonseca C, Spencer-Martins I, Hahn-Hägerdal B: L-arabinose metabolism in
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Santos H, Spencer-Martins I: Use of in vivo 13C nuclear magnetic
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Spermidine Suppresses Age-Associated Memory Impairment by Preventing Adverse Increase of Presynaptic Active Zone Size and Release
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OPEN ACCESS * stephan.sigrist@fu-berlin.de Citation: Gupta VK, Pech U, Bhukel A, Fulterer A,
Ender A, Mauermann SF, et al. (2016) Spermidine
Suppresses Age-Associated Memory Impairment
by Preventing Adverse Increase of Presynaptic
Active Zone Size and Release. PLoS Biol 14(9):
e1002563. doi:10.1371/journal.pbio.1002563 Abstract Memories are assumed to be formed by sets of synapses changing their structural or func-
tional performance. The efficacy of forming new memories declines with advancing age,
but the synaptic changes underlying age-induced memory impairment remain poorly under-
stood. Recently, we found spermidine feeding to specifically suppress age-dependent
impairments in forming olfactory memories, providing a mean to search for synaptic
changes involved in age-dependent memory impairment. Here, we show that a specific
synaptic compartment, the presynaptic active zone (AZ), increases the size of its ultrastruc-
tural elaboration and releases significantly more synaptic vesicles with advancing age. These age-induced AZ changes, however, were fully suppressed by spermidine feeding. A
genetically enforced enlargement of AZ scaffolds (four gene-copies of BRP) impaired mem-
ory formation in young animals. Thus, in the Drosophila nervous system, aging AZs seem
to steer towards the upper limit of their operational range, limiting synaptic plasticity and
contributing to impairment of memory formation. Spermidine feeding suppresses age-
dependent memory impairment by counteracting these age-dependent changes directly at
the synapse. Academic Editor: Heidi A. Tissenbaum, University
of Massachusetts Medical School, UNITED
STATES Academic Editor: Heidi A. Tissenbaum, University
of Massachusetts Medical School, UNITED
STATES Received: February 21, 2016
Accepted: August 26, 2016
Published: September 29, 2016 Copyright: © 2016 Gupta 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. SpermidineSuppresses Age-Associated
Memory Impairment by Preventing Adverse
Increase of Presynaptic Active Zone Size and
Release Varun K. Gupta1,2, Ulrike Pech3, Anuradha Bhukel1,2, Andreas Fulterer1,2, Anatoli Ender1,
Stephan F. Mauermann1,2, Till F. M. Andlauer4, Emmanuel Antwi-Adjei1,
Christine Beuschel1, Kerstin Thriene5, Marta Maglione1,2, Christine Quentin1,2,
Rene´ Bushow6, Martin Schwa¨rzel1, Thorsten Mielke6, Frank Madeo7,8, Joern Dengjel5,
Andre´ Fiala3, Stephan J. Sigrist1,2* a1111 1 Institute for Biology/Genetics, Freie Universita¨t Berlin, Berlin, Germany, 2 NeuroCure, Charite´, Berlin,
Germany, 3 Georg-August-Universita¨t Go¨ttingen, Molecular Neurobiology of Behavior, Go¨ttingen,
Germany, 4 Max Planck Institute of Psychiatry, Munich, Germany, 5 Centre for Systems Biological
Analysis, University of Freiburg, Freiburg, Germany, 6 Max Planck Institute for Molecular Genetics, Berlin,
Germany, 7 Institute for Molecular Biosciences, NAWI Graz, University of Graz, Graz, Austria,
8 BioTechMed Graz, Graz, Austria Data Availability Statement: Underlying data for
every analysis is shown in S1 Data. Funding: This work was supported by grants from
the Bundesministerium fu¨r Bildung und Forschung
(Smartage, 01GQ1420A) to SJS and CB, the
Forschungszentrum fu¨r neurodegenerative
Erkrankungen to SJS and AE, and the Deutsche
Forschungsgemeinschaft to SJS and AB (Exc 257,
TP A3 and A6 SFB 958; SFB 740 TP C09). UP and
AF were supported by the German Research
Foundation (SFB 889/B04) and the German RESEARCH ARTICLE * stephan.sigrist@fu-berlin.de Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Ministry of Research and Education via the
Bernstein Center for Computational Neuroscience
Go¨ttingen (grant number 01GQ1005A). FM is
grateful to the Austrian Science Fund FWF for
grants P23490-B12, P24381, P 27893, I1000 and
grant ‘SFB Lipotox’ and to BMWFW and the Karl-
Franzens University for grant ‘Unkonventionelle
Forschung’. The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript. functional changes during formation and retrieval of memories. Though alterations in
synaptic performance are believed to accompany aging, the causal relationship between
age-dependent memory impairment and synaptic changes remains largely unknown. Using the fly Drosophila melanogaster as a model, we found that feeding them spermidine
—a polyamine compound—suppresses age-induced decline in olfactory memory, provid-
ing us with a tool to further decipher mechanisms associated with age-dependent memory
impairment. In this study, we investigated the relationship between synaptic changes and
age-dependent memory impairment by studying the olfactory circuitry. We observedan
age-related increase in the levels of the synaptic proteins Bruchpilot and Rim-binding pro-
tein, which caused an enlargement of the presynaptic active zone—the complex of proteins
that mediate neurotransmitter release—and enhanced synaptic transmission. Interest-
ingly, feeding of spermidine was sufficient to abolish these age-associated presynaptic
changes, further emphasizing the relationship between presynaptic performance and age-
dependent memory impairment. Furthermore, flies engineered to express an excess of the
core active zone protein Bruchpilot showed a premature impairment in memory forma-
tion in young flies. Based on our data, aging plausibly steers the synapses towards the
upper limit of their operational range, limiting synaptic plasticity and contributing to
impairment of memory formation. Competing Interests: The authors have declared
that no competing interests exist. Abbreviations: AMI, age-dependent memory
impairment; ARM, anesthesia-resistant memory;
ASM, anesthesia-sensitive memory; AZ, active
zone; BRP, Bruchpilot; EM, electron microscopy;
GFP, green fluorescent protein; HDAC6, histone
deacetylase-6; ITM, intermediate-term memory;
KC, kenyon cell; MBON, mushroom body output
neuron; MCH, 4-methylcyclehexanol; PN,
projection neuron; RBP, rim-binding protein; STED,
stimulated emission depletion microscopy; STM,
short-term memory; SV, synaptic vesicle; Syb,
Synaptobrevin; SynpH, SynaptopHluorin; 2xBRP,
two-copy BRP; 3d, 3-day old flies; 3-Oct, 3-
Octanol; 4xBRP, four-copy BRP; 30d, 30-day-old
flies; 30dSpd, 30-day-old flies treated with
spermidine. PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 Author Summary Neurons communicate by sending impulses, in the form of secretion of neurotransmitters,
across small spaces called synapses. It is these synapses that undergo structural and 1 / 34 PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 Results It is known that the ability to acquire new memories declines with advancing age. Based on pre-
vious study [10], one plausible explanation for this observation might be the increase in the
threshold required for memory formation with age. In fact, when we analyzed olfactory condi-
tioning in aged flies (30-d-old flies or 30d) we found that greater number of exposures to the
unconditioned stimulus in order to attain saturated levels of memory scores, which, however,
never reached the same maximal learning scores found in young flies (3-d-old flies or 3d), indi-
cating that the dynamic range of memory formation is altered with advancing age (S1 Fig). Multiple lines of evidence suggest that presynaptic plasticity processes are responsible for form-
ing olfactory associative memory in Drosophila [11–13]. Therefore, we set out to determine the
role of age-induced changes in the organization and function of synapses in AMI, using dietary
supplementation with spermidine as a tool to identify synaptic changes that can potentially
contribute to AMI. Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Therefore, we set out to determine the role of age-induced changes in the organization and
function of synapses in AMI, using dietary supplementation with spermidine as a tool to iden-
tify synaptic changes that can potentially contribute to AMI. To accomplish this, we analyzed age-induced changes in the ultrastructural,molecular, and
functional organization of synapses within the olfactory system of flies by comparing aged flies
fed with normal food to aged flies fed with spermidine-supplemented food. We found that
aging is associated with an increase in the average size of active zone (AZ) scaffolds, structures
recently shown to scale with synaptic vesicle (SV) release. Consistent with this, optophysiologi-
cal analysis showed that more SVs are released in response to natural odor stimuli in aged flies. Interestingly, these age-associated changes were suppressed by spermidine feeding, indicating
that these changes might be causally relevant to AMI. In fact, genetic manipulation provoking
an increase of T-bar size in young animals was sufficient to induce a premature decline in
memory performance. We suggest that a cumulative increase in the size and function of pre-
synaptic AZ scaffolds might reduce the operational range of synaptic plasticity processes, and
thus, hamper the formation of new memories with age. Additionally, levels of postsynaptic
neurotransmitter receptors and postsynaptic Ca2+ signals remained largely unaffected with
age, suggesting that homeostatic adaptations might be involved in increasing the threshold for
memory formation with advancing age. PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 Introduction Age-dependent memory impairment (AMI), which is associated with both psychiatric and
neurodegenerative disorders, starts in midlife and worsens with advancing age, suggesting that
the greatest driving factor is age itself. The lack of effective treatments that prevent, halt, or
reverse the condition is contributing to a diminishing quality of life for many senior citizens. Therefore, animal models that allow one to monitor physiological changes across their lifespan
and to test for a causal character of age-induced changes might be helpful in exploring the
mechanistic basis of AMI. D. melanogaster, with its short lifespan of around 60 d and advanced
molecular genetic tools, provides an efficient experimental model to unravel mechanisms
underlying AMI. Additionally, the olfactory nervous systems of insects and mammals exhibit
many similarities, suggesting that the mechanisms for olfactory learning may be shared [1]. Moreover, aversive short-, intermediate-, and long-term olfactory memories have been found
to be subject to age-induced decline in Drosophila, with an onset at about 10 d of age and pla-
teaus at about 30 d of age [2–6]. Notably, we recently found a simple dietary supplementation
of spermidine, a polyamine that specificallyprotects from AMI in Drosophila. External stimuli are believed to be represented in the brain as spatiotemporal patterns of
neural activity within a set of neuronal connections. Changes in synaptic communication
(“plasticity”) within certain neuron populations are meant to ultimately encode behavioral
adaptations such as learning and memory. Thus, dysfunctioning of synaptic plasticity might
well be relevant to age-dependent deterioration of learning and memory [7,8]. One of the fun-
damental problems of studying AMI, however, is the inability to differentiate causative changes
from adaptive or protective changes. Moreover, the brain undergoes changes at multiple levels
with advancing age, including alterations in circuits, individual neurons, and single synapses,
further complicating the situation [8]. Nonetheless, recent work has linked AMI to subtle syn-
aptic alterations in the hippocampus and other cortical brain areas, rather than to the loss of
neurons [7,9]. At the same time, the age-associated modulation of molecular entities underly-
ing learning and memory that define and change synapse function remain poorly understood. 2 / 34 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes photon microscopy. Similar to our previous observations[3], we found no significant difference
in the amplitude or time course of cytosolic GCamp3.0 signals of young (3d) and aged (30d) ani-
mals (S3 Fig). Thus, in the context of odor information processing, odor-evoked action potential
frequency or presynaptic Ca2+ influx remained rather unaffected by the age of the animal. Next, we asked whether the release of SVs was altered with advancing age and analyzed the
odor-driven SVs release. To this end, we used SynaptopHluorin (SynpH), a pH-sensitive green
fluorescent protein (GFP) fused to the luminal side of the SV membrane protein Synaptobrevin
(Syb) [16]. SynpH is nonfluorescent at the acidic pH inside SVs; however, when SVs are
released, SynpH is exposed to the neutral extracellular space, and the presynaptic terminal
becomes brightly fluorescent. Following endocytosis,SVs become reacidified,and the cycle can
start again [17]. SynpH was expressed within PNs, and the release of SVs in response to two
odors was monitored, again, at PN-to-KC synapses (Fig 1A–1H). We found a profound
increase in the amplitude of SynpH signals in aged (30d) animals when compared to young
(3d) flies (Fig 1A–1H). In contrast, spermidine administration to 30d flies prevented this age-
dependent increase of odor-induced SynpH signals (30dSpd; Fig 1A–1H). Alterations in the
endocytoticclearance of newly released SVs might, per se, explain the increase in SynpH sig-
nals observed;however, the decay constants of the poststimulus SynpH signal remained essen-
tially unchanged with aging (S4 Fig), indicating that the endocyticclearance cannot be
responsible for the difference in odor-driven SynpH signals observedin aged animals. In addi-
tion, neither the basal expression of SynpH before odor stimulation nor the maximal SynpH
signal determined by high-molar KCl treatment showed systematic differences between young
and aged cohorts (S2B and S5 Figs). These experiments, thus, indicate that the exocytosis of
SVs underlies the increase in SynpH response with advancing age. In addition to measuring the SV release at the PN presynaptic terminals within calyx, we also
measured odor-evoked changes within the axonal projections of KCs within the mushroom body
horizontal lobes by expressing SynpH using mb247-Gal4. Though relative signals were smaller
(when signal was normalized to the whole mushroom body horizontal lobe), likely reflectingthe
well-documentedsparse odor coding of KCs [18], we still observeda substantially higher ampli-
tude of SynpH signals in aged (30d) than in young (3d) flies, and, again, spermidine administra-
tion (30dSpd) protected from this age-dependent increase (S6 Fig). Thus, two major neuron
populations of the olfactory system—PNs and KCs, showed an increase in odor-evoked fluores-
cence changes in response to odor stimuli, indicating higher release of SVs in aged animals. PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 Age-Induced Increase of Odor-Driven Vesicle Release Is Suppressed
by Spermidine Feeding In order to identify synaptic mechanisms plausibly contributing to AMI, we used opto-physio-
logical assays to characterize overall neuronal responses in synaptic terminals of live intact
flies. For these experiments, we focused on projection neuron (PN) to kenyon cell (KC) synap-
ses within the mushroom body calyx of the olfactory system for two reasons: first, aversive
olfactory learning involves coincidence detection of a conditioned stimulus (odor) with an
unconditioned stimulus (electric shock), causing changes in the odor-specific synaptic activity
of second order PNs and third order mushroom body KCs [1,14]; second, the superficial posi-
tion of the calyx within the fly brain enabled us to perform efficient optical analysis [15], since
sensor signals could be retrieved from discrete synaptic bouton areas. We started by expressing cytosolic GCamp3.0 in the PNs (using GH146-Gal4) and found
the basal expression of GCamp3.0 to remain largely unchanged with age (S2A Fig). Next, we
monitored the PN boutons for intracellular Ca2+ responses to two odors typically used for
olfactory conditioning, 3-Octanol (3-Oct) and 4-methylcyclehexanol (MCH), through two- 3 / 34 Spermidine Feeding Specifically Blocks Age-Induced Increases of Core
AZ Components Since Ca2+ influx into presynaptic terminals was apparently not responsible for the profound
age-induced increase in SV release, presynaptic mechanisms downstream of Ca2+ signaling
might be involved. In order to address the molecular and cellular basis of this age-associated
increase in SV release, we started by analyzing proteins directly associated with SVs: Synapsin,
Syb, and Synaptotagmin-1. Synapsin is a SV-associated phosphoprotein important for control-
ling the number of SVs available for release [19], and Syb is a core component of SNARE com-
plex that drives the exocytosis of SVs [20,21]. We found the levels of Synapsin as well as Syb to
remain unchanged with advancing age (comparing aged flies: 30-days old or 30d with young
flies: 3d), regardless of spermidine feeding (30dSpd; Fig 2A–2D and S7 Fig). Synaptotagmin-1 is
a vesicular protein with a central role as a Ca2+ sensor for SNARE-dependent SV fusion [22]. Synaptotagmin-1 decreased slightly with age, and feeding with spermidine had no discernable
influence on this age-dependent change (Fig 2E–2H), indicating that these moderate changes
are seemingly not associated with AMI. 4 / 34 The release of neurotransmitters is a sophisticated process that requires SVs to be in close
vicinity to voltage-gated Ca2+ channels, and this precise spacing is orchestrated by interplay
Fig 1. Imaging of SynpH at PN-to-KC synapses to measure odor-evoked SV release. (a) SynpH expressed in PN boutons and imaged within the
calyx neuropil (GH146 > SynpH). Scale bar: 10 μm. (b–c) False color-coded image of the SynpH activity within the presynaptic terminals of PNs in
response to 3-Oct and MCH shown in (a). Warm colors indicate high levels, and cold colors indicate low levels or no SynpH activity. The color scale on the
right indicates changes in fluorescence (ΔF/F in %). (d) Odor-evoked release of SVs, measured by changes in fluorescence of SynpH of individual flies
over time shown as false colors in presynaptic terminals of PN in the calyx region. The left panel is in response to the odorant 3-Oct and the right panel is
in response to MCH (n = 6–7 flies). (e) Time course of SynpH activity induced by 3-Oct in the presynaptic terminals of PNs within the calyx neuropil of 3d,
30d, and 30dSpd animals (SynpH response averaged across three odor exposures from 6–7 flies). Spermidine Feeding Specifically Blocks Age-Induced Increases of Core
AZ Components (f) Maximum change in SynpH fluorescence (ΔF/F in
%) in response to 3-Oct within the presynaptic terminals of PN boutons of 3d, 30d, and 30dSpd flies (SynpH response averaged across three odor
exposures from 6–7 flies; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (g) Time course of
SynpH activity induced by MCH in the presynaptic terminals of PNs within the calyx region of 3d, 30d, and 30dSpd animals (SynpH response averaged
across three odor exposures from 6–7 flies) (h) Maximum change in SynpH fluorescence (ΔF/F in %) in response to MCH within the presynaptic terminals
of PN boutons of 3d, 30d, and 30dSpd flies (SynpH response averaged across three odor exposures from 6–7 flies; Kruskal-Wallis test with Dunn’s
multiple comparison test, p-values were subject to Bonferroni correction). * p < 0.05, ** p < 0.01, ns = not significant, p 0.05. Underlying data is shown
in S1 Data. doi:10.1371/journal.pbio.1002563.g001
Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes The release of neurotransmitters is a sophisticated process that requires SVs to be in close
vicinity to voltage-gated Ca2+ channels, and this precise spacing is orchestrated by interplay Fig 1. Imaging of SynpH at PN-to-KC synapses to measure odor-evoked SV release. (a) SynpH expressed in PN boutons and imaged within the
calyx neuropil (GH146 > SynpH). Scale bar: 10 μm. (b–c) False color-coded image of the SynpH activity within the presynaptic terminals of PNs in
response to 3-Oct and MCH shown in (a). Warm colors indicate high levels, and cold colors indicate low levels or no SynpH activity. The color scale on the
right indicates changes in fluorescence (ΔF/F in %). (d) Odor-evoked release of SVs, measured by changes in fluorescence of SynpH of individual flies
over time shown as false colors in presynaptic terminals of PN in the calyx region. The left panel is in response to the odorant 3-Oct and the right panel is
in response to MCH (n = 6–7 flies). (e) Time course of SynpH activity induced by 3-Oct in the presynaptic terminals of PNs within the calyx neuropil of 3d,
30d, and 30dSpd animals (SynpH response averaged across three odor exposures from 6–7 flies). Spermidine Feeding Specifically Blocks Age-Induced Increases of Core
AZ Components (f) Maximum change in SynpH fluorescence (ΔF/F in
%) in response to 3-Oct within the presynaptic terminals of PN boutons of 3d, 30d, and 30dSpd flies (SynpH response averaged across three odor
exposures from 6–7 flies; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (g) Time course of
SynpH activity induced by MCH in the presynaptic terminals of PNs within the calyx region of 3d, 30d, and 30dSpd animals (SynpH response averaged
across three odor exposures from 6–7 flies) (h) Maximum change in SynpH fluorescence (ΔF/F in %) in response to MCH within the presynaptic terminals
of PN boutons of 3d, 30d, and 30dSpd flies (SynpH response averaged across three odor exposures from 6–7 flies; Kruskal-Wallis test with Dunn’s
multiple comparison test, p-values were subject to Bonferroni correction). * p < 0.05, ** p < 0.01, ns = not significant, p 0.05. Underlying data is shown
in S1 Data. -KC synapses to measure odor-evoked SV release. (a) SynpH expressed in PN boutons and imaged within the Fig 1. Imaging of SynpH at PN-to-KC synapses to measure odor-evoked SV release. (a) SynpH expressed Fig 1. Imaging of SynpH at PN-to-KC synapses to measure odor-evoked SV release. (a) SynpH expressed in PN boutons and imaged within the
calyx neuropil (GH146 > SynpH). Scale bar: 10 μm. (b–c) False color-coded image of the SynpH activity within the presynaptic terminals of PNs in
response to 3-Oct and MCH shown in (a). Warm colors indicate high levels, and cold colors indicate low levels or no SynpH activity. The color scale on the
right indicates changes in fluorescence (ΔF/F in %). (d) Odor-evoked release of SVs, measured by changes in fluorescence of SynpH of individual flies
over time shown as false colors in presynaptic terminals of PN in the calyx region. The left panel is in response to the odorant 3-Oct and the right panel is
in response to MCH (n = 6–7 flies). (e) Time course of SynpH activity induced by 3-Oct in the presynaptic terminals of PNs within the calyx neuropil of 3d,
30d, and 30dSpd animals (SynpH response averaged across three odor exposures from 6–7 flies). doi:10.1371/journal.pbio.1002563.g001 Spermidine Feeding Specifically Blocks Age-Induced Increases of Core
AZ Components Spermidine feeding suppresses age-associated increase in BRP and rim-binding protein (RBP) levels. (a–c) Adult
brains 3d and 30d w1118 flies, together with 30dSpd w1118 flies immunostained for Synapsin. Scale bar: 50 μm. (d) Quantification of
Synapsin intensity within the central brain region normalized to 3d flies (n = 9–10 independent brains; Kruskal-Wallis test). (e–g)
Adult brains of 3d and 30d w1118 flies, together with 30dSpd w1118 flies immunostained for Synaptotagmin-1 (Syt-1). Scale bar:
50 μm. (h) Quantification of signal intensity of Syt-1 in the central brain region normalized to 3d flies (n = 8–9 independent brains;
Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (i–k) Adult brains of 3d,
30d w1118, and 30dSpd w1118 flies immunostained for BRP (using Nc82 and N-terminal antibodies) and RBP. Scale bar: 50 μm (l–n)
Quantification of BRP (using Nc82 and N-terminal antibodies) and RBP intensities within the central brain region normalized to 3d
flies (n = 14–18 independent brains; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni
correction). ** p < 0.01, *** p < 0.001, ns = not significant, p 0.05. Underlying data is shown in S1 Data. Fig 2. Spermidine feeding suppresses age-associated increase in BRP and rim-binding protein (RBP) levels. (a–c) Adult
brains 3d and 30d w1118 flies, together with 30dSpd w1118 flies immunostained for Synapsin. Scale bar: 50 μm. (d) Quantification of
Synapsin intensity within the central brain region normalized to 3d flies (n = 9–10 independent brains; Kruskal-Wallis test). (e–g)
Adult brains of 3d and 30d w1118 flies, together with 30dSpd w1118 flies immunostained for Synaptotagmin-1 (Syt-1). Scale bar:
50 μm. (h) Quantification of signal intensity of Syt-1 in the central brain region normalized to 3d flies (n = 8–9 independent brains;
Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (i–k) Adult brains of 3d,
30d w1118, and 30dSpd w1118 flies immunostained for BRP (using Nc82 and N-terminal antibodies) and RBP. Scale bar: 50 μm (l–n)
Quantification of BRP (using Nc82 and N-terminal antibodies) and RBP intensities within the central brain region normalized to 3d
flies (n = 14–18 independent brains; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni
correction). ** p < 0.01, *** p < 0.001, ns = not significant, p 0.05. Underlying data is shown in S1 Data. Spermidine Feeding Specifically Blocks Age-Induced Increases of Core
AZ Components doi:10.1371/journal.pbio.1002563.g002 among several proteins that form the AZ scaffold [23,24]. In flies, the ELKS-family protein Bruch-
pilot (BRP) is an essential building block of the AZ scaffold and is neededto effectively cluster Ca2
+ channels as well as regulate the release of SVs [25–28]. When whole-mount brains were stained
for BRP using two different antibodies (BRPNc82 and BRPN-term), we observeda substantial
increase in the levels of BRP with advancing age (Fig 2I, 2J, 2L and 2M). Similarly, Rim-binding
protein (RBP) [29], another structurallyand functionallyimportant component of the AZ scaffold,
was found to be significantly increased in brains of 30d flies compared to 3d animals (Fig 2I, 2J
and 2N). Furthermore, flies analyzed at shorter intervals throughout their lifetime exhibited a pro-
gressive increase in the levels of both BRP and RBP (S8 Fig). Notably, the age-dependent increase
in BRP and RBP signals was suppressed in aged flies fed with spermidine (30dSpd; Fig 2I–2N). The staining efficacycould potentially be influenced by the sheer age of the tissue, e.g., due
to differences in antibody penetration. To rule this out, flies expressing a GFP-tagged genomic
BRP construct (rescuing the lethal brp null mutant [28]) were aged on normal food or food
supplemented with spermidine. We found the endogenous GFP signals to be significantly
increased in 30d flies in comparison to 3d flies, while feeding with spermidine again prevented
this age-related increase (S9 Fig). Since the AZ scaffold has previously been reported to effectively cluster Ca2+ channels [26–
28], we asked whether the age-associated increase in levels of core AZ-proteins might influence
synaptic levels of Ca2+ channels. To address this, we expressed a GFP-labeled genomic con-
struct of α1 subunit Cacophony (Cac), which is the only representative of the mammalian
Cav2.1/2.2 family present in Drosophila [28], and stained the flies for GFP and BRP. We found
the levels of Cac (quantified using an antibody against GFP) to remain unchanged with aging
(S10 Fig). Besides its role in Ca2+ channel clustering, the AZ scaffold has been suggested to cre-
ate a stereotypic arrangement that defines SV release slots by clustering SV release machinery
[28]. In fact, the levels of Unc13, a protein essential for priming SVs by rendering them fusion-
competent [24], were also increased in brains of 30d flies compared to 3d flies (S11 Fig). Again,
spermidine administration suppressed this age-dependent increase (30dSpd; S11 Fig). Spermidine Feeding Specifically Blocks Age-Induced Increases of Core
AZ Components Taken
together, our data suggest that synaptic levels of core AZ scaffold proteins, BRP and RBP, as
well as the levels of critical release factor Unc-13 increased with advancing age. Spermidine Feeding Specifically Blocks Age-Induced Increases of Core
AZ Components (f) Maximum change in SynpH fluorescence (ΔF/F in
%) in response to 3-Oct within the presynaptic terminals of PN boutons of 3d, 30d, and 30dSpd flies (SynpH response averaged across three odor
exposures from 6–7 flies; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (g) Time course of
SynpH activity induced by MCH in the presynaptic terminals of PNs within the calyx region of 3d, 30d, and 30dSpd animals (SynpH response averaged
across three odor exposures from 6–7 flies) (h) Maximum change in SynpH fluorescence (ΔF/F in %) in response to MCH within the presynaptic terminals
of PN boutons of 3d, 30d, and 30dSpd flies (SynpH response averaged across three odor exposures from 6–7 flies; Kruskal-Wallis test with Dunn’s
multiple comparison test, p-values were subject to Bonferroni correction). * p < 0.05, ** p < 0.01, ns = not significant, p 0.05. Underlying data is shown
in S1 Data. 5 / 34 PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 6 / 34 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Fig 2. Spermidine feeding suppresses age-associated increase in BRP and rim-binding protein (RBP) levels. (a–c) Adult
brains 3d and 30d w1118 flies, together with 30dSpd w1118 flies immunostained for Synapsin. Scale bar: 50 μm. (d) Quantification of
Synapsin intensity within the central brain region normalized to 3d flies (n = 9–10 independent brains; Kruskal-Wallis test). (e–g)
Adult brains of 3d and 30d w1118 flies, together with 30dSpd w1118 flies immunostained for Synaptotagmin-1 (Syt-1). Scale bar:
50 μm. (h) Quantification of signal intensity of Syt-1 in the central brain region normalized to 3d flies (n = 8–9 independent brains;
Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (i–k) Adult brains of 3d,
30d w1118, and 30dSpd w1118 flies immunostained for BRP (using Nc82 and N-terminal antibodies) and RBP. Scale bar: 50 μm (l–n)
Quantification of BRP (using Nc82 and N-terminal antibodies) and RBP intensities within the central brain region normalized to 3d
flies (n = 14–18 independent brains; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni
correction). ** p < 0.01, *** p < 0.001, ns = not significant, p 0.05. Underlying data is shown in S1 Data. Fig 2. Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes In order to allow for an unbiased quantification, we applied automated data collection to
acquire more than a thousand transmission electron microscopic images covering nearly a
whole calyx cross-section, which were then “stitched” together into a single high-magnification
image (see Materials and Methods). As describedpreviously [30], PN boutons could be easily
identified, and light-colored boutons containing clear-core SVs were used for analysis. We rec-
ognized that plasma membranes between cellular elements were less aligned, with an increase
in extracellular spacing between cellular elements, in aged (30d) flies when compared to young
(3d) flies (S12 Fig). Spermidine feeding appeared to substantially alleviate this age-related
change (S12 Fig). Driven by the finding that SV release is increased with age, we decided to
analyze the AZs within PN boutons. We found aged animals (30d) to display reduced numbers
of AZs per unit bouton-area in comparison to 3d flies, with no apparent influence of spermi-
dine feeding on this age-dependent decline (Fig 3A–3E). The density of SVs in proximity to the
AZ scaffold appeared unchanged in aged flies (30d as well as 30dSpd), when compared to young
flies (3d; Fig 3F). Additionally, the number of SVs docked at the AZ plasma membrane
appeared essentially unaltered with advancing age (Fig 3G). The AZ scaffold exhibits an electron-dense structure in electron microscopy (EM), and due
to its T-shaped structure in Drosophila, this scaffold is often referred to as a T-bar [24,26,27]. We found the average size of the T-bars to be significantly increased in 30d animals in compar-
ison to 3d flies (Fig 4A–4D). Feeding flies with spermidine suppressed this age-induced
increase in T-bar size (30dSpd; Fig 4A–4D). We have previously introduced STED in the analy-
sis of AZ suborganization [26–28]. At peripheral neuromuscular synapses of Drosophila larvae,
STED allowed us to unmask the “nano-architecture” of AZs where BRP and RBP organize a
scaffold that provides slots for SV release and concentrates Ca2+ channels in the AZ center
[28,29]. When planar AZs were imaged using the BRP C-terminal epitopes at neuromuscular
synapses, they display a ring-shaped structure whose diameter correlated with the EM-derived
physical size of individual T-bar/AZ scaffold [32]. We applied STED to PN-to-KC synapses of
the calyx and found ring-like BRP structures at planar-oriented AZs (S13 Fig). Subsequently,
the analysis of these STED images revealed an increase in the ring diameter of BRP spots with
advancing age, while spermidine treatment was able to suppress this age-associated increase
(Fig 4E–4H). Finally, we performed coimmuno-EM labeling against BRP and RBP on calycal
slices. The number of gold particles positive for BRP as well as RBP was found to increase in
aged flies (30d) in comparison to both young (3d) flies and aged flies fed with spermidine
(30dSpd; Fig 4I–4N). Taken together, the morphological EM, immuno-EM, and STED analysis
consistently show that aged animals display larger AZ scaffolds, plausibly due to an increase in
local amounts of the critical scaffold components: BRP and RBP. Recent in vivo analysis of larval Drosophila neuromuscular junctions has shown that the
local amounts of BRP at a given AZ scale directly with the probability of evoked SV release
[33–37]. Consistent with these studies, we found SV release to increase and AZ scaffolds to
enlarge with age, while importantly both these age-related changes were suppressed by dietary
supplementation with spermidine. Therefore, we next wanted to determine the influence of
these synaptic changes on olfactory memory formation. Age-Induced Enlargement of AZ Scaffolds Is Suppressed by
Spermidine Feeding Next, we asked whether the increase of both BRP and RBP labeling in aged brains reflects an
increase in the number of AZs or just the increase in local amounts of these proteins at individ-
ual AZs. To resolve this, we performed ultrastructuralanalysis on PN-to-KC synapses within
the mushroom body calyx. In contrast to presynaptic terminals of KCs, presynaptic PN termi-
nals within the calyx exhibit a well-definedmorphology [30,31], by which synapse types can be
reliably identified in EM micrographs. Moreover, the superficiality of the calyx enabled us to
perform stimulation emission depletion microscopy (STED) analysis (see below). 7 / 34 PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Fig 3. Ultrastructural analysis of PN-to-KC synapses within the mushroom body calyx. (a) Overview of the calyx neuropil,
obtained by amalgamation of several images over a whole calyx cross-section of a 3d w1118 fly. Scale bar: 10 μm. (b–d) Higher
magnification of PN boutons and dendritic claws of KCs within the calyx of 3d, 30d, and 30dSpd w1118 flies. Scale bar: 2 μm. The
asterisk indicates the PN bouton, and the arrowhead indicates the dendritic claws of KCs. (e) Quantification of AZs normalized to
bouton area (1/pm2) (total of 95–103 boutons across three independent animals, with at least 25 boutons per animal; Kruskal-
Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (f) Quantification of total SVs
within a shell of 150 nm surrounding the AZ scaffold (total of 92–100 electron-micrographs across four independent animals, with
at least 20 electron-micrographs per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to
Bonferroni correction). (g) Quantification of SVs touching the presynaptic plasma membrane (total of 92–100 electron-
micrographs across four independent animals, with at least 20 electron micrographs per animal; Kruskal-Wallis test with Dunn’s
multiple comparison test, p-values were subject to Bonferroni correction). * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not
significant, p 0.05. Underlying data is shown in S1 Data. doi:10.1371/journal.pbio.1002563.g003 doi:10.1371/journal.pbio.1002563.g003 whether genetically provoking an artificial enlargement of AZ scaffolds, independent of the
aging process, might affect memory formation. Since BRP is a major essential building block of
the AZ scaffold in Drosophila [26–28,32], we decided to increase the gene copy number of BRP
from two to four copies by combining two additional genomic copies of brp [28] with two
endogenous copies. As a result, BRP signals increased substantially in 3d flies expressing four-
copy BRP (4xBRP) when compared to 3d flies expressing two-copy BRP (2xBRP; Fig 5A–5E). Additionally, RBP levels also increased concomitantly with BRP (Fig 5A–5D and 5F), consis-
tent with the suggested role of BRP to operate as a “master molecule” in shaping the size (and
functional performance) of the AZ scaffold [28,29,36]. In order to confirm that the increase in
BRP levels resulted in an increase of the average size of AZ scaffolds, we took advantage of
STED imaging. PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 “Early” Memory Impairment after Genetically Enforced Enlargement of
AZ Scaffolds Presynaptic plasticity processes have been reported to be critical for forming olfactory associa-
tive memory in Drosophila [11–13]. Based on our findings, we suggest that the scale-up in the
size and function of AZ scaffolds is likely to change the “operational range” of synaptic plastic-
ity processes and thus change the threshold for memory formation. Thus, we wanted to test 8 / 34 PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 9 / 34 Scale bar: 200 nm. (j–l) Higher magnification of AZ within PN bouton immunostained for BRP (large gold particles) and RBP (small gold
particles) of 3d, 30d, and 30dSpd w1118 flies. Scale bar: 50 nm. (m) Quantification of BRP-positive gold particles per T-bar (total of 94–108 individual T-bars
across three independent animals, with at least 25 T-bars per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to
Bonferroni correction). (n) Quantification of RBP-positive gold particles per T-bar (total of 94–108 individual T-bars across three independent animals, with
at least 25 T-bars per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). * p < 0.05, **
p < 0.01, *** p < 0.001, ns = not significant, p 0.05. Underlying data is shown in S1 Data. Fig 4. High-resolution analysis of PN-to-KC synapses within the mushroom body calyx shows increase in T-bar size. (a–c) Electron micrographs
of calyx region of 3d, 30d, and 30dSpd w1118 animals showing presynaptic specializations in blue (T-bars) at the PN-to-KC synapses. Scale bar: 50 nm. (d)
Quantification representing the average T-bar size in 3d, 30d, and 30dSpd animals (n = 92–100 electromicrographs across four independent animals, with at
least 20 T-bars per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (e–g) STED images of
BRP spots reveal ring-shaped structures (arrowheads) within the calyx of 3d, 30d, and 30dSpd w1118 flies. Scale bar: 500 nm. (h) Comparison of BRP-spot
diameter between 3d, 30d, and 30dSpd flies (total of 94–112 BRP rings across 15 independent animals, with at least 5 BRP rings per animal; Kruskal-Wallis
test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (i) Electron micrographs of PN bouton within the calyx region of
3d w1118 flies. Scale bar: 200 nm. (j–l) Higher magnification of AZ within PN bouton immunostained for BRP (large gold particles) and RBP (small gold
particles) of 3d, 30d, and 30dSpd w1118 flies. Scale bar: 50 nm. (m) Quantification of BRP-positive gold particles per T-bar (total of 94–108 individual T-bars
across three independent animals, with at least 25 T-bars per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to
Bonferroni correction). Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Fig 4. High-resolution analysis of PN-to-KC synapses within the mushroom body calyx shows increase in T-bar size. (a–c) Electron micrographs
of calyx region of 3d, 30d, and 30dSpd w1118 animals showing presynaptic specializations in blue (T-bars) at the PN-to-KC synapses. Scale bar: 50 nm. (d)
Quantification representing the average T-bar size in 3d, 30d, and 30dSpd animals (n = 92–100 electromicrographs across four independent animals, with at
least 20 T-bars per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (e–g) STED images of
BRP spots reveal ring-shaped structures (arrowheads) within the calyx of 3d, 30d, and 30dSpd w1118 flies. Scale bar: 500 nm. (h) Comparison of BRP-spot
diameter between 3d, 30d, and 30dSpd flies (total of 94–112 BRP rings across 15 independent animals, with at least 5 BRP rings per animal; Kruskal-Wallis
test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (i) Electron micrographs of PN bouton within the calyx region of
3d w1118 flies. Scale bar: 200 nm. (j–l) Higher magnification of AZ within PN bouton immunostained for BRP (large gold particles) and RBP (small gold
particles) of 3d, 30d, and 30dSpd w1118 flies. Scale bar: 50 nm. (m) Quantification of BRP-positive gold particles per T-bar (total of 94–108 individual T-bars
across three independent animals, with at least 25 T-bars per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to
Bonferroni correction). (n) Quantification of RBP-positive gold particles per T-bar (total of 94–108 individual T-bars across three independent animals, with
at least 25 T-bars per animal; Kruskal-Wallis test with Dunn’s multiple comparison test p-values were subject to Bonferroni correction) * p < 0 05 **
p
g
p
y
y
pp
g
y
p
g Fig 4. High-resolution analysis of PN-to-KC synapses within the mushroom body calyx shows increase in T-bar size. (a–c) Electron micrographs
of calyx region of 3d, 30d, and 30dSpd w1118 animals showing presynaptic specializations in blue (T-bars) at the PN-to-KC synapses. Scale bar: 50 nm. (d)
Quantification representing the average T-bar size in 3d, 30d, and 30dSpd animals (n = 92–100 electromicrographs across four independent animals, with at
least 20 T-bars per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 (e–g) STED images of
BRP spots reveal ring-shaped structures (arrowheads) within the calyx of 3d, 30d, and 30dSpd w1118 flies. Scale bar: 500 nm. (h) Comparison of BRP-spot
diameter between 3d, 30d, and 30dSpd flies (total of 94–112 BRP rings across 15 independent animals, with at least 5 BRP rings per animal; Kruskal-Wallis
test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (i) Electron micrographs of PN bouton within the calyx region of
3d w1118 flies. Scale bar: 200 nm. (j–l) Higher magnification of AZ within PN bouton immunostained for BRP (large gold particles) and RBP (small gold
particles) of 3d, 30d, and 30dSpd w1118 flies. Scale bar: 50 nm. (m) Quantification of BRP-positive gold particles per T-bar (total of 94–108 individual T-bars
across three independent animals, with at least 25 T-bars per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to
Bonferroni correction). (n) Quantification of RBP-positive gold particles per T-bar (total of 94–108 individual T-bars across three independent animals, with
at least 25 T-bars per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). * p < 0.05, **
p < 0.01, *** p < 0.001, ns = not significant, p 0.05. Underlying data is shown in S1 Data. doi:10 1371/journal pbio 1002563 g004 o-KC synapses within the mushroom body calyx shows increase in T-bar size. (a–c) Electron micrographs
18 Fig 4. High-resolution analysis of PN-to-KC synapses within the mushroom body calyx shows increase in T-bar size. (a–c) Electron micrographs
of calyx region of 3d, 30d, and 30dSpd w1118 animals showing presynaptic specializations in blue (T-bars) at the PN-to-KC synapses. Scale bar: 50 nm. (d)
Quantification representing the average T-bar size in 3d, 30d, and 30dSpd animals (n = 92–100 electromicrographs across four independent animals, with at
least 20 T-bars per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (e–g) STED images of
BRP spots reveal ring-shaped structures (arrowheads) within the calyx of 3d, 30d, and 30dSpd w1118 flies. Scale bar: 500 nm. (h) Comparison of BRP-spot
diameter between 3d, 30d, and 30dSpd flies (total of 94–112 BRP rings across 15 independent animals, with at least 5 BRP rings per animal; Kruskal-Wallis
test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (i) Electron micrographs of PN bouton within the calyx region of
3d w1118 flies. Again, a considerable increase in the ring diameter of BRP spots was observed
in 2xBRP flies with advancing age (Fig 5G–5K). Meanwhile, we found young flies (3d) express-
ing 4xBRP to have increased BRP ring diameters when compared to age-matched control flies
(2xBRP), and the ring diameter of BRP spots in 4xBRP flies remained rather unchanged with
age (Fig 5G–5K). Having created a genetic state wherein levels of AZ core scaffold proteins increased prema-
turely in young animals, we decided to investigate the influence of this manipulation on mem-
ory formation. Before doing so, however, we wanted to ascertain whether the innate behavior
was affected in 4xBRP flies. Thus, we measured naïve odor response and shock reactivity and
found 4xBRP flies to show odor avoidance and shock reactivity scores that were indistinguish-
able from 2xBRP age-matched control flies (2xBRP; S1 Table). Subsequently, we started by
measuring short-term memory (STM), and found 4xBRP flies to exhibit lower memory scores
“already” at a young age (3d), and their memory scores declined only negligibly with age (Fig
5L). In contrast, control flies (2xBRP) exhibited normal AMI (Fig 5L). As mentioned earlier, intermediate-term memory (ITM) has also been reported to decline
with age [2–4]. Consistently, we found that 30d 2xBRP flies show substantially reduced ITM
scores (measured 3-h post-training) when compared to 3-d 2xBRP flies (Fig 5M). By contrast,
the 4xBRP flies showed lower ITM scores at a young age (3 d) and, again, the ITM scores did
not decrease further in 30-d 4xBRP flies (Fig 5M). In fact, the learning performance of 3-d
4xBRP flies was comparable to that of 30-d 2xBRP flies. Based on distinct genetic mutants and
specific pharmacological sensitivities [2,4,38,39], the ITM can be dissected into anesthesia-sen-
sitive memory (ASM) and anesthesia-resistant memory (ARM) components. The ASM, unlike
the ARM, has been shown to be strongly impaired with aging [3,4]. The ASM can be calculated
by subtracting ARM scores, measured after amnestic cooling, from ITM. Consistent with previ-
ous studies [2–4,40], we found ARM in 2xBRP and 4xBRP flies to remain relatively unaffected
with age (Fig 5M). In contrast, ASM was nearly absent in 30-d 2xBRP flies when compared to
3-d 2xBRP flies. Reaffirmingour idea, we found the young (3-d) 4xBRP flies to show lower
ASM scores in comparison to age-matched control (2xBRP) flies, while their ASM scores PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 10 / 34 (n) Quantification of RBP-positive gold particles per T-bar (total of 94–108 individual T-bars across three independent animals, with
at least 25 T-bars per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). * p < 0.05, **
p < 0.01, *** p < 0.001, ns = not significant, p 0.05. Underlying data is shown in S1 Data. doi:10.1371/journal.pbio.1002563.g004 PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 11 / 34 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Fig 5. Increase in levels of core AZ proteins BRP and RBP leads to early memory impairment. (a–d) Adult brains of 3d and 30d flies expressing
4xBRP together with age-matched controls brp (2xBRP), immunostained for BRP (using Nc82 and BRP N-terminal antibody) and RBP. Scale bar: 50 μm. (e, f) Quantification of BRP (using N-terminal antibody) as well as RBP intensity within the central brain region normalized to 3d flies (n = 12–13
independent brains; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (g–j) STED images of BRP
label within the calyx region of 3d and 30d flies expressing 4xBRP as well as 2xBRP. Ring-shaped structures are indicated (arrowheads). Scale bar: 500
nm. (k) Quantification of BRP ring diameter in 3d and 30d 4xBRP flies along with age-matched 2xBRP flies (total of 47–68 BRP rings across eight
independent animals, with at least six BRP rings per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni
correction). (l) Aversive associative memory performance 3 min after training (short-term memory; STM) markedly reduced in 3d 4xBRP flies in
comparison to 3d wild-type 2xBRP flies (n = 10–16; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni
correction). (m) Aversive associative memory performance at 3 h after training (intermediate-term memory; ITM), anesthesia-resistant memory (ARM), an
anesthesia-sensitive memory (ASM) of 3d and 30d 4xBRP flies compared to age-matched control (2xBRP) flies (n = 7 independent experiments; Kruskal-
Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (n) Aversive olfactory memory performance 3 min after
training (STM) higher in appl-gal4 > histone deacetylase-6 (HDAC6) RNAi in comparison to age-matched controls (n = 13–21; Kruskal-Wallis test with
Spermidine Protects from Age Dependent Memory Decline by Suppressing Presynaptic Change Fig 5. Increase in levels of core AZ proteins BRP and RBP leads to early memory impairment. PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes declined negligibly with age (Fig 5M). These experiments indicate that a genetically provoked
“up-scaling” of the average AZ scaffold size is sufficient to induce an “early” decline in mem-
ory, similar to AMI, which physiologically occurs over a time course of 20–30 d. A reduction in BRP levels, per se, might be expected to slow down the onset of AMI. To
address this possibility, we removed a single gene copy of brp, and found BRP heterozygotes
(brp69/+ or 1xBRP) to exhibit a considerable reduction in the levels of both BRP and RBP
(S14A–S14F Fig), indicating that our antibody stainings can detect subtle changes and reaf-
firming that BRP levels can directly steer the local amounts of other AZ components in the
Drosophila brain. We found that 3d flies expressing only one BRP copy (brp69/+) displayed
memory scores comparable to 3d control flies (2xBRP); however, these brp69/+ flies still exhib-
ited a normally-occurringAMI (30d; S14G Fig). AZ scaffold-dependent control of neuronal
plasticity is undoubtedly a complex process [24,41], and other mechanisms, operating in paral-
lel to modulations in the amounts of scaffold proteins, might well contribute to the pace and
extent of AMI. Lysine-acetylation of BRP was recently identified as a major node to control the
SV release at larval AZs [42,43]. In particular, the loss of histone deacetylase-6 (HDAC6) was
found to cause hyperacetylation of BRP and provoke a reduction in evoked SV release at AZs
[43]. Interestingly, using immunoprecipitation followed by mass spectroscopic analysis, we
found at least 13 lysine sites within BRP to be target for (de)acetylation, (S15 Fig). Next, we
asked whether loss of HDAC6 might affect memory. While the odor avoidance and shock reac-
tivity were mainly unaffected by knockdown of hdac6 (S1 Table), memory scores of both
young and aged flies with pan-neuronal knockdown of hdac6 were higher than those of age-
matched driver controls (Fig 5N). These findings are consistent with the idea that driving
down the AZs towards the lower limit of their operational range might facilitate memory for-
mation in aged animals. Though any implications of acetylation of BRP or potentially other
AZ scaffold proteins with respect to aging process still require extensive analysis, this result
shows that BRP-directed modifications, reported to reduce SV release, can in fact increase the
efficacyof memory formation in aged animals. PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 (a–d) Adult b Fig 5. Increase in levels of core AZ proteins BRP and RBP leads to early memory Fig 5. Increase in levels of core AZ proteins BRP and RBP leads to early memory impairment. (a–d) Adult brains of 3d and 30d flies expressing
4xBRP together with age-matched controls brp (2xBRP), immunostained for BRP (using Nc82 and BRP N-terminal antibody) and RBP. Scale bar: 50 μm. (e, f) Quantification of BRP (using N-terminal antibody) as well as RBP intensity within the central brain region normalized to 3d flies (n = 12–13
independent brains; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (g–j) STED images of BRP
label within the calyx region of 3d and 30d flies expressing 4xBRP as well as 2xBRP. Ring-shaped structures are indicated (arrowheads). Scale bar: 500
nm. (k) Quantification of BRP ring diameter in 3d and 30d 4xBRP flies along with age-matched 2xBRP flies (total of 47–68 BRP rings across eight
independent animals, with at least six BRP rings per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni
correction). (l) Aversive associative memory performance 3 min after training (short-term memory; STM) markedly reduced in 3d 4xBRP flies in
comparison to 3d wild-type 2xBRP flies (n = 10–16; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni
correction). (m) Aversive associative memory performance at 3 h after training (intermediate-term memory; ITM), anesthesia-resistant memory (ARM), and
anesthesia-sensitive memory (ASM) of 3d and 30d 4xBRP flies compared to age-matched control (2xBRP) flies (n = 7 independent experiments; Kruskal-
Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (n) Aversive olfactory memory performance 3 min after
training (STM) higher in appl-gal4 > histone deacetylase-6 (HDAC6) RNAi in comparison to age-matched controls (n = 13–21; Kruskal-Wallis test with
Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant, p 0.05. Underlying data is shown in S1 Data. doi:10.1371/journal.pbio.1002563.g005 PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 12 / 34 Homeostasis of Odor-Driven Neuronal Ca2+ Signals in Aged Flies Finally, we asked how the postsynaptic compartment might respond to these age-associated pre-
synaptic structuraland functional changes. To address this question, we used GCaMP3.0 fused
to the postsynaptic protein Homer [15] and found the basal expression of Homer-GCamp3.0 to
be largely unaffectedwith age (S2C Fig). Moreover, the sensor was found to be effectively targeted
to the postsynaptic density of the PN::KC synapses, as manifested by its specific enrichment
within the postsynaptic specializations formed by claw-like dendritic endings of multiple KCs
surrounding a single PN bouton (Fig 6A). However, postsynaptic Ca2+ signals did not increase
with age. Rather, a slight tendency towards a decrease of postsynaptic Ca2+ signals was observed
in normally aged animals when compared to young controls (Fig 6A–6H). At the same time,
aged flies treated with spermidine (30dSpd) produced signals more similar to untreated 3d-
Homer-GCaMP3.0 flies than to untreated aged animals (Fig 6A–6H). In order to be certain that
Homer-GCamp3.0 signals were not saturated, we used high-molar KCl treatment to determine
the maximal postsynaptic Ca2+ response. Unlike the odor-evoked maximum change in Homer-
GCamp3.0 fluorescence of about 55%, KCl stimulation resulted in a substantially higher ΔF/F0
value of more than 300% (S16 Fig), suggesting that sensor sensitivity was not a limiting factor for
the postsynaptic Ca2+ signals. Meanwhile, when the cumulative postsynaptic Ca2+ activity was
critically analyzed during the odor stimulation, we found that the Ca2+ responses reduced signifi-
cantly in aged (30d) flies relative to young flies, while the Ca2+ signals were comparable between
young flies and spermidine-fedaged animals (30dSpd; S17 Fig). PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 13 / 34 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Fig 6. Imaging of Homer-GCamp3.0 within the dendritic claws of KCs to measure odor-evoked Ca2+ activity. (a) Expression of Homer-GCamp3.0 in
the dendritic claws of KCs and imaged within the calyx region (mb247 > Homer-GCamp3.0). Scale bar: 10 μm. (b–c) False color-coded image of Homer-
GCamp3.0 activity within the postsynaptic terminals of KCs in response to 3-Oct and MCH shown in (a). Warm colors indicate high activity and cold colors
indicate low or no Ca2+ activity. The numbers indicate changes in fluorescence (ΔF/F in %). (d) Odor-evoked postsynaptic Ca2+ activity, measured by
changes in fluorescence of Homer-Gamp3.0, of an individual fly over time, shown as false colors in dendritic claws of KCs in the calyx region. Homeostasis of Odor-Driven Neuronal Ca2+ Signals in Aged Flies The left panel
is in response to the odorant 3-Oct, and the right panel is in response to MCH (n = 10 flies). (e) Time course of Ca2+ activity induced by 3-Oct in the dendritic
terminals of KCs within the calyx region of 3d, 30d, and 30dSpd animals (GCamp3.0 response averaged across three odor exposures from ten flies). (f)
Maximum change in GCamp3.0 fluorescence (ΔF/F in %) in response to 3-Oct within dendritic claws of KCs of 3d, 30d, and 30dSpd flies (GCamp3.0
response averaged across three odor exposures from ten flies; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to
Bonferroni correction). (g) Time course of Ca2+ activity induced by MCH in the dendritic terminals of KCs of 3d, 30d, and 30dSpd animals (GCamp3.0
response averaged across three odor exposures from ten flies). (h) Maximum change in GCamp3.0 fluorescence (ΔF/F in %) in response to MCH within
dendritic claws of KCs of 3d, 30d, and 30dSpd flies (GCamp3.0 response averaged across three odor exposures from ten flies; Kruskal-Wallis test with Fig 6. Imaging of Homer-GCamp3.0 within the dendritic claws of KCs to measure odor-evoked Ca2+ activity. (a) Expression of Homer-GCamp3.0 in Fig 6. Imaging of Homer-GCamp3.0 within the dendritic claws of KCs to measure odor-evoked Ca2+ activity. (a) Expression of Homer-GCamp3.0 in
the dendritic claws of KCs and imaged within the calyx region (mb247 > Homer-GCamp3.0). Scale bar: 10 μm. (b–c) False color-coded image of Homer-
GCamp3.0 activity within the postsynaptic terminals of KCs in response to 3-Oct and MCH shown in (a). Warm colors indicate high activity and cold colors
indicate low or no Ca2+ activity. The numbers indicate changes in fluorescence (ΔF/F in %). (d) Odor-evoked postsynaptic Ca2+ activity, measured by
h
i fl
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l Fig 6. Imaging of Homer-GCamp3.0 within the dendritic claws of KCs to measure odor-evoked Ca2+ activity. (a) Expression of Homer-GCamp3.0 in
the dendritic claws of KCs and imaged within the calyx region (mb247 > Homer-GCamp3.0). Scale bar: 10 μm. Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). The grey bars indicate the duration of the odor stimuli. * p < 0.05, **
p < 0.01, ns = not significant, p 0.05. Underlying data is shown in S1 Data. doi:10.1371/journal.pbio.1002563.g006 doi:10.1371/journal.pbio.1002563.g006 PNs provide cholinergic input to the KCs within the calyx [44]. We used a fusion of mush-
room body-specificenhancer mb247 to the Dα7 subunit of the acetylcholine receptor (mb247::
Dα7GFP) to explicitly visualize postsynaptic acetylcholine receptors. We showed previously
that expression of Dα7-GFP from KCs localized specificallyto the KC postsynaptic densities,
where it closely matched the AZs of the PNs [45]. While we observedan age-related increase in
BRP in 30d mb247::Dα7GFP flies in comparison to 3d mb247::Dα7GFP flies, the levels of Dα7
subunit (quantified using an antibody against GFP fused to the α7 subunit of acetylcholine
receptors) did not change with age, and spermidine feeding had no effect on the level of the α7
subunit of acetylcholine receptors (Fig 7A–7E). Similarly, when we stained for endogenous
Drep2, a postsynaptic scaffold protein that is known to express strongly within the postsynap-
tic densities of PN::KC synapses [46], we also found Drep2 to remain unchanged with age
(S18 Fig). g
At first glance, the increase in release of SVs might be expected to translate into increased
postsynaptic responses; however, ample evidence from various studies in different model
organisms, including Drosophila, support the existence of homeostatic controls, allowing neu-
rons to remain within a certain range of excitation [47,48] in order to avoid epileptic states and
Ca2+-induced degeneration. In an attempt to directly examine the existence of such homeo-
static controls, we wanted to determine whether an increase in the amount of depolarization
required to trigger an action potential might influence the architecture of the apposed AZ scaf-
fold. To achieve this, we used dORK1ΔC, a constitutively open K+ selective pore that causes
hyperpolarization of neurons and subsequent inactivation of neuronal function [45,49]. dORK1ΔC was specificallyexpressed in the KCs, and presynaptic terminals of PNs within the
calyx were analyzed for BRP levels (Fig 7F–7I). Indeed, we found a substantial increase in the
levels of BRP in the calyces of both 3d as well as 10d mb247>dORK1ΔC flies, when compared
to age-matched controls (Fig 7J). Thus, a drop in postsynaptic excitability can drive a homeo-
static increase in presynaptic AZ scaffolds, leading to a potential increase in SV release at olfac-
tory synapses—a finding similar to the one we found at aging synapses. Though the exact
mechanisms allowing for homeostatic compensation of the elevated presynaptic release remain
to be further worked out, it is tempting to speculate that homeostatic mechanisms coupling
postsynaptic excitability to presynaptic release function might drive aging synapses towards
the upper limit of their operational range and be critically involved in AMI (see model in
Fig 7K) Homeostasis of Odor-Driven Neuronal Ca2+ Signals in Aged Flies (b–c) False color-coded image of Homer-
GC
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ti it
ithi th
t
ti t
i
l
f KC i
t 3 O t
d MCH h
i ( ) W
l
i di
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ti it
d
ld
l n the dendritic claws of KCs to measure odor-evoked Ca2+ activity. (a) Expression of Homer-GCamp3.0 in Cs to measure odor-evoked Ca2+ activity. (a) Expression of Homer-GCamp3.0 in g 6. Imaging of Homer-GCamp3.0 within the dendritic claws of KCs to measure odor-evoked Ca2+ activity. (a Fig 6. Imaging of Homer GCamp3.0 within the dendritic claws of KCs to measure odor evoked Ca
activity. (a) Expression of Homer GCamp3.0 in
the dendritic claws of KCs and imaged within the calyx region (mb247 > Homer-GCamp3.0). Scale bar: 10 μm. (b–c) False color-coded image of Homer-
GCamp3.0 activity within the postsynaptic terminals of KCs in response to 3-Oct and MCH shown in (a). Warm colors indicate high activity and cold colors
indicate low or no Ca2+ activity. The numbers indicate changes in fluorescence (ΔF/F in %). (d) Odor-evoked postsynaptic Ca2+ activity, measured by
changes in fluorescence of Homer-Gamp3.0, of an individual fly over time, shown as false colors in dendritic claws of KCs in the calyx region. The left panel
is in response to the odorant 3-Oct, and the right panel is in response to MCH (n = 10 flies). (e) Time course of Ca2+ activity induced by 3-Oct in the dendritic
terminals of KCs within the calyx region of 3d, 30d, and 30dSpd animals (GCamp3.0 response averaged across three odor exposures from ten flies). (f)
Maximum change in GCamp3.0 fluorescence (ΔF/F in %) in response to 3-Oct within dendritic claws of KCs of 3d, 30d, and 30dSpd flies (GCamp3.0
response averaged across three odor exposures from ten flies; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to
Bonferroni correction). (g) Time course of Ca2+ activity induced by MCH in the dendritic terminals of KCs of 3d, 30d, and 30dSpd animals (GCamp3.0
response averaged across three odor exposures from ten flies). (h) Maximum change in GCamp3.0 fluorescence (ΔF/F in %) in response to MCH within
dendritic claws of KCs of 3d, 30d, and 30dSpd flies (GCamp3.0 response averaged across three odor exposures from ten flies; Kruskal-Wallis test with PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 14 / 34 PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 Homeostasis at PN::KC synapses of aged flies. (a–c) Mushroom body calyx of 3d and 30d mb247::Dα7GFP flies and 30dSpd mb247:: Dα7GFP flies
immunostained for GFP-labeled Dα7 as well as BRP (corresponding single z-planes are shown). Scale bar: 10 μm. Arrows indicate the recurrent
presynapses of KCs that remain unopposed to acetylcholine-receptor rings within calycal neuropil; these KCs presynapses are spatially separated from the
sites of cholinergic input onto KCs. (d, e) Quantification of signal intensity of Dα7 (using anti-GFP) and BRP (using Nc82) in the calyx region normalized to
3d flies (n = 8–10 independent calyces; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). *
p < 0.05, ** p < 0.01, ns = not significant, p 0.05. (f–i) BRP immunostained within mushroom body calyx from adult brains of 3d and 10d flies expressing
UAS-dORK1 ΔC in the KCs compared to age-matched controls. (j) Quantification of signal intensity of BRP (using Nc82) in the calyx region normalized to
3d flies (n = 10–12 independent calyces; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (k)
Model showing the age-induced synaptic changes (in red). In the aged brain, the lowering of postsynaptic response with age, due to decrease in
membrane excitability or Ca2+ homeostasis, might steer retrograde changes in the architecture of AZs. As a result, the AZ characterized by T-bar in flies
enlarges in size, leading to higher release of SVs and causing aged synapses to function near the top of their presynaptic plasticity range, leaving little
room for additional synaptic strengthening, and possibly impeding further learning. Underlying data is shown in S1 Data. Fig 7. Homeostasis at PN::KC synapses of aged flies. (a–c) Mushroom body calyx of 3d and 30d mb247::D doi:10.1371/journal.pbio.1002563.g007 odors used for learning experiments. Importantly, spermidine feeding was able to “protect”
from both the functional and structural changes at aged AZs, arguing in favor of specific synap-
tic changes to be causally relevant for AMI. Indeed, installing 4xBRP not only increased the
size of BRP rings in young flies, similar to those found in aged animals, but also provoked
memory impairment in young flies. Notably, a reduction of BRP levels has previously been reported to affect ARM but not ASM
[50]. Here, we report that an increase in BRP levels (by changing the gene copy number of BRP
from two to four copies) severely affected ASM. Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Fig 7. Homeostasis at PN::KC synapses of aged flies. (a–c) Mushroom body calyx of 3d and 30d mb247::Dα7GFP flies and 30dSpd mb247:: Dα7GFP flies
immunostained for GFP-labeled Dα7 as well as BRP (corresponding single z-planes are shown). Scale bar: 10 μm. Arrows indicate the recurrent
presynapses of KCs that remain unopposed to acetylcholine-receptor rings within calycal neuropil; these KCs presynapses are spatially separated from the
sites of cholinergic input onto KCs. (d, e) Quantification of signal intensity of Dα7 (using anti-GFP) and BRP (using Nc82) in the calyx region normalized to
3d flies (n = 8–10 independent calyces; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). *
p < 0.05, ** p < 0.01, ns = not significant, p 0.05. (f–i) BRP immunostained within mushroom body calyx from adult brains of 3d and 10d flies expressing
UAS-dORK1 ΔC in the KCs compared to age-matched controls. (j) Quantification of signal intensity of BRP (using Nc82) in the calyx region normalized to
3d flies (n = 10–12 independent calyces; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). (k)
Model showing the age-induced synaptic changes (in red). In the aged brain, the lowering of postsynaptic response with age, due to decrease in
membrane excitability or Ca2+ homeostasis, might steer retrograde changes in the architecture of AZs. As a result, the AZ characterized by T-bar in flies
enlarges in size, leading to higher release of SVs and causing aged synapses to function near the top of their presynaptic plasticity range, leaving little
room for additional synaptic strengthening, and possibly impeding further learning. Underlying data is shown in S1 Data. m body calyx of 3d and 30d mb247::Dα7GFP flies and 30dSpd mb247:: Dα7GFP flies
gle z-planes are shown). Scale bar: 10 μm. Arrows indicate the recurrent
gs within calycal neuropil; these KCs presynapses are spatially separated from the
of Dα7 (using anti-GFP) and BRP (using Nc82) in the calyx region normalized to
multiple comparison test, p-values were subject to Bonferroni correction). *
ned within mushroom body calyx from adult brains of 3d and 10d flies expressing
tification of signal intensity of BRP (using Nc82) in the calyx region normalized to
multiple comparison test, p-values were subject to Bonferroni correction). (k)
ain, the lowering of postsynaptic response with age, due to decrease in Fig 7. Discussion The aging process, causing progressive deterioration of an organism, is subject to a complex
interplay of regulatory mechanisms. One of the primary aims of aging research is to use the
understanding of this process to delay or prevent age-related pathologies, including AMI. We
previously showed that restoration of polyamine levels by dietary supplementation with sper-
midine suppressed AMI in fruit flies [3], providing us with a protective paradigm to identify
candidate processes that might be functionally associated with AMI. As an insight towards the
synaptic basis of AMI, we describe an age-induced increase in the levels of core AZ proteins,
BRP, and RBP and of the functionally critical release factor Unc13, together with a shift
towards an enlargement of AZ scaffolds within the olfactory system. In addition, based on
SynpH experiments, we observeda substantial increase in the release of SVs at aged synapses
(PN-to-KC and KC-to-mushroom body output neuron [MBON] synapses) in response to PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 15 / 34 These findings suggest that the two PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 16 / 34 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes complementary forms of memory (ARM and ASM) might rely on the recruitment of distinct
presynaptic “functional modules.” The loss of brp has been shown to severely reduce release
function in response to single low frequency, but not in response to high-frequencystimulation
[27], indicating that SV release at low-frequency stimulation might be particularly relevant for
forming ARM, a memory component that develops gradually after training. On the other
hand, mobilization of the SVs during high frequency stimulation has been suggested to be criti-
cal for formation of ASM [50], a memory component that predominates early memory and
decays with age. Thus, the increase in the size of the AZ scaffolds might potentially contribute
directly to AMI by interfering with mechanisms facilitating SV availability in the course of
forming ASM. Though the exact mechanisms underlying age-induced synaptic changes remain to be fully
worked out, a reduction in autophagy-mediated protein degradation might well be involved
[51–53]. Autophagy is a cellular digestion pathway that involves the sequestration of cyto-
plasmic components within a double-membrane vesicle called autophagosome, which fuses
with lysosomes (autolysosomes) to degrade autophagic cargo by acidic hydrolases [52]. Inter-
estingly, spermidine was shown to induce autophagy in several model systems, including
rodent tissues and cultured human cells [51,54,55]. Moreover, amelioration of a-synuclein neu-
rotoxicity due to spermidine administration was accompanied by autophagy induction [56]. Of
note, we also found that spermidine feeding prevented accumulation of poly-ubiquitinated
proteins by plausibly halting normally occurringage-induced decline of autophagic clearance
[3,57]. The gene atg7 encodes an E1-like enzyme required for activation of both Atg8 and
Atg12, a step critical for the completion of the autophagic pathway [53]. We found that atg7-
mutant flies (atg7-/-) exhibit reduced memory scores at a young age (3d), which declined fur-
ther with age (20-d of age or 20d) [3]. Concurrently, spermidine-mediatedprotection from
memory impairment was eliminated in atg7−/−flies (for both 3d- and 20d-flies) [3,57]. There-
fore, we wondered whether the decrease in the autophagic pathway might, per se, provoke
increase of AZ scaffold components. When staining for BRP in atg7-mutant brains (atg7-/-), we
found a brain-wide increase in levels of BRP (for both BRPNc82 and BRPN-term antibodies), and
spermidine feeding was unable to prevent this age-related increase (S19 Fig). PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes gyrus as well as the CA1 area of the brain [8,65,66]. Additionally, the “unitary” intracellular-
evoked amplitude elicited by minimal stimulation protocols has been found to be greater in old
than in young rodents [67], suggesting that the “surviving synapses” are stronger [68]. It is of
note that the induction threshold for long-term potentiation, considered to be a synaptic corre-
late of learning, has been reported to increase in aged rodents [10]. Similarly, an age-related
increase in the amplitude of endplate potentials evoked has been reported at mouse neuromus-
cular synapses [69,70]. By contrast, a study at neuromuscular junctions of C. elegans revealed
that aged motor neurons undergo a progressive reduction in synaptic transmission [71]. In
flies, however, an age-related increase in the amplitude of the excitatory postsynaptic potential
at adult neuromuscular junctions has been reported recently; this increase was suggested to
tune the response of the homeostatic signaling system and establish a new homeostatic set
point [72]. Collectively, these findings suggest that the dynamic range of synaptic plasticity
may change with advancing age and, thus, contribute to AMI. y
g
g g
Why would an increase in the odor-evoked SV release and ultrastructural size of AZ scaf-
folds impair the efficacy of forming new memories? Synapses appear to display a “finite ceil-
ing and floor” that define a synaptic operating range [73]. In rodents, the formation of new
memories seems to drive synaptic strength to the upper limit of a fixed operating range,
thereby creating an imbalance [73]. As a result, if the synapses are not returned to the mid-
point of the synaptic modification range, then additional strengthening required for new
memory formation might be blocked, and the system is driven to employ homeostatic com-
pensatory mechanisms to balance the change [74]. In our experiments, we found dendritic
Ca2+ signals and postsynaptic receptor levels to remain largely unchanged with age, suggest-
ing the existence of homeostatic mechanisms that might allow the up-scaling of presynaptic
release to be compensated by lowering the postsynaptic response to a given amount of neuro-
transmitter released. On the other hand, this upscaling of presynaptic structure and function
might also be a homeostatic response to a reduction in postsynaptic excitability or Ca2+
homeostasis, steering retrograde enlargement of AZ scaffold and higher release of SVs (Fig
7K). PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 The finding that
spermidine feeding in atg7−/−flies neither blocked the increase in BRP levels (S19 Fig) nor sup-
pressed memory impairment [3] suggests that the integrity of the autophagic system is crucial
for the spermidine-mediatedprotection from age-associated increase in AZ scaffold compo-
nents. Spermidine effects were recently shown to involve widespread changes of both nuclear
and cytosolic protein acetylation [58,59]. In primary neurons, autophagosomes have previously been observedto form at the distal
end of the axon, indicating compartmentalization and spatial regulation of autophagosome
biogenesis [60,61]. More recently, autophagosomes were demonstrated to form directly near
synapses and were found to be required for presynaptic assembly at developing synaptic termi-
nals of Caenorhabditis elegans [62]. Moreover, the crucial release factor Unc13 was found to
accumulate under conditions of defective endosomal microautophagy (a specializedform of
autophagy) at developing neuromuscular synapses of Drosophila, suggesting Unc13 to be a
substrate of this form of autophagy [63]. Interestingly, we have shown recently that the synap-
tic levels of Unc13-A isoform scale tightly with the levels of the BRP/RBP scaffold [64]. Thus, it
is conceivable that some of the AZ proteins, whose levels increase with age (BRP/RBP/Unc13),
might be direct substrates of “pre-synaptic autophagy,” and that spermidine feeding might aug-
ment effective autophagic degradation of these proteins at aging synapses. We also observeda moderate decrease in synapse numbers in aging Drosophila brains, a
phenotype that was unaffected by spermidine feeding. Our data compare favorably with studies
in mammals. For example, loss of synapses in aged rodents has been reported in the dentate PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 17 / 34 Behavioral Assays Behavioral experiments were performedin dim red light at 25°C and 80% relative humidity with
3-Oct (1:150 dilution in mineral oil presented in a 14 mm cup) and MCH (1:100 dilution in min-
eral oil presented in a 14 mm cup) servingas olfactorycues, and 120V AC current servingas a
behavioral reinforcer. Standard single-cycle olfactoryassociative memory was performedas previ-
ously described[3,4,46,84,85], with minor modifications.Briefly, about 60–80 flies received one
training session, during which they were exposedsequentially to one odor (conditioned stimulus,
CS+; 3-Oct or MCH) paired with electricshock (unconditionedstimulus, US) and then to a second
odor (CS−; MCH or 3-Oct) without US for 60 s with 30 s rest interval betweeneach odor presenta-
tion. During testing, the flies were exposedsimultaneously to the CS+ and CS−in a T-maze for 30 s. The conditioned odor avoidance was tested immediately after training for STM (memory
tested immediately after odor conditioning). Subsequently, flies were trapped in either T-maze
arm, anesthetized, and counted. From this distribution, a performance index was calculated as
the number of flies avoiding the shocked odor minus the number avoiding the nonshocked odor
divided by the total number of flies and, finally, timed by 100. A 50:50 distribution (no learning)
yielded a PI of zero, and a 0:100 distribution away from the CS+ yielded a PI of 100. A final per-
formance index was calculated by the average of both reciprocal indices for the two odors. For ITM, flies were trained as describedabove, but tested 3 h after training. As a component
of ITM, ARM was separated from ASM by cold-amnestic treatment, during which the trained
flies were anesthetized 90 s on ice at 30 min before testing. In the end, ASM was calculated by
subtracting the performance index of ARM from that of ITM for each training session on the
same day, respectively. Animal Rearing and Fly Strains All fly strains were reared under standard laboratory conditions [79] at 25°C and 70%
humidity, with constant 12:12 h light/dark cycle. Flies from an isogenizedw1118 strain were
used as the wild-type control for all experiments. Flies carrying P(acman) cacGFP, P(acman)
brp83GFP and P(acman) brp83 [28] and mb247::Dα7GFP [45] were describedpreviously. The
generation of UAS-homer-GCaMP3.0 flies are describedelsewhere [15]. Briefly, cDNA of dho-
mer was amplified from w 1118 flies and inserted with a C-terminal linked GCaMP336 into
pUAST. Both UAS-GCamp3.0 (on the 3rd chromosome) [80] and UAS-SynpH [81] were
kindly provided by Gero Miesenböck.Atg7d14 and Atg7d77 flies were kind gifts from Thomas
Neufeld [53]. In addition, mb247-Gal4 [82] and gh146-Gal4 [83] were used. As previously described[3], the fly food was prepared according to Bloomington media rec-
ipe (www.flystocks.bio.indiana.edu/Fly_Work/media-recipes/media-recipes.htm) with minor
modification,which was called Spd−or normal food. Spermidine (Sigma Aldrich) was prepared
as a 2 M stock solution in sterile distilled water, aliquoted in single-use portions and stored at
−20°C. After food had cooled down to 40°C, Spermidine was added to normal food to a final
concentration of 1 mM or 5 mM Spd, and called Spd1mM+ or Spd5mM+, respectively. Parental
flies mated on either Spd−or Spd5mM+ food for all experiments, and their progeny were allowed
to develop on the respective food. Flies used in all experiments were F1 progeny. The flies were
collected once a day for aging, as a results-specific age indicated is day ± 24 h. In fact, the influx of postsynaptic Ca2+ through glutamate receptors at the peripheral
glutamatergic synapses of Drosophila has been reported to control presynaptic assembly by
retrograde signalling [47,48,75]. While the exact nature of homeostatic controls connecting
pre- with postsynaptic neurons in the olfactory system remains to be resolved, changes in
plasma membrane excitability, a change in postsynaptic neurotransmitter sensitivity, or an
increase in inhibitory GABAergic drive are obvious candidate processes. Taken together, we
propose these synaptic changes steer the presynaptic AZs to function towards the upper limit
of their operational range, making these synapses unable to react adequately to conditioning
stimuli and provoke potentiation or depression of synapses in order to encode memory for-
mation [11,12,76]. Sleep is widely believed to be critical for formation and consolidation of memories [77]. In
sleep-deprived animals, neuronal circuits would exceed available space and/or saturate, thereby
affecting an individual’s ability to learn [77]. Importantly, sleep deprivation has also been asso-
ciated with widespread increases of BRP levels in the Drosophila brain [78]. Notably, we also
observeda brain-wide increase in BRP levels in aged brains. It is tempting to speculate that
both sleep deprivation and aging change the operational range over several synaptic relays and
thereby affect memory formation—a topic that deserves further investigation in future. Taken together, our data show that upscaling of presynaptic structure and function contrib-
ute to an AMI in Drosophila. Furthermore, and restoration of polyamine levels prevents these
age-associated alterations as well as AMI. Thus, spermidine feeding provides a unique opportu-
nity to further the molecular and functional dissection of the mechanisms underlying AMI
with the ultimate goal of restoring memory function in older humans. 18 / 34 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Staining Protocol Whole-mount adult brains. Adult brains were dissected in HL3 (which contains 70 mM
NaCl, 5 mM KCl, 20 mM MgCl2, 10 mM NaHCO3, 5 mM Trehalose, 115 mM Sucrose, 5 mM 19 / 34 PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Hepes, added to 500 ml H2O; pH adjusted to 7.2) on ice and immediately fixed in cold 4%
Paraformaldehyde (v/v) for 20 min at 20–30°C. After fixation, the brains were incubated in 1%
PBT (phosphate-buffered saline (PBS) containing 1% Triton X-100; v/v) for 20 min and then
preincubated in 0.3% PBT (PBS containing 0.3% Triton X-100) with 10% normal goat serum
(NGS; v/v) at 20–30°C. For primary antibody treatment, samples were incubated in 0.3% PBT
containing 5% NGS and the primary antibodies for 48 h at 20–30°C. After primary antibody
incubation, brains were washed in 0.3% PBT, four times for 30 min at 20–30°C, and then over-
night at 4°C. All samples were then incubated in 0.3% PBT with 5% NGS containing the sec-
ondary antibodies for 24 h at 20–30°C. Brains were washed again four times for 30 min at 20–
30°C, then overnight at 4°C. Brains were finally mounted in Vectashield overnight before con-
focal scanning (Vector Laboratories). Image acquisition, processing, and analysis. The images were acquired, processed, and
analyzed as previously described[46,85], with minor modifications. Briefly, conventional con-
focal images were acquired at room temperature with a Leica Microsystems TCS SP8 confocal
microscope using a 63x 1.4 NA oil objective for detailed scans and a 20x 0.7 NA oil objective
for overview scans. All images were acquired using Leica LCS AF software. Lateral pixel size
was approximately 300 nm for overview scans, approximately 100 nm for detailed scans. Typi-
cally, 1,024 x 1,024 images were scanned at 400 Hz using 4x line averaging. Images of calyces
for cryostat sections were acquired at room temperature with a Leica Microsystems TCS SP5
CW STED microscope in confocal mode using a 100x 1.4 NA oil objective. STED images were
acquired on a Leica TCS STED CW. Images were deconvolved using the built-in deconvolution
algorithms of the Leica LAS-AF software. The PSF was generated by using a 2-D Lorentz func-
tion with the full-width half-maximum set to 60 nm (as calculated on the image using the Wie-
ner filter algorithm; regulation parameter: 0.05). Staining Protocol In order to analyze the brain scans, the signal intensity within a neuropil of interest (whole
central brain, or CB; antennal lobes, or AL; and calyx) was determined using Amira software
(Amira 5.3.3, FEI Visualization Sciences Group, Oregon, US). The region of interest within the
3-D image stack was masked using the tool Segmentation Editor by interpolating manual selec-
tions between slices. Average intensity values were calculated for all pixels within each mask
for each channel separately. For STED analysis, deconvolved BRP spots (stained with monoclonal Nc82 antiboby) were
processed in ImageJ. The diameters of planar oriented BRP rings were measured using the line
tool of ImageJ. The distance from intensity maximum to intensity maximum was acquired in
the plot window of individual hand-drawn lines and transferred to Microsoft Excel. PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Following embedding,sections of 60 nm, each, were cut using a Leica Ultracut E ultramicro-
tome equipped with a 2 mm diamond knife. Sections were collected on 100 mesh copper grids
(Plano GmbH, Germany) coated with 0.1% Pioloform resin. Contrast was enhanced by placing
the grids in 2% uranyl acetate for 30 min, followed by washing with water three times and,
then, incubation in lead citrate for 2 min. The grids were washed three times with water and
dried. Images were acquired fully automatically on a FEI Tecnai Spirit transmission electron
microscope operated at 120 kV equipped with a FEI 2K Eagle CCD camera using Leginon [86]. Regions of interest were first selected at 560x nominal magnification and then successively
imaged at 4,400x and 26,000x nominal magnification, respectively. Series of more than 1,000
TEM images were then stitched to a single montage covering nearly the full calyx region using
the TrakEM2 software [87] implemented in Fiji [88]. Immuno-EM Brains were dissected in HL3 solution and fixed for 20 min at room temperature with 4%
paraformaldehyde and 0.2% Glutaraldehyde in a buffer containing 50 mM Sodium Cacody-
late and 50 mM NaCl at pH 7.5. Afterwards, brains were washed twice in the buffer and
dehydrated through a series of increasing alcohol concentrations. Samples were embedded in
London-Resin (LR)-Gold resin by incubating them in Ethanol/LR-Gold 1:1 solution over-
night at 4°C, followed by Ethanol/LR-Gold 1:5 solution for 4 h at room temperature. Thereaf-
ter, the samples were washed first with LR-Gold/0.2% Benzil overnight, a second time for 4
h, and then again overnight. Finally, the brains were placed in BEEM capsules covered with
LR-Gold/0.2% Benzil resin and placed under a UV lamp at 4°C for 5 d to allow for polymeri-
zation of the resin. Following embedding, sections 70–80 nm, each, were cut using a Leica Ultracut E ultrami-
crotome equipped with a 2 mm diamond knife. Sections were collected on 100 mesh nickel
grids (Plano GmbH, Germany) coated with 0.1% Pioloform resin and transferred to a buffer
solution (20 mM Tris-HCl, 0.9% NaCl, pH 8.0). Prior to staining, sections were blocked for 10
min with 0.04% BSA in buffer. Sections were incubated with the primary antibody (guinea pig-
anti RBPSH3II+III and rabbit-anti BRPlast200, 1:500 dilution) in blocking solution overnight at
4°C. After washing four times in buffer, the sections were incubated in buffer containing the
secondaryantibody (goat anti-guinea pig 10 nm colloidal gold, goat anti-rabbit 5 nm colloidal
gold British Biocell, 1:100) for 2–3 h at room temperature. Finally, the sections were washed
four times in buffer and three times in distilled water. Contrast was enhanced by placing the
grids in 2% uranyl acetate for 30 min, followed by washing three times with water and, after-
wards, incubation in lead citrate for 2 min. Afterwards, the grids were washed three times with
water and dried. Images were acquired on a FEI Tecnai Spirit, 120 kV transmission electron
microscope equipped with a FEI 2K Eagle CCD camera. PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 EM Conventional EM. Brains were dissected in HL3 solution and fixed for 20 min at room
temperature with 4% paraformaldehyde and 0.5% glutaraldehyde in a PBS. Subsequently, the
brains were incubated overnight at 4°C with 2% glutaraldehyde in buffer containing 0.1 M
sodium cacodylate at pH 7.2. Brains were then washed three times in cacodylate buffer for 10
min at 20–30°C. Afterwards, the brains were incubated with 1% Osmium tetroxide and 0.8%
KFeCn (in 0.1 M cacodylate buffer) for 90 min on ice. Brains were then washed with cacodylate
buffer for 10 min on ice and then three quick washes with distilled water. The brain were
stained with 1% Uranylacetate (w/v) for 90 min on ice and dehydrated through a series of
increasing alcohol concentrations. Samples were embedded in EPON resin by incubation
sequentially in ethanol/EPON 1:1 solution for 45 min and 90 min at 20–30°C, then in pure
EPON overnight at 15–20°C. Thereafter, the resin was changed once and brains were embed-
ded in a single block at 60°C to allow for polymerization of the resin. 20 / 34 Optophysiological Imaging of GCaMP3.0, Homer-GCamp3.0 and
SynaptopHlourin (SynpH) Female 3d or 30d flies were briefly anesthetized on ice and immobilized in a small chamber
under thin sticky tape. A small window was cut through the sticky tape and the cuticle of the
head capsule using a splint of a razor blade. Trachea were carefully removed and the brain was
covered with Ringer’s solution (5 mM HEPES, 130 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM
CaCl2, pH = 7.3). Imaging was performed using an LSM 7 MP two-photon microscope (Carl
Zeiss) equipped with a mode-lockedTi-sapphire Chameleon Vision II laser (Coherent), a 500–
550 nm bandpass filter, and a Plan-Apochromat 20×1.0 NA water-immersion objective (Carl
Zeiss). A custom-built device to supply odorous air with a constant flow rate of 1 ml/s directly
to the fly’s antennae was attached to the microscope. Odor stimulation (MCH or 3-Oct, diluted
1:100 or 1:150, respectively, in mineral oil or pure mineral oil) was controlled using a custom-
written LABVIEW program (National instruments). GCamp3.0, homer-GCaMP, and SynpH
were excited at 920 nm and fluorescence monitored at an image acquisition rate of 5 Hz. The
odorants were presented with a 20 s break between stimulation, and each fly was exposed to
five to six repetitive experiments. The images were aligned to reduce small shifts in the X–Y direction using a custom written
ImageJ plugin. The mean intensity within the region of interest of five images before stimulus
onset was used as baseline fluorescence (F0). The difference in intensity (ΔF) was calculated by
subtracting F0 from the fluorescence intensity value within the ROI of each image (Fi) and, sub-
sequently, divided by the baseline fluorescence. ΔF/F0 values of three or more repetitions were
averaged for each fly. Odor-induced fluorescence changes of SynpH were considered in calycal PN boutons show-
ing ΔF/F0 values more than twice the standard deviation of the baseline fluorescence. The bou-
tons with the five highest odor-induced ΔF/F0 amplitudes were considered for further analysis. We found SynpH to exhibit rapid photo-bleaching, therefore, bleaching correction was per-
formed on its ΔF/F0 values. For this, first, ΔF/F0 values from the onset of the stimulus until the
decay of the signal were removed and then the best least square fit was obtained using the
remaining ΔF/F0 values (second order polynomial decay function). Subsequently, this decay
function was subtracted from the entire original ΔF/F0 curve, and the new modifieddata are
the bleaching corrected data. Antibodies Used The following primary antibodies were used: MαBRPNc82 (ref. 9, 10; 1:100), GPαBRPN-term
(1:800) [25,27], RbαRBPC-term (1:800) [29], MαSynapsin (1:20) [89], RatαSyb (1:100) [90],
RbαSynaptotagmin-1C-term (1:500) [91], RbαGFP (Molecular Probes; 1:500), RbαDrep2C-term
(1:500) [46], RbαUnc13C-term (1:500) [63], RbαBRPlast200 (1:500), and GPαRBPSH3II+III (1:500). The following secondary antibodies were used: GαM Alexa 488 (Molecular Probes; 1:400),
GαR Alexa 488 (Molecular Probes; 1:500), GαGP Alexa 555 (Invitrogen; 1:800), GαM Cy3
(Dianova; 1:500), and GαR Cy5 (Invitrogen; 1:400). The following primary antibodies were used: MαBRPNc82 (ref. 9, 10; 1:100), GPαBRPN-term
(1:800) [25,27], RbαRBPC-term (1:800) [29], MαSynapsin (1:20) [89], RatαSyb (1:100) [90],
RbαSynaptotagmin-1C-term (1:500) [91], RbαGFP (Molecular Probes; 1:500), RbαDrep2C-term
(1:500) [46], RbαUnc13C-term (1:500) [63], RbαBRPlast200 (1:500), and GPαRBPSH3II+III (1:500). The following secondary antibodies were used: GαM Alexa 488 (Molecular Probes; 1:400) The following secondary antibodies were used: GαM Alexa 488 (Molecular Probes; 1:400),
GαR Alexa 488 (Molecular Probes; 1:500), GαGP Alexa 555 (Invitrogen; 1:800), GαM Cy3
(Dianova; 1:500), and GαR Cy5 (Invitrogen; 1:400). For Immunoprecipitation, BRPlast200 and IgG were used at final amount of 50 ug per 500 ul. For western blots, secondary antibody was used at a dilution 1:1,000. 21 / 34 PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 D. Melanogaster Head Extract and Immunoprecipitation For the identification of (de)acetylated residues of BRP, we did “conventional” protein extrac-
tions from Drosophila heads combined with BRP immunoprecipitations. The protocol could
be divided into four main sections. 1) Precoupling of antibodies to matrix (50 ug antibody per
reaction): 3 LoBind cups (2 ml; Eppendorf)containing Affiprep Protein A matrix were pre-
pared: 1 X 30ul for specific antibody, 1 X 30ul for IgG control, 1 X 60 ul for head extract pre-
clearing. The cups were washed 3 X with 500 ul H-buffer (25 mM HEPES pH 8.3 (NaOH), 150
mM NaCl, 1 mM MgCl2, 1 mM EGTA, 10% Glycerol) by inverting several times, followed by
centrifugation 1,000 gmax (3,000 rpm) for 1 min. 500 ul H-buffer (+ BRPlast200 or IgG) per cou-
pling was prepared. 500 ul antibody solution (= 50 ug IgG) was added per 30 ul washed Protein
A-beads. Beads were incubated with antibody solution for 2 h on the wheel at 4°C. The Affi-
prep beads-antibody were collected by centrifugation for 3 min at 1,000 gmax. Affiprep beads-
Antibody were washed 3 X by inverting tubes and 3 X for 10 min on wheel with IP buffer. 2)
Homogenizing fly heads from stored fly heads [–80°C]. Fly heads were transferred with a clean
spatula into 1 ml glass homogenizer. For 300 ul frozen fly heads, 300 ul Homogenization buffer
(without detergent) was added, and heads were sheared at 900 rpm using an electronic over-
head stirrer. Samples were collected in LoBind cups (2 ml; Eppendorf).2 X 300 ul was added to
rinse pestle and homogenizer (Total volume in cups ~1,100–1,200 ul). Sodium-deoxycholic
Acid (DOC) was added to a final concentration of 0.4% (28 ul of 10% stock spiked into homog-
enate (1:25 v/v)). Triton X-100 was added to a final concentration of 1% (35 ul of 20% stock
spiked into homogenate (1:20 v/v)). The samples (Homogenate) were incubated for 60 min at
4°C at level 8 (slow) on wheel. 20 ul of homogenate was stored for SDS-PAGE analysis for
monitoring antigen during extraction/pull-downprocedure. Homogenate (H) was centrifuged
for 15 min at 17,000 gmax. Supernatant (yellow in color) was transferred to a fresh LoBind cup. Centrifugation of S1 was repeated 4X to get rid of fat and remaining head debris. After final
centrifugation step, remaining supernatant was diluted 1:1 with H-buffer (without detergent). Optophysiological Imaging of GCaMP3.0, Homer-GCamp3.0 and
SynaptopHlourin (SynpH) Fluorescence emission of cytosolic GCamp was determined within specific boutons in the
calyx that respond to the odor stimulus, and only the boutons showing ΔF/F0 values of more
than 100% in four to five stimulations were averaged for each fly and considered for final anal-
ysis. Fluorescence changes of mb247-Gal4; UAS-homer-GCamp flies were averaged over the
five most responsive microglomerularstructures, as anatomically defined by basal
fluorescence. False color-coded images were obtained by subtracting the image just before stimulus onset
from the image at the maximum of the intensity difference (i.e., at 2 s after odor onset) and
divided by the baseline fluorescence. The KCl experiments were performed using a fluorescence microscope (Zeiss) equipped
with a xenon lamp (Lambda DG-4, Sutter Instrument), a 14-bit CCD camera (Coolsnap HQ,
Photometrics) and a 20 × NA = 1 water-immersion objective. Images were acquired at 5 Hz
using Metafluor (Visitron Systems). After recording some initial frames, KCl was added to the
Ringer’s solution covering the fly brain (final concentration 0.05 M). Fluorescence changes
were determined in a circular region covering the calyx (d = 20 μm), and background fluores-
cence determined outside the calyx was subtracted. 22 / 34 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes D. Melanogaster Head Extract and Immunoprecipitation Total volume of Input was ~1,400 ul and of following composition: 25 mM Hepes pH 8.05
(NaOH), 150 mM NaCl, 0.5 mM MgCl2, 0.5 mM EGTA, 5% Glycerol, 0.2% DOC, 0,55% Tri-
ton X-100. 3) Preclearing of fly head extract on Protein A-IgG beads: Diluted fly head extract
was applied to preclearing beads and incubated for 60 min at 4°C while rotating on wheel. Pre-
cleared extract was separated by centrifugation for 3 min at 1,000 gmax. Supernatant (IP input)
was recovered. 4) Precipitation: Precleared extract (IP input) was applied to antibody-bead
matrix (600 ul to specific Antibody-beads,600 ul to control IgGs) and antibody–antigen bind-
ing was performed overnight at 4°C. Immunoprecipitates were collected by centrifugation at
1,000 gmax for 4 min at 4°C. Affiprep Beads-Antibody-Antigen were washed 3 X with a quick
rinse followed by 2 X 20 min with 1 mL IP Buffer (H-buffer + 0.5% Triton-X 100 + 0.2% Na-
DOC).Affiprep Beads-Antibody-Antigenwere resuspended in 1,000 ul IP buffer and trans-
ferred to a clean LoBind cup (2 ml; Eppendorf).Affiprep Beads-Antibody-Antigenwere centri-
fuged, and most of the supernatant was removed (without removing beads). 4.) Elution: For
elution, 100 ul of 2X Laemmeli Buffer was added to Affiprep Beads-Antibody-Antigenand
heated for 10 min at 95°C, 600 rpm, followed by centrifugation for 5 min at 1,000 gmax. Super-
natant (IP eluate) was transferred into a fresh LoBind Cup (2 ml; Eppendorf).Immunoprecipi-
tation was verified with western blot. Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes separated on 4%–12% gradient SDS-PAGE (NuPAGE, Invitrogen). The gel lanes were cut into
ten equal slices, the proteins were in-gel digested with trypsin (Promega) [92], and the resulting
peptide mixtures were processed on STAGE tips [93] and analyzed by LC-MS/MS. Mass spectrometric(MS) measurements were performed on an LTQ Orbitrap XL mass
spectrometer (Thermo Fisher Scientific) coupled to an Agilent 1200 nanoflow–HPLC (Agilent
Technologies GmbH, Waldbronn, Germany) [94]. HPLC–column tips (fused silica) with
75 μm inner diameter (New Objective, Woburn, MA, USA) were self-packed with Reprosil–
Pur 120 ODS–3 (Dr. Maisch, Ammerbuch, Germany) to a length of 20 cm. Samples were
applied directly onto the column without a precolumn. A gradient of A (0.5% acetic acid (high
purity, LGC Promochem, Wesel, Germany) in water and B (0.5% acetic acid in 80% acetonitrile
(LC–MS grade, Wako, Germany) in water) with increasing organic proportion was used for
peptide separation (loading of sample with 2% B; separation ramp: from 10%–30% B within 80
min). The flow rate was 250 nl/min and for sample application 500 nl/min. The mass spec-
trometer was operated in the data-dependent mode and switched automatically between MS
(maximum of 1 x 106 ions) and MS/MS. Each MS scan was followed by a maximum of five
MS/MS scans in the linear ion trap using normalized collision energy of 35% and a target value
of 5,000. Parent ions with a charge state from z = 1 and unassigned charge states were excluded
for fragmentation. The mass range for MS was m/z = 370–2,000. The resolution was set to
60,000. MS parameters were as follows: spray voltage 2.3 kV; no sheath and auxiliary gas flow;
ion transfer tube temperature 125°C. Identification of proteins and protein ratio assignment using MaxQuant. The MS raw
data files were uploaded into the MaxQuant software version 1.4.1.2 [95] for peak detection,
generation of peak lists of mass error corrected peptides, and for database searches. A full-
length UniProt D. melanogaster database additionally containing common contaminants such
as keratins and enzymes used for in-gel digestion (based on UniProt Drosophila FASTA ver-
sion December 2013) was used as reference. Carbamidomethylcysteinewas set as fixed modifi-
cation, methionine oxidation, protein amino-terminal acetylation, and lysine acetylation were
set as variable modifications, and label-free was chosen as quantitation mode. Three missed
cleavages were allowed, enzyme specificity was trypsin/P, and the MS/MS tolerance was set to
0.5 Da. The average mass precision of identified peptides was in general less than 1 ppm after
recalibration. Peptide lists were further used by MaxQuant to identify and relatively quantify
proteins using the following parameters: peptide and protein false discovery rates, based on a
forward-reverse database, were set to 0.01, minimum peptide length was set to seven, mini-
mum number of peptides for identification and quantitation of proteins was set to two, of
which one must be unique, minimum ratio count was set to two, and identified proteins were
requantified. The “match-between-run” option (2 min) was used. To analyze acetylation status of BRP, the data was processed using the freely available Per-
seus software (Cox et al, 2011). For each IP, average acetylation intensity was calculated out of
intensities of all sites identified in each replicate normalized to the respective protein intensity
of BRP. Sample Preparation and Mass Spectrometry For identification of (de)acetylated lysine residues in BRP, IP eluate was heated in SDS-PAGE
loading buffer, reduced with 1 mM DTT (Sigma-Aldrich) for 5 min at 95°C and alkylated
using 5.5 mM iodoacetamide(Sigma-Aldrich) for 30 min at 20°C. The protein mixtures were 23 / 34 PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 S1 Fig. Aversive olfactorymemory as a function of stimulation intensity in aged flies. STM index plotted against shock number as experiencedduring training sessions with 120 V
DC in 3d (light blue bars) and 30d (dark blue bars) wild-type w1118 flies (n = 6–8; Kruskal-Wal-
lis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correction). p < 0.05, p < 0.01, p < 0.001, ns = not significant, p 0.05. Underlying data is shown
in S1 Data. S2 Fig. Basal fluorescenceof different sensors (within the calyx neuropil) used for optoge-
netic analysis. (a) Quantification of levels of GCamp3.0 in the PN terminals within the calyx
region normalized to 3d flies (n = 6–7 independent calyces; Kruskal-Wallis test). (b) Quantifi-
cation of levels of SynaptopHlourin (SynpH) in the PN terminals within the calyx region nor-
malized to 3d flies (n = 7–12 independent calyces; Kruskal-Wallis test). (c) Quantification of
levels of Homer-GCamp3.0 in the dendritic claws of KCs within the calyx region normalized to
3d flies (n = 10–12 independent calyces; Kruskal-Wallis test). ns = not significant, p 0.05. Underlying data is shown in S1 Data. (T
) S3 Fig. Ca2+-imaging in the PNs within the calyx region in response to odors in aged flies. (a) Expression of GCaMP3.0 in the PNs and imaged within the calyx neuropil. (b, c) False
color-coded image of Ca2+ activity within the presynaptic terminals of PNs in response to
3-Oct and MCH shown in (a). Warm colors indicate high levels, while cold colors low levels or
no Ca2+ activity. The numbers indicate changes in fluorescence (ΔF/F in %). Scale bar: 10 μm. (d) Odor-evoked Ca2+ activity, measured by changes in fluorescence of Gamp3.0, of an individ-
ual fly over time, shown as false colors in the presynaptic terminal of PNs in calyx region, in
response to the odorants 3-Oct and MCH. (GCamp3.0 response averaged across three odor
exposures from 6–7 animals). (e) Time course of Ca2+ activity induced by 3-Oct (averaged
across three odor exposure) in the presynaptic terminals of PNs within calyx neuropil of 3d
and 30d, together with 30dSpd flies (GCamp3.0 response averaged across three odor exposures
from 6–7 animals). (f)Maximum change in GCamp3.0 fluorescence (ΔF/F in %) in response to
3-Oct in PN boutons of 3d and 30d as well as 30dSpd flies (GCamp3.0 response averaged across
three odor exposures from 6–7 animals; Kruskal-Wallis test). Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes order to avoid being biased by outliers, which are represented by solid circles. All p-values that
are reported have been subject to Bonferroni correction for the number of comparisons. Addi-
tional relevant information is indicated in the figure legends. The data for the behavioral stud-
ies were collected with the investigator blind to the genotypes, treatment, and age of genotypes. There was no blinding in the other experiments. The data were collected and processed side by
side in randomized order for all experiments. In order to analyze the difference in Homer-
GCaMP3.0 responses (Fig 6 and S17 Fig), two-sided Kolmogorov-Smirnov tests were con-
ducted in R, and the GCaMP3 responses only during odor stimulation and were compared. Supporting Information S1 Data. Excel spreadsheetcontaining, in separate sheets,numericaldata underlying panels
1e–1h, 2d, 2h, 2l–2n, 3e–3g, 4d, 4h, 4m–4n, 5e–5f, 5k, 5m–5n, 6e–6h, 7d–7e, 7j, S1, S2a–
S2c, S3e–S3h, S4a–S4f, S5b–S5c, S6a–S6d, S7d, S8e–S8g, S9d, S10d–S10e, S11d–S11f,
S14e–S14g, S16b–S16c, S17, S18d–S18f, S19e–S19f, S1 Table. (XLSX) PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 Statistics Data were analyzed in R v3.1.2 using the additional CRAN package dunn.test v1.2.2. Asterisks
are used in the figures to denote significance: p < 0.05, p < 0.01, p < 0.001, ns = not sig-
nificant. Nonparametric methods were used because of the small sample sizes and because of
failure of tests for normality for parts of the data (Shapiro-Wilk test). Unless indicated other-
wise, the different groups in each figure were first compared using the Kruskal-Wallis test, fol-
lowed by Dunn’s test for posthoc multiple comparisons. Nonparametric tests were used in 24 / 34 PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 S1 Fig. Aversive olfactorymemory as a function of stimulation intensity in aged flies. (g) Time course of Ca2+ activity
induced by MCH (averaged across three odor exposure) in the presynaptic terminals of PNs
within calyx neuropil of 3d and 30d flies, together with 30dSpd flies (GCamp3.0 response PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 25 / 34 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes averaged across three odor exposures from 6–7 animals). (h) Maximum change in GCamp3.0
fluorescence (ΔF/F in %) in response to MCH in PN boutons of 3d, 30d, and 30dSpd flies
(GCamp3.0 response averaged across three odor exposures from 6–7 animals; Kruskal-Wallis
test). The grey bars indicate the duration of the odor stimuli. ns = not significant, p 0.05. Underlying data is shown in S1 Data. (TIF) S4 Fig. Decay time constant (τ) of odor-evoked SynpH response. (a) Overall decay time con-
stant (τ) of SynpH signal in response to 3-Oct (3-Octonal). (b) Fast component of decay time
constant (τ) of SynpH signal in response to 3-Oct. (c) Slow component of decay time constant
(τ) of SynpH signal in response to 3-Oct. (d) Overall decay time constant (τ) of SynpH signal
in response to MCH. (e) Fast component of decay time constant (τ) of SynpH signal in
response to MCH. (f)Slow component of decay time constant (τ) of SynpH signal in response
to MCH. (n = 6–7 flies; Kruskal-Wallis test). ns = not significant, p 0.05. Underlying data is
shown in S1 Data. (TIF) S5 Fig. KCl-induced changes in fluorescenceof SynpH within the calyx neuropil. (a) SynpH
expressed in the PNs and imaged within the calyx region. The two rings indicate the region of
interest (calyx neuropil) and background region used for analysis. Scale bar: 50 μm. (b) KCl-
induced release of SVs, measured by changes in fluorescence (ΔF/F in %) of SynpH of a single
fly over time. (c) Maximum change in fluorescence (ΔF/F in %) of SynpH response to KCl in
3d, 30d, and 30dSpd flies (n = 5–6 flies; Kruskal-Wallis test). ns = not significant, p 0.05. Underlying data is shown in S1 Data. (TIF) S6 Fig. Imaging of SynpH at KC-to-MBON synapses to measure odor-evoked SV release. (a) Time course of SynpH activity induced by 3-Oct in the presynaptic terminals of KCs within
the horizontal lobe of mushroom body of 3d, 30d, and 30dSpd animals (SynpH response aver-
aged across three odor exposures from 6–7 flies). S8 Fig. BRP and RBP increase progressively with age. (a–d) Adult brains of 3d, 10d, 20d, and
30d w1118 flies immunostained for BRP (using Nc82 and N-terminal antibodies) and RBP. S1 Fig. Aversive olfactorymemory as a function of stimulation intensity in aged flies. Analysis of the endogenous expression of BRPGFP in adult brains. (a–c) Adult brains
of 3d- and 30d-BRP(83-ex13)GFP flies, and 30dSpd- BRP(83-ex13)GFP flies (BRPGFP). Brains
were fixed in 5% PFA and scanned for GFP signal. Scale bar: 50 μm. (d) Quantification of GFP
signal within the central brain region normalized to 3d flies (n = 9–18 independent brains;
Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni
correction). p < 0.05, p < 0.01, p < 0.001. Underlying data is shown in S1 Data. (TIF) S10 Fig. Analysis of Ca2+ channel and BRP in wild-typebrains. (a–c) Mushroom body calyx
of 3d, 30d, and 30dSpd flies expressing GFP-labeled genomic construct of α1 subunit Cacoph-
ony (CacGFP) and immunostained for GFP as well as BRP (corresponding single z-planes are
shown). Scale bar: 10 μm. (d,e) Quantification of signal intensity of CacGFP (using anti-GFP)
and BRP (using Nc82) in the calyx region normalized to 3d flies (n = 7–9 independent calyces;
Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni
correction). p < 0.01, ns = not significant, p 0.05. Underlying data is shown in S1 Data. (TIF) S11 Fig. Analysis of for Unc13 and BRP in wild-typebrains. (a–c) Adult brains of 3d and
30d w1118 flies, together with 30dSpd w1118 flies, immunostained for BRP (using Nc82 and N-
terminal antibody) and Unc13. Scale bar: 50 μm. (d–f)Quantification of signal intensity of the
proteins in the central brain region normalized to 3d flies (n = 10–15 independent brains;
Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni
correction). p < 0.05, p < 0.01, p < 0.001. Underlying data is shown in S1 Data. (TIF) S12 Fig. EM of PN-to-KC synapses.Electron micrographs revealed that the alignment of the
plasma membrane, with evident increase in extracellular spacing between cellular elements, to
be affected in 30d w1118 flies, when compared to 3d or 30dSpd w1118 flies. Scale bar: 500 nm. The
arrowheads point to the alignment of the plasma membrane between subcellular entities. (TIF) S13 Fig. STED analysis of BRP ring diameter at PN-to-KC synapses.Examples of confocal
and STED images of BRP spots within the calyx region of 3d and 30d w1118 flies, together with
30dSpd w1118 flies. Scale bar: 500 nm. S1 Fig. Aversive olfactorymemory as a function of stimulation intensity in aged flies. (b) Maximum change in SynpH fluorescence
(ΔF/F in %) in response to 3-Oct within the presynaptic terminals of KCs of 3d, 30d, and
30dSpd flies (SynpH response averaged across three odor exposures from 6–7 flies; Kruskal-
Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correc-
tion). (c) Time course of SynpH activity induced by MCH in the presynaptic terminals KCs
within the horizontal lobe of mushroom body of 3d, 30d, and 30dSpd animals (SynpH response
averaged across three odor exposures from 6–7 flies) (d) Maximum change in SynpH fluores-
cence (ΔF/F in %) in response to MCH within the presynaptic terminals of KCs of 3d, 30d, and
30dSpd flies (SynpH response averaged across three odor exposures from 6–7 flies; Kruskal-
Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni correc-
tion). p < 0.05, p < 0.01, ns = not significant, p 0.05. Underlying data is shown in S1
Data. (
) S7 Fig. Analysis of Syb in wild-typebrains. (a–c) Adult brains of 3d, 30d and 30dSpd w1118
flies immunostained for Syb. Scale bar: 50 μm. (d) Quantification of Syb intensity within the
central brain region normalized to 3d flies (n = 6–9 independent brains; Kruskal-Wallis test). ns = not significant, p 0.05. Underlying data is shown in S1 Data. (TIF) S7 Fig. Analysis of Syb in wild-typebrains. (a–c) Adult brains of 3d, 30d and 30dSpd w1118
flies immunostained for Syb. Scale bar: 50 μm. (d) Quantification of Syb intensity within the
central brain region normalized to 3d flies (n = 6–9 independent brains; Kruskal-Wallis test). ns = not significant, p 0.05. Underlying data is shown in S1 Data. (TIF) S8 Fig. BRP and RBP increase progressively with age. (a–d) Adult brains of 3d, 10d, 20d, and
30d w1118 flies immunostained for BRP (using Nc82 and N-terminal antibodies) and RBP. 26 / 34 PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Scale bar: 50 μm (e–g) Quantification of BRP (using Nc82 and N-terminal antibodies) and
RBP intensities within the central brain region normalized to 3d flies (n = 10–12 independent
brains; Kruskal-Wallis test with Dunn’s multiple comparison test, p-values were subject to
Bonferroni correction). p < 0.01, p < 0.001, ns = not significant, p 0.05. Underlying
data is shown in S1 Data. S9 Fig. PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes type (2xBRP) flies (n = 7–12; Kruskal-Wallis test with Dunn’s multiple comparison test, p-val-
ues were subject to Bonferroni correction). p < 0.05, p < 0.01, ns = not significant,
p 0.05. Underlying data is shown in S1 Data. (TIF)
S15 Fig. Acetylation and mass spectroscopy. (a) BRP sequence with acetylated peptide frag-
ments (yellow) and lysine sites positive for acetylation (red) identified through mass spectros-
copy. (b) Position of possible lysine residues that undergo (de)acetylation within BRP. (TIF) type (2xBRP) flies (n = 7–12; Kruskal-Wallis test with Dunn’s multiple comparison test, p-val-
ues were subject to Bonferroni correction). p < 0.05, p < 0.01, ns = not significant,
p 0.05. Underlying data is shown in S1 Data. (TIF) type (2xBRP) flies (n = 7–12; Kruskal-Wallis test with Dunn’s multiple comparison test, p-val-
ues were subject to Bonferroni correction). p < 0.05, p < 0.01, ns = not significant,
p 0.05. Underlying data is shown in S1 Data. (TIF) S15 Fig. Acetylation and mass spectroscopy. (a) BRP sequence with acetylated peptide frag-
ments (yellow) and lysine sites positive for acetylation (red) identified through mass spectros-
copy. (b) Position of possible lysine residues that undergo (de)acetylation within BRP. (TIF) S15 Fig. Acetylation and mass spectroscopy. (a) BRP sequence with acetylated peptide frag-
ments (yellow) and lysine sites positive for acetylation (red) identified through mass spectros-
copy. (b) Position of possible lysine residues that undergo (de)acetylation within BRP. (TIF) S16 Fig. KCl-inducedchanges in fluorescenceof Homer GCamp3.0 within the calyx neuropil. (a) Homer GCamp3.0 expressed in the dendritic claws of KCs and imaged within the calyx region. The two rings indicate the region of interest (calyx neuropil) and background region used for analy-
sis. Scalebar:50 μm. (b) KCl-induced influx of postsynaptic Ca2+ ion, measured by changes in fluo-
rescence (ΔF/F in %) of Homer GCamp3.0 of a single fly over time. (c) Maximum change in
fluorescence(ΔF/F in %) of Homer GCamp3.0 response to KCl in 3d, 30d, and 30dSpd flies (n = 8–9
flies;Kruskal-Wallis test). ns = not significant,p 0.05. Underlying data is shown in S1 Data. (TIF) S17 Fig. Empirical cumulative distribution functions for postsynaptic Homer-GCaMP3.0
response during odor stimulation. Empirical cumulative distribution functions for 3-Oct and
MCH, as used in the Kolmogorov-Smirnov test. Only the GCaMP3 response during odor pre-
sentation (seconds 1–3, grey bars in Fig 3K and 3M) was used. Two-sided Kolmogorov-Smir-
nov tests were conducted for the analysis of difference. The differences for 3-Oct were not
significant. The differences for MCH between 3d and 30d (), as well as between 30d and
30dSpd () were significant after Bonferroni correction for three groups. p < 0.05, p < 0.01. Underlying data is shown in S1 Data. (TIF) S18 Fig. Calyx neuropil from wild-typebrains immunostained for Drep2 and BRP. (a–c)
Mushroom body calyx from adult brains of 3d and 30d w1118 flies, together with 30dSpd w1118
flies immunostained for Drep2 and BRP (using Nc82 as well as N-terminal antibodies) (corre-
sponding single z-planes are shown). Scale bar: 10 μm. (d–f)Quantification of signal intensity
of these proteins in the calyx region normalized to 3d flies (n = 10 independent brains; Krus-
kal-Wallis test with Dunn’s multiple comparison test, p-values were subject to Bonferroni cor-
rection). p < 0.01, p < 0.001, ns = not significant, p 0.05. Underlying data is shown in
S1 Data. S19 Fig. Autophagy required for spermidine-mediatedsuppression of age-associated
increase in BRP levels. (a–d) Comparison of BRP signal intensity (using Nc82 and N-terminal
antibodies) in brains of 3d w1118 control animals, 3d and 20-d old (20d) atg7-/- flies, raised
either on normal or spermidine-supplemented food. Scale bar: 50 μm. (e–f)Quantification of
BRP within the central brain region of young (3d) and old (20d) atg7-/- mutants normalized to
3d w1118 flies (n = 9–12 independent brains; Kruskal-Wallis test with Dunn’s multiple compari-
son test, p-values were subject to Bonferroni correction). p < 0.01, ns = not significant,
p 0.05. Underlying data is shown in S1 Data. (TIF) S1 Fig. Aversive olfactorymemory as a function of stimulation intensity in aged flies. These calyces were also stained for Drep2, a protein found
highly enriched in dendritic claws of KCs, allowing the quantification of the diameter of BRP
spots that mark the synapse between PNs and KCs. (TIF) S14 Fig. Effect of removing one-copy of BRP on memory formation. (a–d) Adult brains of
3d and 30d brp69/+ (1xBRP) flies together with age-matched controls (2xBRP), immunos-
tained for BRP (using Nc82 and N-terminal antibody), and RBP. Scale bar: 50 μm. (e, f)Quan-
tification of BRP (using N-terminal antibody) and RBP intensity within the central brain
region normalized to 3d flies (9–10 independent brains; Kruskal-Wallis test with Dunn’s multi-
ple comparison test, p-values were subject to Bonferroni correction). (g) Aversive associative
memory performance 3 min after training (STM) of brp69/+ (1xBRP) flies compared to wild- PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 27 / 34 S1 Table. Aversive odor avoidance and shock reactivity in different genotypes. Underlying
data is shown in S1 Data.
(TIF) 28 / 34 PLOS Biology | DOI:10.1371/journal.pbio.1002563
September 29, 2016 Spermidine Protects from Age-Dependent Memory Decline by Suppressing Presynaptic Changes Writing – original draft: VKG FM SJS. Writing – original draft: VKG FM SJS. Acknowledgments We would like to thank T. Neufeld (University of Minnesota), G. Miesenböck (University of
Oxford), and the Bloomington Stock Center for fly stocks. We are also grateful to N. E. Reist
and Hugo J. Bellen, as well as R. Robin Hiesinger and E. Buchner for Synaptotagmin-1, Syb,
and Synapsin antibodies, respectively. We would like to thank M. Böhme for his technical help
in STED imaging. We further thank M. G. Holt and S. T. Sweeney for reading the manuscript
critically. Formal analysis: VKG UP AB AF SFM AE TFMA EAA MM SJS.
Investigation: VKG UP AB AF SFM AE KT CB CQ EAA MM RB.
Resources: TM MS JD FM AF. Writing – original draft: VKG FM SJS. PLOS Biology | DOI:10.1371/journal.pbio.1002563
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STAT5BN642H is a driver mutation for T cell neoplasia
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The Journal of clinical investigation/The journal of clinical investigation
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Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 R E S E A R C H A R T I C L E Conflict of interest: The authors have declared that no conflict of interest exists.
Submitted: April 12, 2017; Accepted: October 5, 2017.
License: This work is licensed under the Creative Commons Attribution 4.0 Inter-
national License. To view a copy of this license, visit http://creativecommons.org/
licenses/by/4.0/.
Reference information: J Clin Invest. 2018;128(1):387–401.
https://doi.org/10.1172/JCI94509. Introduction due to differences in the level of STAT5 proteins and possible dis-
tinct chromatin-remodeling capabilities as a result of interactions
with other transcriptional regulatory proteins (10–12). Recently,
the hSTAT5BN642H mutation was described as a gain-of-function
(GOF) mutation in leukemic patients that causes enhanced and
prolonged tyrosine phosphorylation (13–27). This mutation is
associated with a poor prognosis and an increased risk of relapse,
despite the use of combination chemotherapy (13). The N642H
mutation is located in the center of the STAT5B SH2 domain, the
phosphotyrosine-binding domain that is essential for the forma-
tion of parallel STAT5 dimers and efficient nuclear transloca-
tion (1). STAT5BN642H has been found in more than 90 patients
with 7 types of aggressive leukemia or lymphoma including γδ T
cell–derived lymphoma, hepatosplenic T cell lymphomas, large
granular lymphocytic (LGL) leukemia, T cell acute lymphoblas-
tic leukemia (T-ALL), T cell prolymphocytic leukemia, NK/T cell
lymphoma, and enteropathy-associated T cell lymphoma (13–25). The 2 signal transducer and activator of transcription 5 proteins,
STAT5A and STAT5B, encoded by 2 different genes with 89%
DNA sequence homology, are downstream targets of cytokines
and growth factors (1, 2). STATs are highly expressed and/or
hyperactivated by tyrosine and serine phosphorylation in numer-
ous hematopoietic cancers (3–6). The 2 STAT5 proteins have been
reported to have redundant functions largely due to overlapping
genome binding sites (7–9). There are different phenotypes upon
genetic loss or somatic point mutation resulting in hyperactivation
of STAT5A or STAT5B. STAT5B has a dominant role in immune
cells, as suggested by its higher expression levels compared with
STAT5A in NK or T cell subsets (7–9). Interestingly, mutations
in cancer patients have predominantly been found in the Src
homology 2 (SH2) domain of human STAT5B (hSTAT5B). This
suggests that differences in hematopoietic transformation are Studies in mice have implicated STAT5 signaling in the expan-
sion of CD8+ T cells as well as the development of lymphoblastic
lymphoma (28). Nevertheless, there is no evidence of whether the
hSTAT5BN642H mutation is capable of driving the development and
progression of leukemia. Drug-sensitivity tests on hSTAT5BN642H-
expressing leukemic blasts from T-ALL patients indicated that the
mutation provides a survival advantage in leukemic cells (17). STAT5BN642H is a driver mutation for T cell neoplasia Ha Thi Thanh Pham,1,2 Barbara Maurer,1,2 Michaela Prchal-Murphy,3 Reinhard Grausenburger,3 Eva Grundschober,3
Tahereh Javaheri,1,2 Harini Nivarthi,4 Auke Boersma,5 Thomas Kolbe,6,7 Mohamed Elabd,1 Florian Halbritter,4 Jan Pencik,1
Zahra Kazemi,8,9 Florian Grebien,1 Markus Hengstschläger,10 Lukas Kenner,1,11,12 Stefan Kubicek,4 Matthias Farlik,4
Christoph Bock,4,8,13 Peter Valent,14,15 Mathias Müller,2 Thomas Rülicke,5 Veronika Sexl,3 and Richard Moriggl1,2,8 1Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria. 2Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna, Vienna, Austria. 3Institute of Pharmacology and
Toxicology, University of Veterinary Medicine Vienna, Vienna, Austria. 4CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria. 5Institute of Laboratory Animal
Science, and 6Biomodels Austria (Biat), University of Veterinary Medicine Vienna, Vienna, Austria. 7IFA-Tulln, University of Natural Resources and Life Sciences, Tulln, Austria. 8Medical University of Vienna,
Vienna, Austria. 9Center of Physiology and Pharmacology, Vienna, Austria. 10Center of Pathobiochemistry and Genetics, Institute of Medical Genetics, Medical University of Vienna, Vienna, Austria. 11Clinical
Institute of Pathology, Medical University of Vienna, Vienna, Austria. 12Unit of Pathology of Laboratory Animals, University of Veterinary Medicine Vienna, Vienna, Austria. 13Max Planck Institute for Informatics,
Saarbrücken, Germany. 14Department of Internal Medicine I, Division of Hematology and Hemostaseology, and 15Ludwig Boltzmann-Cluster Oncology, Medical University of Vienna, Vienna, Austria. STAT5B is often mutated in hematopoietic malignancies. The most frequent STAT5B mutation, Asp642His (N642H),
has been found in over 90 leukemia and lymphoma patients. Here, we used the Vav1 promoter to generate transgenic
mouse models that expressed either human STAT5B or STAT5BN642H in the hematopoietic compartment. While STAT5B-
expressing mice lacked a hematopoietic phenotype, the STAT5BN642H-expressing mice rapidly developed T cell neoplasms. Neoplasia manifested as transplantable CD8+ lymphoma or leukemia, indicating that the STAT5BN642H mutation drives
cancer development. Persistent and enhanced levels of STAT5BN642H tyrosine phosphorylation in transformed CD8+ T cells
led to profound changes in gene expression that were accompanied by alterations in DNA methylation at potential histone
methyltransferase EZH2-binding sites. Aurora kinase genes were enriched in STAT5BN642H-expressing CD8+ T cells, which
were exquisitely sensitive to JAK and Aurora kinase inhibitors. Together, our data suggest that JAK and Aurora kinase
inhibitors should be further explored as potential therapeutics for lymphoma and leukemia patients with the STAT5BN642H
mutation who respond poorly to conventional chemotherapy. jci.org Volume 128 Number 1 January 2018 Results (TET2) and DNA methyltransferase 3α (DNMT3A) affecting
DNA methylation are frequently found in T cell lymphoma (29). TET1/2 was also shown to interact with STAT5 in Tregs that are
strictly dependent on STAT5 because of direct regulation of the
STAT5 target genes FOXP3 and CD25 (30). Interestingly, the
DNMT3A gene was shown to be controlled by STAT5 in AML cells
(31). Drugs interfering with epigenetic changes are powerful tools
in cancer drug development and have found entry into therapeu-
tic strategies (29). A key role of STAT5 is to support the process of
histone acetylation and methylation in T cells, which was shown
for the TCR locus (32, 33). Furthermore, the histone methyltrans-
ferase EZH2 and histone deacetylase 1 (HDAC1) were shown to be
recruited via STAT5 binding (34, 35). hSTAT5BN642H is an activating mutation in hematopoietic cells. Somatic
mutations of STAT5B, many of which are located in the SH2 domain,
have been found in patients with lymphoid neoplasia (Figure 1A)
(13–26, 36). To investigate the impact of these somatic mutations on
hSTAT5B activity, we analyzed different potential GOF mutations
in 293T cells using tyrosine phosphorylation of STAT5 (pY-STAT5)
as a correlation for activity. We observed high pY-STAT5 levels
under steady-state conditions in cells expressing the N642H muta-
tion, the most frequent STAT5 mutation in patients with leukemia
or lymphoma. The 2 SH2 domain variants Y665H and Y665F also
showed enhanced activity in the absence of cytokine stimulation
(Figure 1B). We observed a similar pattern of pY-STAT5B upon
expression of the N642H mutant in the murine pro–B cell line Ba/
F3 and the murine myeloid cell line 32D (Figure 1C). In contrast,
expression of hSTAT5B at comparable levels failed to induce signif-
icant pY-STAT5 in the absence of IL-3 stimulation (Figure 1C and
Supplemental Figure 1A; supplemental material available online
with this article; https://doi.org/10.1172/JCI94509DS1). Impor-
tantly, hSTAT5BN642H rendered Ba/F3 and 32D cells cytokine inde-
pendent, supporting the finding of a proto-oncogenic potential of
hSTAT5BN642H (15) (Supplemental Figure 1B). Here, we investigated the oncogenic potential of the
hSTAT5BN642H mutation compared with the nonmutated hSTAT5B
using Vav1-driven transgenic mouse models. In contrast to WT
hSTAT5B, moderate hSTAT5BN642H expression levels triggered leu-
kemia or lymphoma development, which manifested as a transplant-
able CD8+ T cell disease. Related Commentary: p. 113 Conflict of interest: The authors have declared that no conflict of interest exists. Submitted: April 12, 2017; Accepted: October 5, 2017. License: This work is licensed under the Creative Commons Attribution 4.0 Inter-
national License. To view a copy of this license, visit http://creativecommons.org/
licenses/by/4.0/. Reference information: J Clin Invest. 2018;128(1):387–401. https://doi.org/10.1172/JCI94509. Epigenetic abnormalities are major drivers of hematopoiet-
ic malignancies. Mutations in Tet methylcytosine dioxygenase 2 jci.org Volume 128 Number 1 January 2018 3 8 7 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 The Journal of Clinical Investigation R E S E A R C H A R T I C L E Figure 1. hSTAT5BN642H is an activating mutation. (A) Schematic of STAT5B mutations identified in leukemia and lymphoma patients. Each dot represents
1 patient. (B) WB analysis of pY-STAT5, total STAT5 protein, and HSC70 in 293T cells that were transiently transfected with different hSTAT5B (hS5B)
variants using a pMSCV-IRES-GFP vector, with or without growth hormone (GH) stimulation. (C) WB analysis of pY-STAT5, STAT5, FLAG, and HSC70 in
hSTAT5B- or hSTAT5BN642H-expressing (N642H) Ba/F3 cells with or without IL-3 stimulation. (B and C) Nontransfected and pMSCV-transfected cells are
shown as controls. Data presented in B and C are representative of 3 independent experiments. Samples were run on parallel gels for B and C, and a load-
ing control is provided for each gel. Figure 1. hSTAT5BN642H is an activating mutation. (A) Schematic of STAT5B mutations identified in leukemia and lymphoma patients. Each dot represents
1 patient. (B) WB analysis of pY-STAT5, total STAT5 protein, and HSC70 in 293T cells that were transiently transfected with different hSTAT5B (hS5B)
variants using a pMSCV-IRES-GFP vector, with or without growth hormone (GH) stimulation. (C) WB analysis of pY-STAT5, STAT5, FLAG, and HSC70 in
hSTAT5B- or hSTAT5BN642H-expressing (N642H) Ba/F3 cells with or without IL-3 stimulation. (B and C) Nontransfected and pMSCV-transfected cells are
shown as controls. Data presented in B and C are representative of 3 independent experiments. Samples were run on parallel gels for B and C, and a load-
ing control is provided for each gel. Results (A) Survival curve shows Figure 2. Moderate Vav1-driven expression of hSTAT5BN642H in mice leads to HSC expansion. (A) Survival curve shows the percentages of disease-free
survival of hSTAT5BN642H (N642H) mice (n = 21) compared with that of hSTAT5B (hS5B) (n = 20) and WT (n = 10) mice. (B) WB analysis of pY-STAT5, total
STAT5, and HSC70 in the LNs and spleens of WT mice and hSTAT5BN642H- and hSTAT5B-transgenic mice. Quantification of the WB was performed using
ImageJ. Data are representative of 3 independent experiments. (C) Flow cytometric analysis of the percentage of LSKs, LT-HSCs (CD150+CD48–), ST-HSCs
(CD150+CD48+), MPPs (CD150–CD48+), (D and E) common lymphoid progenitors (lineage−Sca1+IL-7R+AA4+), MPCs (lineage−Sca1–IL-7R–c-Kit+), and CD3+ cells
in the BM of WT, hSTAT5B, and hSTAT5BN642H mice. Analyses in C–E included 7-week-old WT (n = 7), hSTAT5B (n = 5), and hSTAT5BN642H (n = 5) mice. Data
represent the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA with Bonferroni’s correction. Figure 2. Moderate Vav1-driven expression of hSTAT5BN642H in mice leads to HSC expansion. (A) Survival curve shows the percentages of disease-free
survival of hSTAT5BN642H (N642H) mice (n = 21) compared with that of hSTAT5B (hS5B) (n = 20) and WT (n = 10) mice. (B) WB analysis of pY-STAT5, total
STAT5, and HSC70 in the LNs and spleens of WT mice and hSTAT5BN642H- and hSTAT5B-transgenic mice. Quantification of the WB was performed using
ImageJ. Data are representative of 3 independent experiments. (C) Flow cytometric analysis of the percentage of LSKs, LT-HSCs (CD150+CD48–), ST-HSCs
(CD150+CD48+), MPPs (CD150–CD48+), (D and E) common lymphoid progenitors (lineage−Sca1+IL-7R+AA4+), MPCs (lineage−Sca1–IL-7R–c-Kit+), and CD3+ cells
in the BM of WT, hSTAT5B, and hSTAT5BN642H mice. Analyses in C–E included 7-week-old WT (n = 7), hSTAT5B (n = 5), and hSTAT5BN642H (n = 5) mice. Data
represent the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA with Bonferroni’s correction. Analysis of WBC counts in hSTAT5BN642H mice revealed an
increase of approximately 20-fold compared with that detected in
hSTAT5B and WT mice (Figure 3C). The WBC count in hSTAT5B
mice only increased slightly with age but remained within a phys-
iological range (Supplemental Figure 3B). The drastic increase in
the WBC count in STAT5BN642H mice was correlated with an expan-
sion of CD8+ T cells (Figure 3C). Results Transcriptome and DNA methylome anal-
yses illustrated profound changes in gene expression and reduced
DNA methylation of potential EZH2-binding sites, with Aurora kinas-
es being one of the most altered genes in hSTAT5BN642H-transgenic
animals. In line with this, we found that Aurora kinase and JAK inhib-
itors were effective in blocking neoplastic T cell expansion and organ
infiltration driven by hSTAT5BN642H. This suggested that inhibitors of
Aurora kinases and JAK have potential as a treatment for patients suf-
fering from hSTAT5N642H-driven lymphoma or leukemia. Vav1-driven expression of hSTAT5BN642H induces cancer. Given
that hSTAT5BN642H was found in different hematopoietic cancers,
we expressed hSTAT5B or hSTAT5BN642H in mice during early
hematopoiesis using the Vav1 oncogene promoter. This led to
transgene expression primarily in cells of the hematopoietic sys- 3 8 8 jci.org Volume 128 Number 1 January 2018 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 The Journal of Clinical Investigation The Journal of Clinical Investigation The Journal of Clinical Investigation R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E The Journal of Clinical Investigation
R E S E A R C H A R T I C L E
Figure 2. Moderate Vav1-driven expression of hSTAT5BN642H in mice leads to HSC expansion. (A) Survival curve shows the percentages of disease-free
survival of hSTAT5BN642H (N642H) mice (n = 21) compared with that of hSTAT5B (hS5B) (n = 20) and WT (n = 10) mice. (B) WB analysis of pY-STAT5, total
STAT5, and HSC70 in the LNs and spleens of WT mice and hSTAT5BN642H- and hSTAT5B-transgenic mice. Quantification of the WB was performed using
ImageJ. Data are representative of 3 independent experiments. (C) Flow cytometric analysis of the percentage of LSKs, LT-HSCs (CD150+CD48–), ST-HSCs
(CD150+CD48+), MPPs (CD150–CD48+), (D and E) common lymphoid progenitors (lineage−Sca1+IL-7R+AA4+), MPCs (lineage−Sca1–IL-7R–c-Kit+), and CD3+ cells
in the BM of WT, hSTAT5B, and hSTAT5BN642H mice. Analyses in C–E included 7-week-old WT (n = 7), hSTAT5B (n = 5), and hSTAT5BN642H (n = 5) mice. Data
represent the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA with Bonferroni’s correction. Figure 2. Moderate Vav1-driven expression of hSTAT5BN642H in mice leads to HSC expansion. Results (D) CD8/CD4 T cell ratios in LNs
were determined using flow cytometry. Analyses included 7-week-old WT (n = 5), hSTAT5B (n = 5), and hSTAT5BN642H (n = 5) mice. (E) Quantification of the
absolute number of CD4+ and CD8+ T cells, myeloid cells (CD11b+Gr1+), and B cells (CD19+) in spleens from hSTAT5BN642H- and hSTAT5B-transgenic mice and
WT mice. Analyses included 7-week-old WT (n = 13), hSTAT5B (n = 6), and hSTAT5BN642H (n = 6 and 11) mice. (F) CD3+CD8+ splenic cells were analyzed by flow
cytometry for their expression of CD25. Analyses included 8-week-old WT (n = 8), hSTAT5B (n = 9), and (n = 6) hSTAT5BN642H mice. (G) CD3+CD8+ splenic cells
were further analyzed for CD62L and CD44 expression. Analyses included WT (n = 8), hSTAT5B (n = 5), and hSTAT5BN642H (n = 5) mice at 8 weeks of age. Data represent the mean ± SD. n ≥ 6. **P < 0.01 and ***P < 0.001, by 1-way ANOVA with Bonferroni’s correction. cells expressed surface markers indicative of mature T cells with
an activated phenotype and high expression of IL-2Rα (CD25), a
direct target gene of STAT5 (41) (Figure 3F). Fifty percent of the
diseased CD8+ T cells also expressed markers reminiscent of cen-
tral memory T cells. Moreover, we found that the percentage of
cells expressing markers for effector memory T cells was elevated
in the diseased mice compared with that observed in WT controls
(Figure 3G). High numbers of proliferating T cells were associated
with splenomegaly and lymphoma formation, and proliferating T
cells were found to heavily infiltrate peripheral organs, leading to
fatal pulmonary obstruction (Figure 4 and Supplemental Figure 4). tion of CD8+ T cells (Figure 5, C and D). Of note, the i.v. injection of
CD8+ T cells from diseased mice into nonirradiated Ly5.1+CD45.1+
recipient mice was sufficient to phenotypically recapitulate the pri-
mary disease, identifying the CD8+ T cells as the malignant cell pool
(Figure 5, E–G, and Supplemental Figure 5, C and D). JAK inhibitors suppress disease progression in the hSTAT5BN642H-
driven disease model. A number of treatment regimens have been
suggested for leukemia and lymphoma patients carrying the
hSTAT5BN642H mutation. However, there is limited knowledge about
the effectiveness of these treatments, partially because of the lack of
a suitable preclinical model (17, 18). Results https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 The Journal of Clinical Investigation The Journal of Clinical Investigation R E S E A R C H A R T I C L E Figure 3. hSTAT5BN642H mice suffer from an aggressive CD8+ T cell lymphoma. (A) Macroscopic comparison of hSTAT5BN642H and hSTAT5B mouse spleens
and LNs with those from WT mice. Scale bars: 1 cm. (B) Modified Wright staining of blood smears from hSTAT5BN642H (N642H), hSTAT5B (hS5B), and WT
mice (original magnification, ×100). (C) WBC count using an animal blood counter (scil Vet ABC). CD8/CD4 ratios in the peripheral blood were determined
using flow cytometry. Analysis included 7- to 10-week-old WT (n = 20), hSTAT5B (n = 15), and hSTAT5BN642H (n = 20) mice. (D) CD8/CD4 T cell ratios in LNs
were determined using flow cytometry. Analyses included 7-week-old WT (n = 5), hSTAT5B (n = 5), and hSTAT5BN642H (n = 5) mice. (E) Quantification of the
absolute number of CD4+ and CD8+ T cells, myeloid cells (CD11b+Gr1+), and B cells (CD19+) in spleens from hSTAT5BN642H- and hSTAT5B-transgenic mice and
WT mice. Analyses included 7-week-old WT (n = 13), hSTAT5B (n = 6), and hSTAT5BN642H (n = 6 and 11) mice. (F) CD3+CD8+ splenic cells were analyzed by flow
cytometry for their expression of CD25. Analyses included 8-week-old WT (n = 8), hSTAT5B (n = 9), and (n = 6) hSTAT5BN642H mice. (G) CD3+CD8+ splenic cells
were further analyzed for CD62L and CD44 expression. Analyses included WT (n = 8), hSTAT5B (n = 5), and hSTAT5BN642H (n = 5) mice at 8 weeks of age. Data represent the mean ± SD. n ≥ 6. **P < 0.01 and ***P < 0.001, by 1-way ANOVA with Bonferroni’s correction. Figure 3. hSTAT5BN642H mice suffer from an aggressive CD8+ T cell lymphoma. (A) Macroscopic comparison of hS Figure 3. hSTAT5BN642H mice suffer from an aggressive CD8+ T cell lymphoma. (A) Macroscopic comparison of hSTAT5BN642H and hSTAT5B mouse spleens
and LNs with those from WT mice. Scale bars: 1 cm. (B) Modified Wright staining of blood smears from hSTAT5BN642H (N642H), hSTAT5B (hS5B), and WT
mice (original magnification, ×100). (C) WBC count using an animal blood counter (scil Vet ABC). CD8/CD4 ratios in the peripheral blood were determined
using flow cytometry. Analysis included 7- to 10-week-old WT (n = 20), hSTAT5B (n = 15), and hSTAT5BN642H (n = 20) mice. jci.org Volume 128 Number 1 January 2018 Results Similarly, CD8+ T cells increased
by 3-fold in the lymph nodes (LNs) of hSTAT5BN642H mice (Fig-
ure 3D), which was confirmed by immunohistochemical staining
(Supplemental Figure 3C). The numbers of CD4+ T cells were also
moderately increased, whereas the percentage, but not the total
number, of CD19+ B cells was reduced in the LNs of hSTAT5BN642H
mice compared with controls (Figure 3E and Supplemental Fig-
ure 3D). Hematocrit levels were comparable in all mouse models
(Supplemental Figure 3E). We also observed a mild expansion of
other hematopoietic cell types such as CD19+ B cells, CD4+ T cells,
and CD11b+Gr1+ myeloid cells in the spleen (Figure 3E and Supple-
mental Figure 3F). tem, including hematopoietic stem cells (HSCs) (37) (Supplemen-
tal Figure 2, A and B). Transgenic mice expressing hSTAT5BN642H
rapidly developed malignant disease leading to death between
40 and 100 days of age. hSTAT5B-transgenic mice showed no
signs of disease when sacrificed at the age of 12 months or older
(Figure 2A). Despite expressing comparable levels of total STAT5,
only hSTAT5BN642H-transgenic mice showed elevated pY-STAT5
signals, indicating strong and persistent tyrosine phosphorylation
(Figure 2B). In line with this observation, Vav1-driven expression
of hSTAT5BN642H, but not hSTAT5B, led to increased numbers
of progenitor cells throughout all early hematopoietic compart-
ments, including lineage–Sca1+c-Kit+ cells (LSKs), long-term HSCs
(LT-HSCs), short-term HSCs (ST-HSCs), and multipotent progen-
itors (MPPs) (CD150+CD48–, CD150+CD48+, CD150–CD48+ frac-
tions) (Figure 2C). Likewise, the numbers of common lymphoid
progenitors (CLPs) and myeloid progenitor cells (MPCs) were sig-
nificantly elevated in mice expressing hSTAT5BN642H (Figure 2D). hSTAT5BN642H mice had 3 times more CLPs than did WT mice,
which translated into expansion of CD3+ T cells, but not CD19+,
B cells in their BM (Figure 2E and Supplemental Figure 2C). The
elevated number of MPCs was also accompanied by a signifi-
cant increase in the percentage of CD11b+Gr1+ cells in the BM of
hSTAT5BN642H mice (Supplemental Figure 2C). We used flow cytometry to analyze markers for T cell acti-
vation (CD25) and surface markers for T cell subpopulations,
including naive CD8+ T cells (CD62LhiCD44lo), central memory
CD8+ T cells (CD62LhiCD44hi), and effector memory CD8+ T cells
(CD62LloCD44hi) (38–40). This analysis showed that the leukemic jci.org Volume 128 Number 1 January 2018 3 8 9 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E Figure 4. Highly proliferative T cells infiltrate into the
peripheral organs of hSTAT5BN642H mice. Histological anal-
ysis using H&E, CD3, and Ki67 staining of the lungs of 8- to
10-week-old hSTAT5B, hSTAT5BN642H, and WT mice. Data
are a representative of 3 independent experiments. Scale
bar: 100 μm. Original magnification, ×20 and ×40 (insets). Figure 4. Highly proliferative T cells infiltrate into the
peripheral organs of hSTAT5BN642H mice. Histological anal-
ysis using H&E, CD3, and Ki67 staining of the lungs of 8- to
10-week-old hSTAT5B, hSTAT5BN642H, and WT mice. Data
are a representative of 3 independent experiments. Scale
bar: 100 μm. Original magnification, ×20 and ×40 (insets). Figure 4. Highly proliferative T cells infiltrate into the
peripheral organs of hSTAT5BN642H mice. Histological anal-
ysis using H&E, CD3, and Ki67 staining of the lungs of 8- to
10-week-old hSTAT5B, hSTAT5BN642H, and WT mice. Data
are a representative of 3 independent experiments. Scale
bar: 100 μm. Original magnification, ×20 and ×40 (insets). lation of 371 genes in T cells derived from hSTAT5BN642H compared
with that observed in WT T cells (FDR-adjusted P < 0.05) (Sup-
plemental Figure 7A). As expected, known STAT5 targets such as
Ccl5, Ccr5, Pim1, Bcl2, and Il2r were among the top upregulated
genes, confirming hSTAT5BN642H transgene specificity (Supple-
mental Figure 7, B and C and Supplemental Tables 2 and 3) (7–9). Importantly, gene set enrichment analysis (GSEA) confirmed that
genes upregulated in CD8+ T cell lymphoma patients were high-
ly enriched, which emphasized the validity of our model (Figure
7B) (43, 44). Additional pathway analysis showed that E2F targets,
the G2M checkpoint, and MYC targets were the most upregulated
pathways, underlining the high proliferation rate of leukemic cells
and indicating hSTAT5BN642H as a driver for cell-cycle progres-
sion (Figure 7C and Supplemental Figure 7, D and E). In contrast,
inflammatory gene pathways or developmental core cancer path-
ways were significantly downregulated (P < 0.05), as analyzed by
pathway analysis (Supplemental Figure 7E). whether cells carrying the hSTAT5BN642H mutation are sensitive to
JAK inhibition. As expected, the FDA-approved JAK inhibitors rux-
olitinib and tofacitinib reduced the activation of STAT5 and cell
viability, with an IC50 of 0.11 μM (ruxolitinib) and 0.12 μM (tofaci-
tinib) and comparable IC50 values for all genotypes (Figure 6, B and
C, and Supplemental Figure 6A). R E S E A R C H A R T I C L E Moreover, other FDA-approved
drugs such as HDAC inhibitors for the treatment of T cell lympho-
ma were tested (42). Entinostat and several other drugs were also
found to be effective in inducing apoptosis in T cells, with an IC50 in
the nanomolar range, but did not exert differential effects between
hSTAT5B- and hSTAT5N642H-expressing cells (Supplemental Figure
6A and Supplemental Table 1). Following the in vitro data, we investigated the effect of rux-
olitinib in vivo by treating hSTAT5BN642H CD8+ T cell recipient
Ly5.1+CD45.1 mice, 60 days after transplantation, with ruxolitinib
(45 mg/kg) for a period of 30 days. The treatment significantly
reduced the size of LNs and spleens (Figure 6, D and E). The WBC
count as well as CD25 expression on donor hSTAT5BN642H CD8+
T cells were also reduced upon ruxolitinib treatment (Figure 6F
and Supplemental Figure 6B). Furthermore, ruxolitinib decreased
the degree of T cell infiltration into the lungs, skin, BM, LNs, and
spleens of treated mice, leading to a substantial reduction in dis-
ease burden (Figure 6G and Supplemental Figure 6C). The treat-
ment did not significantly affect the myeloid cell population in the
hematopoietic organs (Supplemental Figure 6D). Besides its function as a transcription factor, STAT5 can
shape chromatin by interacting with other chromatin-remodeling
enzymes such as EZH2 (35, 45). As changes in DNA methylation
patterns have recently been associated with malignant disease
and particularly with leukemia (46, 47), we questioned wheth-
er the dramatic changes observed in the expression profiles of
hSTAT5BN642H CD8+ T cells would also be reflected by alterations
in the DNA methylome. Using reduced representation bisul-
fite sequencing (RRBS), we found that overall DNA methylation
across CpG islands (CGIs) among hSTAT5BN642H and WT T cells
was highly consistent (Pearson’s r = 0.98), with only 1,380 CGIs
being substantially different (absolute difference ≥5 percentage
points) (Figure 7D) (48). When comparing WT and hSTAT5B CD8+
T cells, we found weaker differences and overlaps (Supplemental
Figure 8A). Combining DNA methylation analysis with mRNA
expression data, we identified a small set of genes with substan-
tial and concordant changes in DNA methylation and expression
of genes within the proximity of differentially methylated CGIs
(Supplemental Figure 8, B and C). Interestingly, the genes with
higher expression in hSTAT5BN642H T cells and concordant loss hSTAT5BN642H CD8+ T cells exhibit substantial changes in gene
expression profile, accompanied by specific changes in DNA methyla-
tion. Results Typically, STAT5 is activated in
response to cytokine signaling, and cells harboring the hSTAT5BN642H
mutant show prolonged pY-STAT5 levels upon stimulation rather
than being constitutively active (18). When we analyzed the level
of pY-STAT5 in primary T cells derived from the LNs of WT and
hSTAT5B- and hSTAT5BN642H-transgenic mice, we detected dras-
tically reduced levels of pY-STAT5 one hour after IL-2 deprivation
in WT and hSTAT5B-expressing T cells. In contrast, low levels of
pY-STAT5 remained detectable up to 4 hours after IL-2 removal
in hSTAT5BN642H-expressing T cells (Figure 6A). The finding that
cytokines efficiently activated hSTAT5BN642H prompted us to test To test whether the T cell disease in hSTAT5BN642H-transgenic
mice was transplantable, we transferred BM cells from mutant or
WT control mice i.v. into nonirradiated, immunocompromised
NSG recipient mice. The recipients of mutant cells became termi-
nally sick approximately 3 months after injection (Figure 5A). Bone
marrow transplantations (BMTs) induced disease, with a phenotype
comparable to that of hSTAT5BN642H-transgenic mice. The disease
was characterized by enlarged spleens and lymphoma formation,
with T cell infiltration into peripheral organs (Figure 5B and Supple-
mental Figure 5, A and B) caused by excessive expansion and infiltra- 3 9 0 jci.org Volume 128 Number 1 January 2018 The Journal of Clinical Investigation
R E S E A R C H A R T I C L E
Figure 4. Highly proliferative T cells infiltrate into the
peripheral organs of hSTAT5BN642H mice. Histological anal-
ysis using H&E, CD3, and Ki67 staining of the lungs of 8- to
10-week-old hSTAT5B, hSTAT5BN642H, and WT mice. Data
are a representative of 3 independent experiments. Scale
bar: 100 μm. Original magnification, ×20 and ×40 (insets). Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 The Journal of Clinical Investigation The Journal of Clinical Investigation R E S E A R C H A R T I C L E (A) Percentage of disease-free survival following hSTAT5BN642H whole BMT into 8-week-
old NSG recipient mice compared with WT BMT. (B) Macroscopic view of LNs and spleen from a hSTAT5BN642H BMT recipient mouse compared with those
from a WT BMT recipient mouse. Scale bar: 1 cm. (C) Flow cytometric analysis shows the quantity of CD3+ cells and CD8/CD4 T cell ratio in the spleens of
BMT recipient mice. (D) Histological analysis of CD3+ cells from the lungs of NSG recipient mice after hSTAT5BN642H or WT BMT. Scale bar: 100 μm. Original
magnification, ×20 and ×40 (insets). (E) Percentage of disease-free survival after hSTAT5BN642H or WT CD8+ T cell transplantation into nonirradiated
8-week-old Ly5.1/CD45.1 recipient mice. (F) Flow cytometric analysis shows the quantity of splenic CD3+CD8+ cells in CD8+ T cell–transplanted mice. (G)
Spleen versus BW ratios of WT and hSTAT5BN642H CD8+ T cell–transplanted Ly5.1/CD45.1 mice. (A–C) n = 4 WT mice and n = 5 hSTAT5BN642H mice; (E–G) n = 6
WT mice and n = 5 hSTAT5BN642H mice. Data represent the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001, by unpaired, 2-tailed Student’s t test. pared with that detected in WT murine STAT5B and hSTAT5B. The mutated STAT5 increased its binding to the Cis promoter and
was also found at the promoter regions of the EZH2 known targets
Cdkn2a and Ccnd2. In addition, it bound to the less methylated
CGI in association with Aurkb (Figure 8C). Although EZH2 bind-
ing was found to be reduced in hSTAT5BN642H CD8+ T cells, EZH2
retained its binding at the same CGI (Figure 8D and Supplemental
Figure 9C). However, hSTAT5BN642H was not shown to have direct
interactions with EZH2 (Figure 8E). of DNA methylation at nearby CGIs included the mitotic check-
point protein KNTC1 (49) and the oncogene topoisomerase type
IIα (TOP2A) (36, 50), which is known to regulate DNA topologi-
cal structure and cell-cycle progression (Supplemental Figure 8B,
right, sector II). None of these genes was substantially affected in
hSTAT5B CD8+ T cells (Supplemental Figure 8B). Specific DNA methylation changes in hSTAT5BN642H reveal targets
for therapy. Location overlap analysis (LOLA) (51) of regions that
lost methylation in T cells expressing hSTAT5BN642H compared
with WT cells revealed significant enrichment for sites known to
bind EZH2 and/or SUZ12 proteins. R E S E A R C H A R T I C L E Given the leukemogenic effect of hSTAT5BN642H, which is not
shared by WT hSTAT5B, we next investigated alterations in gene
expression and epigenetic modifications in T cells derived from
both mouse models. CD8+ T cells were isolated from the LNs of
13-week-old WT and hSTAT5B and hSTAT5BN642H diseased mice,
and mRNA sequencing analysis was performed. While the glob-
al expression patterns of WT and hSTAT5B CD8+ T cells were
comparable, the gene expression signature of cells expressing
hSTAT5BN642H showed a distinct pattern (Figure 7A). We found a
significant upregulation of 564 genes and a significant downregu- 3 9 1 jci.org Volume 128 Number 1 January 2018 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 The Journal of Clinical Investigation
S E A R C H A R T I C L E
wnloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 The Journal of Clinical Investigation R E S E A R C H A R T I C L E Figure 5. hSTAT5BN642H CD8+ T cells are the cancer-initiating cells. (A) Percentage of disease-free survival following hSTAT5BN642H whole BMT into 8-week-
old NSG recipient mice compared with WT BMT. (B) Macroscopic view of LNs and spleen from a hSTAT5BN642H BMT recipient mouse compared with those
from a WT BMT recipient mouse. Scale bar: 1 cm. (C) Flow cytometric analysis shows the quantity of CD3+ cells and CD8/CD4 T cell ratio in the spleens of
BMT recipient mice. (D) Histological analysis of CD3+ cells from the lungs of NSG recipient mice after hSTAT5BN642H or WT BMT. Scale bar: 100 μm. Original
magnification, ×20 and ×40 (insets). (E) Percentage of disease-free survival after hSTAT5BN642H or WT CD8+ T cell transplantation into nonirradiated
8-week-old Ly5.1/CD45.1 recipient mice. (F) Flow cytometric analysis shows the quantity of splenic CD3+CD8+ cells in CD8+ T cell–transplanted mice. (G)
Spleen versus BW ratios of WT and hSTAT5BN642H CD8+ T cell–transplanted Ly5.1/CD45.1 mice. (A–C) n = 4 WT mice and n = 5 hSTAT5BN642H mice; (E–G) n = 6
WT mice and n = 5 hSTAT5BN642H mice. Data represent the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001, by unpaired, 2-tailed Student’s t test. Figure 5. hSTAT5BN642H CD8+ T cells are the cancer-initiating cells. jci.org Volume 128 Number 1 January 2018 R E S E A R C H A R T I C L E These are components of poly-
comb repressor complex 2 (PCR2), which promotes methylation
of histone 3 at lysine 27 (FDR-adjusted P ≤ 0.05, Fisher’s exact
test) (Figure 8A, top, Supplemental Figure 9A, and Supplemental
Table 4). STAT5 has recently been reported to oppose a network
of transcription factors such as NF-κB and IKAROS in B cell acute
lymphoblastic leukemia (52) and to interact with EZH2 (35). Fur-
thermore, TOP2A expression has been previously linked to EZH2
expression in aggressive prostate cancer (53). Consistently, target
genes of EZH2 and SUZ12 were found to be enriched in CD8+ T
cells derived from hSTAT5BN642H mice (Figure 8B, Supplemental
Figure 9B, and Supplemental Table 5). STAT5BN642H-expressing T cells are sensitive to AURKB inhibition. Among EZH2 target genes, the genes encoding Aurora kinase B
(Aurkb) and DNA topoisomerase 2α (Top2a) were significantly upreg-
ulated, and AURKB targets were highly enriched in hSTAT5BN642H-
expressing CD8+ T cells (Figure 8B and Supplemental Figure 10A). Western blot analysis showed that hSTAT5BN642H mice had higher
AURKB activity, and quantitative PCR (qPCR) analysis validat-
ed the hSTAT5BN642H-dependent upregulation of Aurkb levels in
CD8+ T cells (Figure 9A and Supplemental Figure 10B). This led us
to test the dual-specific JAK and Aurora kinase inhibitor AT9283
as a potential therapeutic in hSTAT5BN642H-expressing cells. We
found that hSTAT5BN642H-expressing T cells were exquisitely more
sensitive to AT9382, with a 10-fold lower IC50 compared with that
of hSTAT5B-expressing T cells (Figure 9B), but not to etoposide,
a TOP2A inhibitor (Supplemental Figure 10C). AT9283 was not
effective in reducing STAT5 activation compared with ruxolitinib
but efficiently reduced AURKB activity (Figure 9C). The high sen- To investigate whether STAT5BN642H has a role in the upregula-
tion of these genes, we performed ChIP with isolated CD8+ T cells
from WT, hSTAT5B, and hSTAT5BN642H mice. Given its hyperac-
tivation status, binding of hSTAT5BN642H to DNA increased com- 3 9 2 jci.org Volume 128 Number 1 January 2018 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 The Journal of Clinical Investigation
R E S E A R C H A R T I C L
Figure 6. hSTAT5BN642H-driven diseased T cells can be treated with JAK inhibitors. (A) WB analysis of pY-STAT5 levels in isolated and cultivated LN T cells
from hSTAT5BN642H, hSTAT5B, and WT mice after IL-2 removal. R E S E A R C H A R T I C L E (B) Dose-response curve of WT, hSTAT5BN642H, and hSTAT5B T cells 72 hours after ruxolitinib
treatment, analyzed using CellTiter-Glo (CTG) assay. IC50 values were determined using GraphPad Prism. Error bars indicate the mean ± SEM. DMSO (100%
viability) and 10 μM bortezomib (0% viability) on each plate served as controls. (C) WB of hSTAT5BN642H, hSTAT5B, and WT T cell cultures after 5 hours of
treatment with ruxolitinib or tofacitinib, analyzed for pY-STAT5. (D) Macroscopic view of LNs and spleens from CD8+ T cell–transplanted mice treated with
ruxolitinib compared with vehicle controls. CD8+ T cell–recipient mice were treated with ruxolitinib at the dosage of 45 mg/kg twice a day for 30 days. (E)
Quantification of spleen versus BW ratio of vehicle- and ruxolitinib-treated hSTAT5BN642H CD8+ cell–transplanted mice. (F) WBC counts of vehicle- and
ruxolitinib-treated hSTAT5BN642H CD8+ cell–transplanted mice, measured using a scil Vet ABC animal blood counter. Flow cytometric analysis of CD25
expression in peripheral blood CD8+ T cells. MFI, mean fluorescence intensity. (G) Histological analysis of CD3+ cells in the lungs of recipient mice after
treatment with ruxolitinib compared with the vehicle-treated group. Scale bar: 100 μm. Original magnification, ×20 and ×40 (insets). n = 5 vehicle-treated
mice and n = 4 ruxolitinib-treated mice. Data represent the mean ± SD. n ≥ 6. *P < 0.05, by unpaired, 2-tailed Student’s t test. Data presented in A–C are
representative of 3 independent experiments. Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 The Journal of Clinical Investigation The Journal of Clinical Investigation R E S E A R C H A R T I C L E Figure 6. hSTAT5BN642H-driven diseased T cells can be treated with JAK inhibitors. (A) WB analysis of pY-STAT5 levels in isolated and cultivated LN T cells
from hSTAT5BN642H, hSTAT5B, and WT mice after IL-2 removal. (B) Dose-response curve of WT, hSTAT5BN642H, and hSTAT5B T cells 72 hours after ruxolitinib
treatment, analyzed using CellTiter-Glo (CTG) assay. IC50 values were determined using GraphPad Prism. Error bars indicate the mean ± SEM. DMSO (100%
viability) and 10 μM bortezomib (0% viability) on each plate served as controls. (C) WB of hSTAT5BN642H, hSTAT5B, and WT T cell cultures after 5 hours of
treatment with ruxolitinib or tofacitinib, analyzed for pY-STAT5. (D) Macroscopic view of LNs and spleens from CD8+ T cell–transplanted mice treated with
ruxolitinib compared with vehicle controls. R E S E A R C H A R T I C L E CD8+ T cell–recipient mice were treated with ruxolitinib at the dosage of 45 mg/kg twice a day for 30 days. (E)
Quantification of spleen versus BW ratio of vehicle- and ruxolitinib-treated hSTAT5BN642H CD8+ cell–transplanted mice. (F) WBC counts of vehicle- and
ruxolitinib-treated hSTAT5BN642H CD8+ cell–transplanted mice, measured using a scil Vet ABC animal blood counter. Flow cytometric analysis of CD25
expression in peripheral blood CD8+ T cells. MFI, mean fluorescence intensity. (G) Histological analysis of CD3+ cells in the lungs of recipient mice after
treatment with ruxolitinib compared with the vehicle-treated group. Scale bar: 100 μm. Original magnification, ×20 and ×40 (insets). n = 5 vehicle-treated
mice and n = 4 ruxolitinib-treated mice. Data represent the mean ± SD. n ≥ 6. *P < 0.05, by unpaired, 2-tailed Student’s t test. Data presented in A–C are
representative of 3 independent experiments. Discussion sitivity of AT9283 could be an attribute of Aurora serine/threonine
and JAK tyrosine/serine kinase combinatorial inhibition, as IC50
values of ruxolitinib and tofacitinib were similar in all genotypes
(Figure 6 and Supplemental Figure 6). Combinatory treatment with
ruxolitinib and AZD1152, an AURKB-specific inhibitor, showed an
additive effect, which further supported our hypothesis (Supple-
mental Figure 10D). Although AZD1152 treatment did not affect
STAT5 phosphorylation in all genotypes, it efficiently inhibited
AURKB activity in hSTAT5BN642H-expressing T cells (Figure 9D). Here, we provide evidence that the STAT5BN642H mutation is a
direct driver and not a bystander mutation for lymphoid malig-
nancy. Expression of hSTAT5BN642H triggers the development of
leukemia or lymphoma characterized by highly proliferative and
invasive CD8+ T cells. hSTAT5BN642H activation remains large-
ly cytokine dependent, which renders the diseased cells sensi-
tive to JAK inhibition. When comparing T cells from transgenic
hSTAT5BN642H mice with those from their hSTAT5B counterparts, jci.org Volume 128 Number 1 January 2018 3 9 3 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 The Journal of Clinical Investigation The Journal of Clinical Investigation The Journal of Clinical Investigation R E S E A R C H A R T I C L E Figure 7. hSTAT5BN642H provokes substantial changes in gene expression, accompanied by specific changes in DNA methylation of CD8+ T cells. (A) Heatmap
showing Z scores of rlog-transformed and library size–normalized counts of genes upregulated (red) or downregulated (blue) in hSTAT5B or hSTAT5BN642H
and WT CD8+ T cells (FDR-adjusted P < 0.05). Analyses included 13-week-old WT (n = 5), hSTAT5B (n = 4), and hSTAT5BN642H (n = 5) mice. Each column in the
heatmap represents data from CD8+ T cells from 1 mouse of a given genotype, and each row represents data for a given gene. (B) Enrichment blot of the CD8+
T cell lymphoma expression signature. Barcode blot indicates the position of the gene in the gene set. Red and blue colors represent, respectively, positive
and negative Pearson’s correlations with hSTAT5BN642H CD8+ T cells. The gene set was obtained from published gene signature cytotoxic T cells (43, 44). (C) Top
enriched gene sets are the results of GSEA including E2F target, G2M checkpoint, MYC target, and cell-cycle progression in hSTAT5BN642H CD8+ T cells. P values
in B and C were determined by Kolmogorov-Smirnov test. Discussion (A) Heatmap stantial changes in gene expression, accompanied by specific changes in DNA methylation of CD8+ T cells. (A) Heatmap Figure 7. hSTAT5BN642H provokes substantial changes in gene expression, accompanied by specific changes in DNA me Figure 7. hSTAT5BN642H provokes substantial changes in gene expression, accompanied by specific changes in DNA methylation of CD8+ T cells. (A) Heatmap
showing Z scores of rlog-transformed and library size–normalized counts of genes upregulated (red) or downregulated (blue) in hSTAT5B or hSTAT5BN642H
and WT CD8+ T cells (FDR-adjusted P < 0.05). Analyses included 13-week-old WT (n = 5), hSTAT5B (n = 4), and hSTAT5BN642H (n = 5) mice. Each column in the
heatmap represents data from CD8+ T cells from 1 mouse of a given genotype, and each row represents data for a given gene. (B) Enrichment blot of the CD8+
T cell lymphoma expression signature. Barcode blot indicates the position of the gene in the gene set. Red and blue colors represent, respectively, positive
and negative Pearson’s correlations with hSTAT5BN642H CD8+ T cells. The gene set was obtained from published gene signature cytotoxic T cells (43, 44). (C) Top
enriched gene sets are the results of GSEA including E2F target, G2M checkpoint, MYC target, and cell-cycle progression in hSTAT5BN642H CD8+ T cells. P values
in B and C were determined by Kolmogorov-Smirnov test. (D) Scatterplot contrasting the mean DNA methylation levels in WT and hSTAT5BN642H-mutant T cells
in all CGIs covered in at least 1 sample per genotype (n = 15,209). The density of data points in each plot region is indicated by color intensity, and CGIs with
lower DNA methylation in WT (n = 770) or hSTAT5BN642H (n = 610) cells are indicated by black and red crosses, respectively (absolute difference ≥5 percentage
points, n = 2 per genotype). Analyses included 13-week-old mice. NES, normalized enrichment score. 59, 60). Transgenic mouse models expressing high levels of murine
Stat5a or Stat5b developed lymphoblastic lymphoma at low pene-
trance (5%–25%) and with a late onset (up to 456 days) (28, 61). we found reduced DNA methylation of EZH2-binding sites. This
correlated with an increase in the transcription of STAT5B and
EZH2 target genes including the cell-cycle regulators Top2A and
Aurkb, for which AURKB represents a potential therapeutic target. Discussion (D) Scatterplot contrasting the mean DNA methylation levels in WT and hSTAT5BN642H-mutant T cells
in all CGIs covered in at least 1 sample per genotype (n = 15,209). The density of data points in each plot region is indicated by color intensity, and CGIs with
lower DNA methylation in WT (n = 770) or hSTAT5BN642H (n = 610) cells are indicated by black and red crosses, respectively (absolute difference ≥5 percentage
points, n = 2 per genotype). Analyses included 13-week-old mice. NES, normalized enrichment score. Figure 7. hSTAT5BN642H provokes substantial changes in gene expression, accompanied by specific changes in DNA methylation of CD8+ T cells. (A) Heatmap
showing Z scores of rlog-transformed and library size–normalized counts of genes upregulated (red) or downregulated (blue) in hSTAT5B or hSTAT5BN642H
and WT CD8+ T cells (FDR-adjusted P < 0.05). Analyses included 13-week-old WT (n = 5), hSTAT5B (n = 4), and hSTAT5BN642H (n = 5) mice. Each column in the
heatmap represents data from CD8+ T cells from 1 mouse of a given genotype, and each row represents data for a given gene. (B) Enrichment blot of the CD8+
T cell lymphoma expression signature. Barcode blot indicates the position of the gene in the gene set. Red and blue colors represent, respectively, positive
and negative Pearson’s correlations with hSTAT5BN642H CD8+ T cells. The gene set was obtained from published gene signature cytotoxic T cells (43, 44). (C) Top
enriched gene sets are the results of GSEA including E2F target, G2M checkpoint, MYC target, and cell-cycle progression in hSTAT5BN642H CD8+ T cells. P values
in B and C were determined by Kolmogorov-Smirnov test. (D) Scatterplot contrasting the mean DNA methylation levels in WT and hSTAT5BN642H-mutant T cells
in all CGIs covered in at least 1 sample per genotype (n = 15,209). The density of data points in each plot region is indicated by color intensity, and CGIs with
lower DNA methylation in WT (n = 770) or hSTAT5BN642H (n = 610) cells are indicated by black and red crosses, respectively (absolute difference ≥5 percentage
points, n = 2 per genotype). Analyses included 13-week-old mice. NES, normalized enrichment score. BN642H provokes substantial changes in gene expression, accompanied by specific changes in DNA methylation of CD8+ T c Figure 7. hSTAT5BN642H provokes substantial changes in gene expression, accompanied by specific changes in DNA methylation of CD8+ T cells. 3 9 4
jci.org Volume 128 Number 1 January 2018 Discussion We now show that moderate expression of hSTAT5BN642H,
but not hSTAT5B, is sufficient to trigger an aggressive disease
that causes rapid lethality at a young age, with full penetrance
irrespective of gender, demonstrating the potent oncogenic
role of the hSTAT5BN642H mutation. Despite the Vav1 promoter–
dependent expression of hSTAT5BN642H throughout the entire
hematopoietic system, malignancy evolved in CD8+ T cells. This T cells express considerably more STAT5B than do other cell
types of the hematopoietic system (54–57), suggesting a privileged
role for STAT5B in the T cell compartment (58). Moreover, STAT5B
is the dominant STAT5 protein in effector and regulatory T cells,
and the differences in STAT5A and STAT5B governing T cell func-
tion are largely associated with paralog expression differences (7, 3 9 4
jci.org Volume 128 Number 1 January 2018 3 9 4 jci.org Volume 128 Number 1 January 2018 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 The Journal of Clinical Investigation The Journal of Clinical Investigation R E S E A R C H A R T I C L E ure 8. hSTAT5BN642H-driven DNA methylation changes accompanied by enhanced DNA-binding activity of STAT5 result in the induction of Aurora
ase B. (A) Region set enrichment analysis testing CGIs with lower DNA methylation in hSTAT5BN642H cells than in WT cells (top) or lower DNA methyla-
n in WT cells than in hSTAT5BN642H cells (bottom). Enrichment was determined using LOLA (51). Each dot represents 1 ChIP-seq experiment for a given
nscription factor from the CODEX database. The vertical dashed line represents the significance threshold (FDR-adjusted P ≤ 0.05). (B) Enrichment
t of EZH2 target genes in HSCs, together with their methylation states of EZH2-bound and EZH2-unbound CGIs 100 kb up- and downstream of the
nscriptional start sites (TSSs). Barcode blot indicates the position of the gene in the gene set. Red and blue colors represent, respectively, positive and
gative Pearson’s correlations with hSTAT5BN642H CD8+ T cells. The gene set was obtained from the MSigDB (72). Black circles indicate CGIs overlapping
h EZH2-binding sites. p.p., percentage points. n = 2 per genotype. ChIP with anti-STAT5 (C) or anti-EZH2 (D) in CD8+ T cells isolated from WT (n = 7),
TAT5B (n = 7), or hSTAT5BN642H (n = 4) mice. Discussion Binding of STAT5 to the Cis and Ccnd2 promoters or binding of EZH2 to the promoter regions of Cdkn2A and
nd2 served as positive controls. Horizontal dotted line indicates the threshold for nonspecific binding. (E) ChIP with anti-STAT5, anti-EZH2, or IgG in
AT5BN642H-expressing CD8+ T cells, followed by WB analysis. IB, immunoblot. Data presented in C–E are representative of 2 independent experiments. or bars indicate the mean ± SD. Figure 8. hSTAT5BN642H-driven DNA methylation changes accompanied by enhanced DNA-binding activity of STAT5 re Figure 8. hSTAT5BN642H-driven DNA methylation changes accompanied by enhanced DNA-binding activity of STAT5 result in the induction of Aurora
kinase B. (A) Region set enrichment analysis testing CGIs with lower DNA methylation in hSTAT5BN642H cells than in WT cells (top) or lower DNA methyla-
tion in WT cells than in hSTAT5BN642H cells (bottom). Enrichment was determined using LOLA (51). Each dot represents 1 ChIP-seq experiment for a given
transcription factor from the CODEX database. The vertical dashed line represents the significance threshold (FDR-adjusted P ≤ 0.05). (B) Enrichment
blot of EZH2 target genes in HSCs, together with their methylation states of EZH2-bound and EZH2-unbound CGIs 100 kb up- and downstream of the
transcriptional start sites (TSSs). Barcode blot indicates the position of the gene in the gene set. Red and blue colors represent, respectively, positive and
negative Pearson’s correlations with hSTAT5BN642H CD8+ T cells. The gene set was obtained from the MSigDB (72). Black circles indicate CGIs overlapping
with EZH2-binding sites. p.p., percentage points. n = 2 per genotype. ChIP with anti-STAT5 (C) or anti-EZH2 (D) in CD8+ T cells isolated from WT (n = 7),
hSTAT5B (n = 7), or hSTAT5BN642H (n = 4) mice. Binding of STAT5 to the Cis and Ccnd2 promoters or binding of EZH2 to the promoter regions of Cdkn2A and
Ccnd2 served as positive controls. Horizontal dotted line indicates the threshold for nonspecific binding. (E) ChIP with anti-STAT5, anti-EZH2, or IgG in
STAT5BN642H-expressing CD8+ T cells, followed by WB analysis. IB, immunoblot. Data presented in C–E are representative of 2 independent experiments. Error bars indicate the mean ± SD. STAT5BN642H has been shown previously to render Ba/F3 cells
cytokine independent and to be constitutively active in HeLa cells
(13, 15, 17). jci.org Volume 128 Number 1 January 2018 Discussion IC50 values were determined using
GraphPad Prism. Error bars indicate the mean ± SEM. DMSO (100% viability) and 10 μM bortezomib (0% viability) on each plate served as controls. n = 6
per genotype. (C and D) WB of hSTAT5BN642H, hSTAT5B, and WT T cell cultures after 5 hours of treatment with AT9283 or AZD1152, for determination of
pY-STAT5, total STAT5, p-AURKB, total AURKB, p-H3 (Ser10), and total H3 levels. Data presented in A–D are representative of 3 independent experiments. Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 The Journal of Clinical Investigation The Journal of Clinical Investigation R E S E A R C H A R T I C L E Figure 9. hSTAT5BN642H-driven diseased T cells are sensitive to Aurora kinase B inhibition. (A) WB analysis of p-AURKB, total AURKB, and HSC70 in
LNs from WT and hSTAT5BN642H- and hSTAT5B-transgenic mice. WB quantification (bar graph) was performed using ImageJ. (B) Dose-response curves of
WT, hSTAT5BN642H, or hSTAT5B T cells in response to AT9283 after 72 hours of treatment, analyzed using a CTG assay. IC50 values were determined using
GraphPad Prism. Error bars indicate the mean ± SEM. DMSO (100% viability) and 10 μM bortezomib (0% viability) on each plate served as controls. n = 6
per genotype. (C and D) WB of hSTAT5BN642H, hSTAT5B, and WT T cell cultures after 5 hours of treatment with AT9283 or AZD1152, for determination of
pY-STAT5, total STAT5, p-AURKB, total AURKB, p-H3 (Ser10), and total H3 levels. Data presented in A–D are representative of 3 independent experiments. gested that STAT5 and EZH2 compete for binding to regulatory
sites, as shown in B cells and mammary epithelial cells (52, 75). We observed that, as a consequence of STAT5B hyperactivation,
STAT5BN642H bound more to DNA and subsequently upregulated
many cell-cycle–regulating genes including Top2a and Aurkb. The
fact that the cells were particularly sensitive to Aurora kinase inhi-
bition underlines this observation. tantly, the most upregulated genes were E2F and MYC targets,
which highlights the proliferative nature of the diseased T cells and
explains the upregulation of numerous genes (65). STAT5BN642H
is hyperphosphorylated, and it would be interesting to study its
potential different interactions with CD8+ T cell–specific activators
or repressors compared with the less active WT STAT5B. Discussion Ba/F3 cells have been used to determine the oncogenic
potential of many leukemogenic drivers, however, the expression
level of the oncogene is often very high, and the cells might have
acquired additional mutations as a result of long-term cultivation. In cytokine-independent cell lines such as HeLa or HEK293T,
STAT5 might be activated by other available growth stimuli. Cells
expressing low levels of STAT5BN642H, however, remain dependent
on cytokine stimulation, as shown in our diseased T cell model. This was also observed in NK cells by Küçük and colleagues (18). development could be a result of the CD8+ T cell sensitivity to
the Stat5a/b gene dosage that was described previously in mice
(62). Moreover, it has been reported that CD8+ T cells are more
susceptible to oncogenic drivers, especially when these drivers
are activated by cytokines or triggered via T cell receptors (62,
63). Similarly, hSTAT5BN642H activation remains cytokine depen-
dent, and the upregulation of IL-2Rα, a direct target of STAT5,
resulted in CD8+ T cells becoming more sensitive to low doses
of cytokine stimulation. So far, hSTAT5BN642H mutations have
primarily been found in patients with T cell or NK cell malignan-
cies, pointing toward the sensitivity of these patients to aberrant
STAT5 activation. When STAT5BN642H was identified in CD8+ T
cells in patients, such as those with T cell LGL (T-LGL) or epi-
theliotropic intestinal T cell lymphoma (13, 23, 64), it gave rise to
more aggressive disease (26). The malignant transformation and expansion of CD8+ T cells
in transgenic mice correlated with the upregulation of direct STAT5
target genes such as D-type cyclins, Bcl2 family members, and Pim
kinases, which promote cell-cycle progression and survival. Impor- 3 9 5 jci.org Volume 128 Number 1 January 2018 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 The Journal of Clinical Investigation
R E S E A R C H A R T I C L E
Figure 9. hSTAT5BN642H-driven diseased T cells are sensitive to Aurora kinase B inhibition. (A) WB analysis of p-AURKB, total AURKB, and HSC70 in
LNs from WT and hSTAT5BN642H- and hSTAT5B-transgenic mice. WB quantification (bar graph) was performed using ImageJ. (B) Dose-response curves of
WT, hSTAT5BN642H, or hSTAT5B T cells in response to AT9283 after 72 hours of treatment, analyzed using a CTG assay. jci.org Volume 128 Number 1 January 2018 Methods Plasmid construction/mutagenesis and transfection. hSTAT5B variants
were generated using site-directed mutagenesis (80). Mutagenic PCR
was performed using KOD Polymerase (Novagen). PCR products were
subsequently digested with DpnI enzyme (New England BioLabs) to
remove the methylated template according to the manufacturer’s pro-
tocol. E. coli was transformed with the digested reaction, and positive
clones were selected by Sanger sequencing (81). Plasmid transfection
was performed using Lipofectamine 2000 Reagent (Invitrogen, Ther-
mo Fisher Scientific). Cytokine stimulation of T cells was performed with human IL-2
(100 U/ml; ProleukinÒ; Novartis), murine IL-4 (100 ng/ml; R&D Sys-
tems), or murine IL-7 (10 ng/ml; R&D Systems). The 293T and 32D cell lines were gifts of M. Hengstschläger
(Center of Pathobiochemistry and Genetics, Institute of Medical
Genetics, Medical University of Vienna, Vienna, Austria) and F. Grebien (Ludwig Boltzmann Institute for Cancer Research, Vienna,
Austria), respectively. The Ba/F3 cell line was provided by A. D’An-
drea (Dana-Farber Cancer Institute, Harvard Medical School, Boston,
Massachusetts, USA). The cases of patients harboring the STAT5BN642H mutation were
assembled from previously published work (13–26, 36). Animals and generation of transgenic mice. Transgenic mice were
generated and bred on a C57BL/6NCrl background and maintained
in a specific pathogen–free environment in the experimental mouse
facility at the University of Veterinary Medicine (Vienna, Austria). We used the Vav1-hematopoietic vector Vav1-hCD4 (HS21/45) (37)
to generate several transgenic mouse lines expressing hSTAT5B and
hSTAT5BN642H in the hematopoietic system and selected the lines
B6N-Tg(STAT5B)731Biat and B6N-Tg(STAT5BN/H)726Biat, respec-
tively, for further experiments. The hSTAT5BN642H construct was gen-
erated using overlapping PCR technology as previously described (80)
(forward primer: GAAAGAATGTTTTGGCATCTGATGCCTTTTAC;
reverse primer: GTAAAAGGCATCAGATGCCAAAACATTCTTTC). The construct was digested with the HindIII restriction enzyme and
gel purified for pronuclear injection (82). The transgenic mice were
identified by genotyping PCR (forward primer: ACGCAGGACACA-
GAGAATGAG; reverse primer: GTGATGGTGGCGTTGACCTC). WT
(C57BL/6NCrl) mice and B6-Ly5.1/Cr (B6.SJL-PtprcaPepcb/BoyCrCrl)
mice were purchased from Charles River Laboratories, and NSG
(NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice were purchased from The Jack-
son Laboratory. Given the rapid development and strong phenotype of
the hSTAT5BN642H-transgenic mice, the colony was propagated via in
vitro fertilization with archived sperm cells (83). Transplantation experiments. BM cells (1 × 106) from hSTAT5BN642H
or WT mice were transplanted by lateral tail vein injection into nonirra-
diated NSG mice. Mice were monitored daily and evaluated at the first
sign of disease onset. Methods CD8+ T cells were isolated using a CD8+ Magni
Sort Mouse T Cell Enrichment Kit (eBioscience), and sorted cells were
checked with flow cytometry for their purity. Cells (1 × 106) were inject-
ed i.v. into nonirradiated Ly5.1/CD45.1 mice. IHC. Mouse organs were incubated overnight in 4% phos-
phate-buffered formaldehyde solution (Roti-Histofix; Carl Roth),
dehydrated, embedded, and cut (4-μm-thick sections). For immu-
nohistochemical staining, heat-mediated antigen retrieval was per-
formed in citrate buffer at pH 6.0 (Dako) and stained with antibod-
ies against CD3 (Thermo Fisher Scientific; RM-9107-S0; dilution
1:300); Ki67 (Novocastra, Leica Biosystem; NCL-Ki67p; dilution
1:1,000); and STAT5B (Santa Cruz Biotechnology; sc-1656; dilution
1:200) using standard protocols. Images were taken using a Zeiss
Imager Z.1 microscope. Western blot analysis. Western blotting (WB) was performed using
standard protocols. The antibodies used were: monoclonal rabbit anti-
mouse phosphorylated STAT5 (p-STAT5) (Invitrogen, Thermo Fisher
Scientific; 716900; dilution 1:1,000); purified mouse anti-STAT5 (BD;
610191; dilution 1:2,000); monoclonal mouse anti-mouse HSC70
(Santa Cruz Biotechnology; sc-7298; dilution 1:10,000); monoclonal
mouse anti-Flag M2 ( MilliporeSigma; F3156; dilution 1:1,000); mono-
clonal rabbit anti–p–Aurora A (Thr288), p–Aurora B (Thr232), and
p–Aurora C (Thr198) (Cell Signaling Technology; 2914; 1:1,000);
monoclonal rabbit anti-Aurora B/AIM1 (Cell Signaling Technology;
3094; 1:1,000); monoclonal rabbit anti–histone H3 (anti-H3) (Cell
Signaling Technology; 4499; 1:1,000); monoclonal rabbit anti–p-H3
(Ser10) (Cell Signaling Technology; 53348; 1:1,000); ECL anti-mouse
IgG; HRP-linked whole antibody from sheep (GE Healthcare; NA931V;
dilution 1:10,000); ECL anti-rabbit IgG; and HRP-linked whole anti-
body from sheep (GE Healthcare; NA934V; dilution 1:10,000). WB
quantification was performed using ImageJ software (NIH). (See the
complete unedited blots in the supplemental material.) Hematocytometry and flow cytometry. Mouse blood was collect-
ed into EDTA tubes (Greiner Bio-One Mini-Collect K3EDTA Tubes;
Thermo Fisher Scientific) from the facial vein or from euthanized
mice via cardiac puncture, and blood smears were stained using mod-
ified Wright staining. WBC counts were measured using an animal
blood counter (scil Vet ABC). For flow cytometry, erythrocytes were lysed using Gay’s solution
(10 mM KHCO3 and 75 mM NH4Cl, pH 7.4). Single-cell suspensions
were prepared by mincing organs through a 70-μm cell strainer (BD
Biosciences). HSC staining was performed as previously described
(84). All antibodies used for flow cytometry were purchased from
eBioscience and BD (see Supplemental Table 6 for the list of the anti-
bodies). All analyses were performed on the BD FACSCanto II using
FACSDiva software (BD). Further analysis was performed using
FlowJo software. Discussion Recent work suggested that altered DNA methylation patterns
in T cells are indicative or even causative for T cell transformation
and that methylation of gene bodies was correlated with active
transcription contributing to carcinogenesis (66, 67). Epigenetic
regulators such as EZH2, TET1/2, and HDAC play important roles
in leukemogenesis (68–71) and have been shown to interact with
STAT5 (30, 31, 34, 35). EZH2 has been linked to the long-term
repopulating capability, proliferation, and inhibition of apoptosis
of HSCs (72, 73), all of which are important for transformed cells as
well as for governing peripheral T cell fates (74). We demonstrate
here that the expression of hSTAT5BN642H not only led to transcrip-
tional changes but also changed DNA methylation. Decreased
methylation at EZH2- and SUZ12-binding sites in hSTAT5BN642H
T cells resulted in the upregulation of EZH2 target genes. There
are conflicting reports regarding the interaction between EZH2
and STAT5. In 2011, Mandal and colleagues reported that STAT5
plays an essential part in the recruitment of EZH2 to repress Ig
κ-chain (Igk) transcription in progenitor B cells (35). Others sug- Work by many groups identified STAT5 as an important tar-
get for therapy, since it is essential for JAK2V617F-, Flt3-ITD-, and
BCR/ABL-driven diseases (76–78). Currently, intensive efforts
are being made to inhibit STAT5 by blocking its SH2 domain (79). However, effective targeting of STAT5 remains challenging. Sev-
eral different strategies have been suggested for the treatment
of hSTAT5BN642H-expressing cells including the use of BCL2,
MEK1/2, and JAK inhibitors (17, 18). Although some patients
respond to JAK inhibitors, the lack of sensitivity in other patients
requires broader therapeutic targets (16, 17). We believe that the
hSTAT5BN642H-transgenic mouse model will serve as a valuable
preclinical model. Using this model, we showed that the com-
bined use of Aurora kinase and JAK inhibitors is a potential thera-
peutic strategy to treat lymphoma and leukemia patients with the
STAT5BN642H mutation. We show here that hSTAT5BN642H acts as a driver mutation in
the development of leukemia and lymphoma and propose that 3 9 6 jci.org Volume 128 Number 1 January 2018 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 R E S E A R C H A R T I C L E The Journal of Clinical Investigation upstream inhibition of JAK activation or the chromatin-remodel-
ing partners of STAT5 could be an alternative targeting strategy
for enhanced STAT5 activation. mycin) (all from Gibco, Thermo Fisher Scientific) supplemented with
IL-3 (1 ng/ml; ImmunoTools). IL-3 stimulation was performed with 10
ng/ml IL-3 for 20 minutes. The hSTAT5BN642H, hSTAT5B, and B6N WT T cells were isolated
from LNs and spleens from 8- to 12-week-old mice. Following T cell
activation by anti-CD3 (BD), T cells were grown in complete RPMI
1640 medium containing 10 mM HEPES, 1× MEM nonessential ami-
no acids, 50 μM β-mercaptoethanol (all from Gibco, Thermo Fisher
Scientific), 1 mM sodium pyruvate (MilliporeSigma), and 100 U/ml
human IL-2 (ProleukinÒ; Novartis). R E S E A R C H A R T I C L E After alignment of reads against contam-
inating sequences (mitochondrial and ribosomal DNA), the remain-
ing reads were aligned against GRCm37 using transcriptome-guided
alignment with TopHat, version 1.4.1 (http://ccb.jhu.edu/software/
tophat/index.shtml). Next, the htseq-count (http://htseq.readthedocs. io/en/master/count.html) with mode union was used to obtain gene
counts for union gene models. Then, differentially expressed genes
(log2 fold change >2 and FDR-adjusted q < 0.1) were determined using
DESeq2, version 1.12.4 (Bioconductor). For heatmaps, centered and scaled rlog-transformed library size–
normalized counts were visualized using the heatmap.2 function of
R package gplots, version 3.0.1 (https://www.rdocumentation.org/
packages/gplots/versions/3.0.1). Viability assay. Murine T cells (5 × 104) from hSTAT5BN642H and
WT mice were seeded in triplicate in 96-well plates. JQ1, 5-azacyti-
dine, entinostat, etoposide, AT9283, tofacitinib, and ruxolitinib (all
from Selleckchem) were added and incubated for 72 hours. All com-
pounds were solubilized in DMSO (MilliporeSigma). DMSO and bor-
tezomib (Selleckchem) were used as a negative and positive control,
respectively. CellTiter-Glo reagent (Promega) was used to determine
viability, measured on an EnSpire plate reader (PerkinElmer). IC50 val-
ues were determined by nonlinear regression using GraphPad Prism 6
(GraphPad Software). Gene lists from differential expression analyses were ranked for
the log2 fold changes between hSTAT5BN642H and WT or hSTAT5BN642H
and hSTAT5B CD8+ T cells. Ranking lists were subsequently used for
GSEA via the Broad Institute’s GSEAPreranked tool at the standard
setting. Gene sets were obtained from current publications or from the
Broad Institute’s Molecular Signatures Database (MSigDB). RNA-seq
data and a description of the experimental design are available in the
NCBI’s Gene Expression Omnibus (GEO) database (GEO GSE104557). RRBS and analysis. Genomic DNA from purified CD8+ T cells
was isolated using an AllPrep DNA/RNA Mini Kit (QIAGEN) and
subsequently subjected to RRBS and analysis. RRBS was carried out
as described earlier (85). In brief, 100 ng genomic DNA was digest-
ed for 12 hours at 37°C with 20 units of MspI (New England Bio-
Labs; R0106L) in 30 μl of 1× NEB Buffer 2. Fill-in and A-tailing were
performed by the addition of Klenow Fragment 3′→ 5′ exo- (New
England BioLabs; M0212L) and dNTP mix (10 mM dATP, 1 mM
dCTP, 1 mM dGTP). After ligation to methylated Illumina TruSeq LT
v2 adaptors using Quick Ligase (New England BioLabs; M2200L),
the libraries were size selected by performing a 0.75× clean-up with
AMPure XP beads (Beckman Coulter; A63881). R E S E A R C H A R T I C L E Up to 12 libraries
were pooled in equal amounts on the basis of qPCR data and bisulfite
converted using the EZ DNA Methylation Direct Kit (Zymo Research;
D5020) with the following changes to the manufacturer’s protocol:
the conversion reagent was used at ×0.9 concentration; incubation
was performed for 20 cycles of 1 minute each at 95°C, followed by
10 minutes at 60°C; and the desulfonation time was extended to
30 minutes. Bisulfite-converted libraries were enriched for up to 17
cycles using PfuTurbo Cx Hotstart DNA Polymerase (Agilent Tech-
nologies; 600412). After a 2× AMPure XP clean-up, quality control
was performed using a Qubit dsDNA HS Assay Kit (Life Technolo-
gies, Thermo Fisher Scientific; Q32854) and an Experion DNA 1K
Analysis Kit (Bio-Rad; 700-7107). Sequencing was performed on an
Illumina HiSeq 3000/4000 System using the 5-bp single-end mode. Initial data processing was carried out at the Biomedical Sequencing
Facility of the Medical University of Vienna (http://www.biomed-
ical-sequencing.at) using an in-house pipeline based on Pypiper
(http://databio.org/pypiper) and Looper (http://databio.org/loop- In vivo ruxolitinib treatment. hSTAT5BN642H CD8+ T cell transplant
recipients were treated with ruxolitinib (Chemietek) twice a day by
oral gavage at a dosage of 45 mg/kg. Ruxolitinib was dissolved in
DMSO (MilliporeSigma) and subsequently diluted in 0.5% methylcel-
lulose (w/v) (MilliporeSigma). ChIP. CD8+ T cells (107 cells) from WT, hSTAT5B, and hSTAT5BN642H
mice were isolated using a CD8+ MagniSort Enrichment Kit. Isolat-
ed cells were washed twice with ice-cold PBS supplemented with
inhibitors (1 mM Na3VO4, 1 mM NaF, 1× cOmplete Protease Inhibitor
Cocktail [PIC], Roche) and fixed with DSG (2 mM, 30 min; Thermo
Fisher Scientific). Cells were washed twice with cold PBS supple-
mented with inhibitor and fixed with formaldehyde (1%, 10 min;
MilliporeSigma). Fixation was quenched by incubation with glycine
(125 mM, 5 min; MilliporeSigma). T cells were subsequently harvest-
ed by centrifugation (350 g, 5 min). Cell lysis was performed with
1% SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris [pH 8.1],
1 mM Na3VO4, 1 mM phenylmethylsulphonyl) at 4°C for 30 minutes
and sonicated using a Diagenode Bioruptor (20 cycles with 30 sec-
onds on, 30 seconds off, high magnitude). Sonication was followed
by chromatin dilution (1:10) in dilution buffer (167 mM NaCl, 16.7
mM Tris [pH 8.1], 1.2 mM EDTA, 1.1% Triton-X, 0.01% SDS). Clear
chromatin was harvested by centrifugation (10,000 g, 10 min, 4°C). R E S E A R C H A R T I C L E er). Sequences were trimmed for adapters using Trimmomatic (86)
with the ILLUMINACLIP settings “:2:40:7 SLIDINGWINDOW:4:15
MAXINFO:20:0.50 MINLEN:18.” All reads were aligned to the
GRCm38 (mm10) assembly of the mouse genome using BSMAP in its
RRBS mapping mode (87, 88). DNA methylation levels for individual
CpGs were calculated using custom Python scripts and loaded into
RnBeads (89) for exploratory analysis and to aggregate DNA meth-
ylation estimates per CGI. The aggregated values were loaded into R
for further analysis. Differentially methylated regions (absolute dif-
ference ≥5 percentage points) were compared with ChIP-seq peaks
from the CODEX database (90) using LOLA (51) to find significant
overlaps (FDR-adjusted P ≤ 0.05) with potential regulators and effec-
tors of DNA methylation differences. To compare DNA methylation
at CGIs with genes, each CGI was associated with all genes within a
10-kb window after conversion of the gene coordinates to the mm10
reference genome using the UCSC LiftOver tool (https://genome. ucsc.edu/cgi-bin/hgLiftOver). RRBS sequencing data were deposited
in the NCBI’s GEO database (GEO GSE104557). er). Sequences were trimmed for adapters using Trimmomatic (86)
with the ILLUMINACLIP settings “:2:40:7 SLIDINGWINDOW:4:15
MAXINFO:20:0.50 MINLEN:18.” All reads were aligned to the
GRCm38 (mm10) assembly of the mouse genome using BSMAP in its
RRBS mapping mode (87, 88). DNA methylation levels for individual
CpGs were calculated using custom Python scripts and loaded into
RnBeads (89) for exploratory analysis and to aggregate DNA meth-
ylation estimates per CGI. The aggregated values were loaded into R
for further analysis. Differentially methylated regions (absolute dif-
ference ≥5 percentage points) were compared with ChIP-seq peaks
from the CODEX database (90) using LOLA (51) to find significant
overlaps (FDR-adjusted P ≤ 0.05) with potential regulators and effec-
tors of DNA methylation differences. To compare DNA methylation
at CGIs with genes, each CGI was associated with all genes within a
10-kb window after conversion of the gene coordinates to the mm10
reference genome using the UCSC LiftOver tool (https://genome. ucsc.edu/cgi-bin/hgLiftOver). RRBS sequencing data were deposited
in the NCBI’s GEO database (GEO GSE104557). were enriched using a CD8+ MagniSort Enrichment Kit, and mRNA
was isolated using TRIzol (MilliporeSigma) in combination with
an RNeasy Mini Kit (QIAGEN). mRNA library preparation (SENSE
mRNA-Seq Library preparation) and RNA sequencing (RNA-seq)
was performed with an Illumina HiSeq 2500 at the Vienna Biocenter
Core Facility (VBCF) Next-Generation Sequencing (NGS) Unit (www. vbcf.ac.at). Adapter trimming and removal of low-quality bases were
performed using cutadapt. Methods Cell culture. 293T cells were cultivated with complete DMEM
medium (10% FCS, 2 mM L-glutamine, 10 U/ml penicillin-streptomy-
cin). Ba/F3 and 32D cells were cultivated with complete RPMI 1640
medium (10% FCS, 2 mM L-glutamine, 10 U/ml penicillin-strepto- RNA sequencing and analysis. mRNA was isolated from CD8+ T
cells harvested from LNs from mice of all 3 genotypes. CD8+ T cells 3 9 7 jci.org Volume 128 Number 1 January 2018 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 2015;208(4):115–128. 10. Bamford S, et al. The COSMIC (Catalogue of
Somatic Mutations in Cancer) database and web-
site. Br J Cancer. 2004;91(2):355–358. 20. Ma X, et al. Rare occurrence of a STAT5B N642H
mutation in adult T-cell acute lymphoblastic leu-
kemia. Cancer Genet. 2015;208(1–2):52–53. 2. Zhang Z, Schwartz S, Wagner L, Miller W. A
greedy algorithm for aligning DNA sequences. J Comput Biol. 2000;7(1–2):203–214. 11. Imada K, et al. Stat5b is essential for natural killer
cell-mediated proliferation and cytolytic activity. J Exp Med. 1998;188(11):2067–2074. 21. Jiang L, et al. Exome sequencing identifies somat-
ic mutations of DDX3X in natural killer/T-cell
lymphoma. Nat Genet. 2015;47(9):1061–1066. 3. Nivarthi H, Friedbichler K, Moriggl R. Stat5 as a
Hematopoietic Master Regulator for Differenti-
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in signaling by IL-2 family cytokines. Oncogene. 2000;19(21):2566–2576. 22. López C, et al. Genes encoding members of the
JAK-STAT pathway or epigenetic regulators are
recurrently mutated in T-cell prolymphocytic
leukaemia. Br J Haematol. 2016;173(2):265–273. 13. Rajala HL, et al. Discovery of somatic STAT5b
mutations in large granular lymphocytic leuke-
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and pathologic hematopoiesis. Front Biosci. 2007;12:2807–2820. 23. Nairismägi ML, et al. JAK-STAT and G-protein-
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of CEBPA mutated AML reveals recurrent CSF3R
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One. 2015;10(11):e0141906. 16. Kiel MJ, et al. Author contributions ium chloride wash buffer (0.5 M LiCl2, 50 mM HEPES, 1 mM EDTA,
0.7% sodium deoxycholate, 1% NP-40) and then once in Tris-EDTA
(TE) buffer containing 50 mM NaCl. Chromatin was eluted in 2× 100
μl elution buffer (1% SDS, 50 mM Tris, 10 mM EDTA). Eluted chro-
matin (20 μl) was used for WB analysis. Samples and inputs were
incubated with 8 μl of 5 M NaCl at 65°C overnight and subsequently
incubated with 0.5 M EDTA, 1 M Tris (pH 6.5), and proteinase K (10
mg/ml) for 2 hours at 55°C. RNA was lysed for 1 hour at 37°C using
0.2 mg/ml RNase-A (MilliporeSigma). Chromatin clean-up was per-
formed using a PCR purification kit (QIAGEN). DNA was subjected
to qPCR using GoTaq Real-Time qPCR (Promega), and the amount
of amplification was quantified using standard curves. Primers are
listed in Supplemental Table 7. RM designed and supervised the study. HTTP, BM, MPM, EG, TJ,
HN, ZK, TK, AB, SK, MF, MM, TR, VS, and RM designed and/or
performed experiments. HTTP, RG, FH, and MPM analyzed data. JP and FG contributed to the interpretation of the data. LK inter-
preted IHC results. ME designed and performed experiments. PV,
MH, and CB revised the manuscript with regard to critical intellec-
tual content. HTTP, BM, VS, and RM wrote the manuscript. R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E Cleared chromatin was incubated with rolling at 4°C with 5 μg STAT5
(C-17) (Santa Cruz Biotechnology; sc-835 X), EZH2 (Diagenode;
pAb-039-050), or IgG (Santa Cruz Biotechnology; sc-2027 X) over-
night at 4°C. Diluted chromatin (1%) was kept as the input. Blocked
Dynal Magnetic Beads (65 μl; Life Technologies, Thermo Fisher Sci-
entific) were added per IP the following day and incubated for an
additional 4 hours at 4°C. IP samples were washed 5 times with lith- 3 9 8 jci.org Volume 128 Number 1 January 2018 Downloaded from http://www.jci.org on January 18, 2018. https://doi.org/10.1172/JCI94509 2. Zhang Z, Schwartz S, Wagner L, Miller W. A
greedy algorithm for aligning DNA sequences.
J Comput Biol. 2000;7(1–2):203–214. 1. Stark GR, Darnell JE. The JAK-STAT pathway at
twenty. Immunity. 2012;36(4):503–514. Acknowledgments We would like to thank Gregor Hörmann, Safia Zahma, Graham
Tebb, Michaela Schlederer, Katrin Meissl, Johannes Schmöllerl,
Patricia Stiedl, Helmut Dolznig, Margit Rosner, Thomas Weich-
hart, Claus Vogl, and all members of Ludwig Boltzmann Institute
for Cancer Research (LBI-CR) for their help and support. This
work and RM, HTTP, BM, MM, VS, SK, CB, MF, and PV are sup-
ported by the Austrian Science Fund (FWF) SFB grant F47 and
F61 subprojects F4701-B20, F4704-B20, F4706-B20, F4707-B20,
F6101, F6102, F6105, F6106, and F6107. FG is supported by the
European Research Council (ERC) Starting Grant ONCOME-
CHAML. FH is supported by a postdoctoral fellowship from the
German Research Council (DFG) (HA 7723/1-1). Statistics. Flow cytometric data are reported as the mean ± SD and
were analyzed using GraphPad Prism 6 (GraphPad Software). Differ-
ences were assessed for statistical significance by an unpaired, 2-tailed
Student’s t test and 1-way ANOVA with Bonferroni’s correction. Kaplan-Meier plots were analyzed using a log-rank (Mantel-Cox) test. P values for GSEA were determined using the Kolmogorov-Smirnov
test. A P value of less than 0.05 was accepted as statistically significant. Study approval. All animal experiments were approved by the
institutional ethics committee and the Austrian Ministry BMWFW
authorities under the animal license protocols BMWFW-68.205/
0166-WF/V/3b/2015), BMWFW-68.205/0117-WF/V/3b/2016, and
BMWFW-68.205/0103-WF/V/3b/2015. All mice were bred and main-
tained under standardized conditions at the University of Veterinary
Medicine Vienna. Address correspondence to: Richard Moriggl, Ludwig Boltz-
mann Institute for Cancer Research, Medical University Vien-
na; University of Veterinary Medicine Vienna, Veterinärplatz 1,
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Modelling public attitude towards drone delivery in Germany
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© 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/. Abstract Background Last-mile delivery by drone is expected to be a promising innovation for future urban logistics. How-
ever, in addition to adoption of services by customers, leveraging this delivery method will depend essentially
on a positive public perception of such services in urban airspace. Objective This article provides novel and comprehensive insights into factors driving or impeding citizens’ attitudes
towards drone delivery. Methodology The article develops a structural equation model that derives from a sequential exploratory mixed
methods design. In the first step, factors affecting attitudes towards drone delivery were identified within the scope
of five focus groups and converted into the development of a questionnaire. In the second step, a German pop-
ulation-representative survey was conducted through telephone interviews, which provided reliable data to test
the model (n = 819). Results Expected risks (particularly stress due to traffic in lower airspace, noise, and visual disturbances), as well
as expected benefits (particularly fast and time-flexible delivery), significantly affect attitudes towards drone-based
delivery, while the individual level of technological openness (technophilia) does not have a significant association. Moreover, the model reveals that the expected risks of drone deliveries are stronger associated with public attitude
than with expected benefits. Conclusions The provided framework suggests fashioning policies and drone delivery applications that focus
on mitigating social, spatial, and visual risks while achieving maximum utility for customers. Keywords Drone delivery, Urban air mobility, Public attitude, Acceptance, SEM, Modelling, Expected benefits,
Expected risks, Technophilia Keywords Drone delivery, Urban air mobility, Public attitude, Acceptance, SEM, Modelling, Expected benefits,
Expected risks, Technophilia referred to in this paper, can lift weights of 2–3 kg and
fulfil flight missions within a diameter of about 15 km [3]. Thereby, delivery drones generate plenty of applications
ranging from medical use [4, 5], intralogistical applica-
tions [6] to commercial last mile delivery, e.g. for online
retailing or food delivery [7, 8]. Though still in its infancy,
drone delivery may form a serious option for innovating
future urban and rural transport networks. 1.1 Background Driven by economic motivations of making processes
faster, more flexible, reliable, and paired with the attri-
bution of a “green” transport option, drone delivery has
climbed high on industrial and political agendas world-
wide [1, 2]. Technically, standard delivery drones, as From a customer perspective, drone delivery is said
to address the demand for ever-faster delivery as well as
to open the opportunity for customers to receive their
delivery within a narrow or even well-defined time-
frame [9]. However, while economic and political actors *Correspondence:
Robin Kellermann
robin.kellermann@tu-berlin.de
1 Mobility Research Cluster, Department of Work, Technology
and Participation, Technische Universität Berlin, 10587 Berlin, Germany Kellermann et al.
European Transport Research Review (2023) 15:38
https://doi.org/10.1186/s12544-023-00606-0 Kellermann et al. European Transport Research Review (2023) 15:38
https://doi.org/10.1186/s12544-023-00606-0 European Transport
Research Review Open Access Open Access Modelling public attitude towards drone
delivery in Germany Robin Kellermann1* , Tobias Biehle1 and Hamid Mostofi1 Kellermann et al. European Transport Research Review (2023) 15:38 Page 2 of 14 Page 2 of 14 Nevertheless, acceptance research on consumer adop-
tion of drone delivery and the willingness to pay for drone
delivery services has grown significantly over the past
decade. Drawing on a bibliometric analysis of respective
empirical studies [14], results show that the variables’
intention to use, ‘attitude’, ‘risk perception’, and ‘personal
innovativeness’ are being used most frequently to analyse
the acceptance of drone delivery. Moreover, the devel-
opment of research hypotheses most often derives from
modifications of the Technology Acceptance Model [15],
the Theory of Reasoned Action [16], and the Diffusion
of Innovation model [17]. For example, Yoo et al. [18]
presented an acceptance model with a specific focus on
delivery drones, which applied the Diffusion of Innova-
tion model [17] and the Technology Acceptance Model
to develop their hypotheses. By testing their model, they
showed that the delivery speed, the ease of use, as well
as individual characteristics such as the personal level of
innovativeness, are central in determining the attitude
towards the adoption of drone delivery. The latter is also
confirmed by Hwang and colleagues, who did extensive
research on the adoption of drone-based food delivery in
South Korea [19].i increasingly pursue ambitions to enable deliveries in low-
level airspace, the creation of a ‘third dimension’ for cargo
transport may not only become a disruptive moment for
customers but may also mark a turning point in people’s
perception of space and traditional notions of land use. Given the potential of wider diffusion of drone delivery in
retail business and the respective possible sizes of drone
fleets [10], it remains a relevant question of how citizens
position themselves regarding the collective exposure to
traffic in low-level airspace as a consequence of using the
technology [11]. Thus, the vision of drone delivery not
only involves an individual service choice for customers
but also entails an inherently public dimension. In cur-
rent acceptance research, however, this comprehensive
understanding appears rather underrepresented, posing
a risk to private sector ambitions and the jurisdiction of
policymakers alike. Against this background, this paper presents and tests
a structural equation model (SEM) aiming to explain
the public’s attitude towards the deployment of delivery
drones for consumer goods in public space. As citizens
are both users and bystanders, the model incorporates
relevant aspects of consumer preferences as well as novel
societal and spatial dimensions, which are also expected
to influence attitudes. The selected factors are subdivided
into three latent variables (expected benefits, expected
risks, technophilia) and derive from a sequential explora-
tory mixed methods design. Therefore, in the first step,
a set of focus group discussions enabled to qualitatively
explore and extract genuine acceptance factors. In a sec-
ond step, these factors were investigated in a population-
representative survey conducted in Germany in early
2020. In the third step, a large sample (n = 819) was tested
in our model. Regarding the potential benefits of drone delivery from
a customer point of view, the notion of (food) delivery
services being “green” have shown a relevant positive
association towards the attitude of respondents [20, 21]. Interestingly, lower delivery fees have not been found to
significantly influence the attractiveness of drone deliv-
ery in general [22] but are suggested to be moderated by
the specific context and circumstance, such as the parcel
value or the availability of drop-off locations [23]. From a
consumer risk perception, the functionality of the service
is in question, including risks of technical delivery fail-
ures [24], low service reliability [18], unattended deliver-
ies, and theft [25]. 1.2 Literature review Public support for commercial drone delivery currently
appears low. For example, results of a representative pop-
ulation survey in Germany conducted in early 2020 show
that only 25% of the respondents support or strongly sup-
port the idea of using the technology for the delivery of
consumer goods, while 55% of the respondents disagree
or strongly disagree [12]. Those results correspond to a
cross-European study by EASA [13], in which partici-
pants were asked to rank their three most useful drone
applications out of fourteen possible. Drone delivery of
goods ordered through online shopping was a priority
for about 20% of the respondents. Moreover, only 18%
of respondents considered drone-based food delivery in
urban environments to be one of the three most useful
applications [13]. In sharp contrast to medical and emer-
gency applications, the delivery of consumer goods thus
doesn’t appear to be perceived as a key necessity. However, Zhu et al. [26] in particular, have shown that
such delivery-related risks are not adequately reflecting
the overall risk belief systems of the public towards drone
delivery. Lateral and public risk dimensions, such as the
risks of drones making the sky less pleasant to look at or
causing property damage, can be strongly intervened to
those believes [26]. Regarding the public dimension of
drone delivery, especially safety and security concerns
are broadly anticipated issues [27, 28]. Researchers also
show that respondents perceive privacy risks as a sig-
nificant concern in relation to the public application of
drone delivery [21, 29]. In addition to that, noise has pre-
dominantly been surveyed as a central barrier to public
acceptance of drones for transportation [30]. In addition,
Kähler et al. [31] conducted an experiment exploring the
salience of drones to the observer in various environ-
ments and applications, suggesting that the perception Kellermann et al. European Transport Research Review (2023) 15:38 Page 3 of 14 Fig. 1 Sequential mixed-methods approach Fig. 1 Sequential mixed-methods approach SEM includes the structural model and measure-
ment model. The measurement model (Factor analysis)
indicates how observable variables measure the latent
variables. The latent variables represent intangible or
psychological concepts such as attitudes, behaviors, and
emotions, which cannot be directly measured through
single items [34]. The structural model (path analysis)
indicates the association between the latent variables
and examines the hypothesized associations. Regression
models test the strength and direction of relationships
between predictors and a dependent variable. 1.2 Literature review However,
SEM includes regression relationships simultaneously
among latent variables and between observed and latent
variables. In contrast to most statistical methods, SEM
can analyze linear associations among variables at the
same time by counting the measurement error. and aesthetic evaluation of drones is a relevant accept-
ance factor. Consequently, concerns regarding visual pol-
lution from drones should be considered more strongly
in future research [31]. From an empathetic social per-
spective, also the potential replacement of post officers
generated by automated drone delivery might be a rel-
evant factor to consider, as both Aydin [32] as well as Al
Haddad et al. [33] demonstrate a linkage between con-
cerns about possible job losses and lower acceptance of
drones. Reviewing the extensive body of research, we can state
that studies currently either focus on consumer adoption
or the influence of contextual aspects of drone delivery
(environment, aesthetics, and noise). However, accept-
ance research on drone delivery strongly demands an
integrated and applicable approach that incorporates
aspects of both individual dimensions of consumer
preferences and the various contextual aspects of drone
delivery that refer to its public and societal dimen-
sions. Against this background, the article is motivated
to achieve an integrated perspective through a model-
oriented expansion of current attitudinal research, com-
bining aspects of drone deliveries’ consumer and public
dimensions. By this, we will provide more comprehensive
as well as statistically robust insights on relevant fac-
tors driving or impeding attitudes towards drone deliv-
ery. Beyond the modeling approach creating conceptual
relevance for attitudinal and social science technology
acceptance in the field, the results can be of specific rel-
evance for market research, the private sector, and poli-
cymakers with respect to future jurisdiction and the
development of drone-related transport policies. Factor analysis tests hypotheses on how well sets of
observed variables in an existing dataset measure latent
construct (i.e., factors). For this reason, in the litera-
ture, factor analysis is also known as a measurement
model. The constructs’ reliability was tested by using
the value of Cronbach’s alpha for the constructs. Com-
posite reliability (sometimes called construct reliabil-
ity) is a measure of internal consistency in scale items,
much like Cronbach’s alpha [35]. α =
N · c
v + (N −1) · c where, N = the number of items, c = average covariance
between item pairs, v = average variance. 2.1 Structural equation model (SEM) Regarding the research purpose of this paper, we applied
the approach of SEM. SEM contains concurrent statisti-
cal approaches, such as analysis of variance, covariance,
regression, path analysis, and factor analysis [34]. SEM
contains the comparisons between-group and within-
group variance, generally related to the ANOVA analysis. These analyses are usually performed by applying data in
means or correlations and covariances (i.e., unstandard-
ized correlations). 1.2 Literature review A high level of alpha means that the observed vari-
ables are highly correlated and can be representative of
a latent variable. Many studies suggest that the value of
Cronbach’s alpha should be above 0.70 to indicate the
reliability of the constructs [35]. 2.2 Data set To develop and test the presented model, a sequential
exploratory mixed methods design was applied [36, To develop and test the presented model, a sequential
exploratory mixed methods design was applied [36,
37]. In this approach (Fig. 1), genuine qualitative data
was collected in the first step using focus groups. To
guide the development of a model structure, results Page 4 of 14 Kellermann et al. European Transport Research Review (2023) 15:38 Fig. 2 Structural model and hypotheses Fig. 2 Structural model and hypotheses Fig. 2 Structural model and hypotheses Participants were chosen according to a pre-screening
questionnaire aiming to exclude participants who worked
in the drone industry and those who had never heard
about drones before. Given that technology acceptance
is often age-related [38–40], four of the five groups were
separated into a younger (18–44) and an older (45–65)
age-group. Since some studies also demonstrated a rela-
tionship between technology acceptance and gender [41,
42], gender balance was assured. In addition, different
levels of education, income and household sizes were
analyzed to avoid selection effects [43]. Following the
methodological procedure proposed by Benighaus and
Benighaus [44], eight to ten participants of each group
were supplied with information on delivery drones and,
in the following, were guided through a group discussion
by a professional moderator. Each group discussion was
initiated by the moderator providing a 10-min presen-
tation, which was compiled beforehand by the research
team. The presentation defined the concept of "drone"
and showed different use cases in a neutral and objective were evaluated using content analysis and interpreted
within the theoretical framework of technology accept-
ance research. Building on this identification and clas-
sification of relevant variables that possibly influence
the perception and attitudes towards drone delivery,
quantitative data was gathered in a second step by con-
ducting a telephone survey. The relevant variables were
queried in a standardized questionnaire, allowing to
build and test the SEM (Fig. 2). 2.2.2 Model and hypothesesh manner. In addition, a short video clip was shown, which
demonstrated what a parcel delivery by drone might
look like in practice. Based on a pre-developed guide-
line, the participants then discussed the subject of fully
automated delivery drones as a possible element of future
urban logistics. The proposed attitudinal model consists of 17 observed
variables that are entirely based on the qualitative results
of the five focus group discussions. Grounded on these
results and informed by key concepts from technol-
ogy acceptance theories, the four latent constructs of
expected benefits, expected risks, technophilia, and atti-
tudes were defined. The following sections present the
four latent constructs in more detail, while Fig. 2 shows
the structural model. The focus groups were analyzed within the theoreti-
cal framework of technology acceptance theory [45, 46]
and by applying a qualitative content analysis [47]. The
different attitudinal dimensions and acceptance factors
were generated inductively throughout the analysis of the
transcripts [48]. Firstly, this assured the invariable identi-
fication of all factors that were considered relevant by the
discussants. Secondly, this allowed for the identification
of factors that had not been identified in previous stud-
ies. A comprehensive review of the focus group results is
provided in Kellermann and Fischer [49]. More precisely,
four key results can be depicted, which informed the sub-
sequent development of a structural model and the for-
mulation of hypotheses: 2.2.2.1 Attitude Attitude represents a central construct
in the original Technology Acceptance Model (TAM)
presented by Davis [15] and describes a person’s posi-
tive or negative perceptions regarding the performance
of an action [50]. In drone-related acceptance research,
attitude has already been proposed as a dependent vari-
able, mostly to predict the intention to use drones or
drone-related services [18, 51, 52]. In line with that, the
construct of attitude was derived from the focus group
discussions. Since the discussions were not only related
to individual interests in using drone delivery services but
primarily to the technology’s expected influence on urban
societies, the term public attitude is used. Consequently,
in this research, attitude represents the target variable,
and hypotheses are tested to explain the formation of
(public) attitudes towards drone delivery. 1. 2.2.2 Model and hypothesesh The focus groups unveiled contrasting attitudes
regarding the participants’ assessments of whether
drone delivery would contribute to a higher quality of
life in cities, if these new services would be useful or if
they are rather to be seen as a threat to public safety
and the common vision of sustainable societies.h In the survey, public attitude was measured by asking
respondents how much they would agree to the state-
ments of drone delivery (1) bringing advantages in the
respondents’ everyday life, (2) being generally safe, (3)
having a positive effect on the quality of life in cities, and
(4) being more environmentally friendly than current
transport alternatives. 2. The focus groups strongly emphasized the adverse
impacts of automated drone delivery. Anticipated
were potential job losses of delivery personal and
intensified trends of social isolation, especially of the
elderly. The expected impact of drone delivery on
urban sound and landscape was a key issue in all the
focus groups. What is more, there were broad discus-
sions on how the quality of life in cities would change
with respect to this new form of urban air traffic. 2.2.2.2 Expected risks The concept of risk was introduced
to attitudinal and behavioral research to explain the con-
sequences of an action or the circumstances surrounding
it cause uncertainty, uneasiness, or anxiety [53]. In drone-
related research, the variable of (perceived) risk has been con-
ceptualized early onwards [52] and proved as a significant
predictor of attitude formation in prior models on drone
delivery [18]. In this research, expected risks are understood
as concerns with respect to the future introduction of drone
delivery in urban areas, directly affecting the attitude. fi
3. Participants positively assessed the outlook of drones
to provide the possibility to determine a package
drop-off by time and location. Other stated expec-
tations of potential benefits were the high reliability
of drone delivery and the option to receive packages
faster. Furthermore, the eventuality of drone delivery
being environmentally friendly was highlighted posi-
tively, thus forming a central aspiration. 4. Finally, discussions about drone delivery tended to be
strongly influenced by participants’ opinions about
technology in general. On the one hand, some partici-
pants expressed enthusiastic technological optimism,
often coupled with a fascination for new technolo-
gies. In stark contrast to that stood the technological
skepticism or even technophobia expressed by other
participants. 2.2.1 Focus groupsh The variables included in the presented model are derived
from the analysis of five focus group discussions, which
scrutinized delivery drones from a societal perspective. They were conducted in the German capital Berlin and
in the state capitals, Stuttgart and Erfurt, in September
and October 2019. The aim of these focus groups was to
explore the public’s basic perception as well as the asso-
ciated beneficial expectations and concerns towards
automated drone delivery as a possible element of future
urban logistics. Kellermann et al. European Transport Research Review (2023) 15:38 Page 5 of 14 2.3 Survey Based on the focus group findings, the survey questionnaire
was designed and administered in a fully structured com-
puter-assisted telephone interview (CATI) with 1000 inter-
views in January 2020. In the beginning, respondents were
informed that the survey thematizes the future of urban
transport. All items were then asked with the agreement or
disagreement of different statements on a five-point Likert
scale. In addition, "don’t know/no answer" was included as
a response option. To minimize distortion of the response
behavior due to fixed item sequences, the corresponding
item batteries were randomized [58]. Moreover, to mini-
mize Acquiescence Response Bias [59], the questionnaire
followed a query of factors by an alternation of positively
and negatively formulated items. The composition of the
sample corresponds to the structure of the total German-
speaking population in Germany aged 18 and over. This
means that the original dataset is representative. 2.2.2.3 Expected benefits Following TAM, the perceived
usefulness of a new technology has a direct correlation to
respondents’ attitudes towards the object [15]. The same
concept was already adopted in drone-related research
[51, 54]. In this research, the variable of expected benefits
describes a similar concept and refers to personal but also
public benefits that a widespread introduction of drone
delivery might contribute to. A direct correlation between
expected benefits on attitude is proclaimed. H2 There is a significant association between the sub-
ject’s expectation of benefits and its attitude to delivery
drones. As potential predictors for the latent variable of
expected benefits, in the survey, it was asked how impor-
tant it would be for the respondent that parcel deliveries
with drones would be (1) reliable, (2) fast, (3) environ-
mentally friendly, (4) flexible in delivery time, and how
important it would be (5) that drop-off locations can be
determined by the customer. H3 There is a significant association between a subject’s
technophilia and its attitude to delivery drones. H3 There is a significant association between a subject’s
technophilia and its attitude to delivery drones. The latent variable of technophilia was measured by
evaluating the respondents’ affinity to new technologies. The survey design drew on established questionnaires in
the field, which already measured technophilia as a latent
variable [57]. Accordingly, to measure technophilia, (1)
the respondents’ subjective level of information about
technologies, (2) their ability to get easily enthusiastic for
new technology, and (3) their general interest in technol-
ogy were surveyed. 2.2.2 Model and hypothesesh H1 There is a significant association between a subject’s
expectation of risks and its attitude to delivery drones. Expected risks were measured by how problematic
respondents considered (1) the noise from parcel deliv-
eries with drones, (2) the potential decrease of personal Kellermann et al. European Transport Research Review (2023) 15:38 Page 6 of 14 Page 6 of 14 contacts between recipients and deliverers/drivers, (3)
caused stress due to delivery drones flying around, (4)
potential job losses of deliverers/drivers due to automa-
tion of flights, (5) a potentially blocked view to the sky. 2.4 Sample After having removed entries with missing values from
the initial dataset, a sample size of 819 respondents was
employed for the statistical analysis of the model. The
characteristics of the sample are indicated in Appendix
1. Around 29% were aged 18 to 39. The age group 40 to
59 was represented by about 40% of the respondents, and
around 30% of persons were age 60+ years. About 39%
of respondents had completed a technical or university
degree. About 23% held a secondary school leaving cer-
tificate or technical college entrance qualification. Only
37% of respondents were either retired or unemployed
while most respondents reported a monthly net house-
hold income of 4500 euros or more (about 27%). Spa-
tially, about 21.1% of respondents lived in major cities of
500,000 or more inhabitants, while with 50%, most of the
respondents originated from smaller to mid-size towns
with a population between 5000 and 100,000 inhabitants. 2.2.2.4 Technophilia While external variables, such as
demographics, have already been part of TAM [15], lat-
ter extensions to the model emphasized more strongly
on individual dispositions [42]. In this research, a person
having a technophile disposition is considered to evalu-
ate and perceive novel technologies in a positive way to
improve his or her own life as well as society as a whole
[55]. Constructs such as personal innovativeness [18] or
cognitively motivated consumer innovativeness [21] were
already emphasized by acceptance research studies in the
field [18],Lin [56]. Henceforth, a direct association of tech-
nophilia with attitude is suggested. Despite the removal of entries with missing values hav-
ing reduced the sample size, the sample of 819 respond-
ents still closely corresponded to the socio-demographics
of Germany (Statistisches [60–63]. Larger deviations
from the socio-demographics of Germany exist, however,
regarding gender distribution, as around 51% of the pop-
ulation in Germany are women [64] while in the survey,
they account for only 45.5%. 3 Data analysis and results Job loss
…that parcel deliveries with drones would block your free view of the sky
Blocked sky
Expected
benefits
How important would be for you…
…that parcel deliveries with drones would be reliable
Reliability
0.789
…that you would get your parcels delivered quickly
Fast delivery
…that parcel deliveries with drones would be environmentally friendly
Environmentally friendly
…that you could use drones to have parcels delivered to a place of your choice, e.g.,
balcony or garden
Spatial flexibility
…that your parcels would be delivered at an exact time of your choice
Time flexibility
Attitude
How much would you agree to the following statement:
I think that parcel deliveries with drones are environmentally friendly than package
deliveries with a delivery van
Environment
0.834
I imagine parcel deliveries with drones to be safe
Safety
Parcel deliveries with drones would have a positive effect on the quality of life in cities
Quality of life
Parcel deliveries with drones would bring me advantages in my everyday life
Utility Variable names
Cronbach alpha The comparative fit index (CFI) analyses the model fit
by evaluating the discrepancy between the data and the
hypothesized model, and its value is in the range from 0
to 1, and the larger values indicate better fit. A CFI value
of 0.90 or larger is considered to indicate an accept-
able model fit. The calculated CFI in this model is 0.936. Tucker–Lewis index (TLI), also known as the non-nor-
med fit index, is an incremental fit index. Bentler and
Bonett [68] recommended that TLI > 0.90 indicates an
acceptable fit. In this model TLI is 0.923. RMSEA (root
mean square error of approximation) is one of the most
applied measures to check structural equation models. RMSEA is the root mean square error of approxima-
tion, indicating how well the model, with unknown but
optimally chosen parameters, would fit the populations’
covariance matrix [69]. It is “one of the most informa-
tive fit indicators because of its sensitivity to the number
of estimators in the model” [70]: 85). The values of 0.01,
0.05 and 0.08 indicate excellent, good, and mediocre fit
respectively. In this model, RMSEA is around 0.05 which
indicates good fit. threshold (0.7), which confirmed the high reliability of
the constructs. The result of the Cronbach test is indi-
cated in Table 1. 3 Data analysis and results Before examining the structural model,
we checked the multicollinearity assumption by using the
value of the variance inflation factor (VIF). All the con-
structs were considered predictors of one of the constructs
and calculated the VIF scores. The VIF scores are less than
2.00, which is less than the recommended value of 10, sug-
gesting minimal collinearity [65]. SEM was then utilized to
estimate the hypothesized relationships. The analyses pro-
vided acceptable fit indices for the structural model. 3 Data analysis and results For the statistical analysis of the model, we employed the
above-mentioned 819 samples. All the values of Cron-
bach’s alpha and CR were above the recommended Kellermann et al. European Transport Research Review (2023) 15:38 Page 7 of 14 Table 1 Reliability of constructs by Cronbach Alpha Test
Latent variable
Observed variable
Variable names
Cronbach alph
Technophilia
How much would you agree to the following statement:
In general, I am well informed about new technologies
Well informed
0.798
I get easily enthusiastic about new technologies
Enthusiastic
I am always interested in new technologies
Interested
Expected
risks
How bad it would be for you…
…the noise from parcel deliveries with drones
Noise
0.778
…the fact that parcel deliveries with drones would mean the personal contact
between recipient and delivery driver would disappear? Loss of personal contact
…the stress caused by drones flying around to deliver packages
Stress
…the fact that delivery drivers would lose their jobs as a result of parcel deliveries
with drones? 3 Data analysis and results Job loss
…that parcel deliveries with drones would block your free view of the sky
Blocked sky
Expected
benefits
How important would be for you…
…that parcel deliveries with drones would be reliable
Reliability
0.789
…that you would get your parcels delivered quickly
Fast delivery
…that parcel deliveries with drones would be environmentally friendly
Environmentally friendly
…that you could use drones to have parcels delivered to a place of your choice, e.g.,
balcony or garden
Spatial flexibility
…that your parcels would be delivered at an exact time of your choice
Time flexibility
Attitude
How much would you agree to the following statement:
I think that parcel deliveries with drones are environmentally friendly than package
deliveries with a delivery van
Environment
0.834
I imagine parcel deliveries with drones to be safe
Safety
Parcel deliveries with drones would have a positive effect on the quality of life in cities
Quality of life
Parcel deliveries with drones would bring me advantages in my everyday life
Utility Table 1 Reliability of constructs by Cronbach Alpha Test
Latent variable
Observed variable
Variable names
Cronbach al
Technophilia
How much would you agree to the following statement:
In general, I am well informed about new technologies
Well informed
0.798
I get easily enthusiastic about new technologies
Enthusiastic
I am always interested in new technologies
Interested
Expected
risks
How bad it would be for you…
…the noise from parcel deliveries with drones
Noise
0.778
…the fact that parcel deliveries with drones would mean the personal contact
between recipient and delivery driver would disappear? Loss of personal contact
…the stress caused by drones flying around to deliver packages
Stress
…the fact that delivery drivers would lose their jobs as a result of parcel deliveries
with drones? 3.2 Factor loadings Each of the four constructs in the model is measured by
at least three observed variables. The load factors are
indicated in Table 2 and visualized in Fig. 3. Results sug-
gest which observed variables most reliably measure the
respective construct. i
Therefore, the first relevant result of our research
is the robust composition of attitude as a target vari-
able. In addition to individual aspects (personal utility
of drone delivery), the composition includes a strong
community-oriented character (positive environmen-
tal effects, quality of urban life, public safety), which is
why the here-used term "public" attitude seems appro-
priate. The standardized estimates show that especially
the impact of drone delivery on the urban quality of life
(0.798) serves as a good predictor for public attitude. The standardized estimates between the latent variable
attitude and its measurement indicate that the observed
variable of the environment has the least standardized
estimates (0.67), while the other three measurements
have almost similar load factors, which are quality of life
(0.8), Safety (0.74), utility (0.79). Testing of the research hypotheses showed mixed
results. Among the measurements for the latent variable
expected benefits, the observed variables of fast deliv-
ery and flexibility in time show the highest standardized
load factor by 0.76 and 0.74, respectively. The influence
of delivery drones’ relative advantage of environmental
friendliness (0.40) is considerably weaker, nevertheless
significant. i. The model reveals a significant association of atti-
tude with expected risks (H1). These results are
generally consistent with previous risk studies [50,
71, 72], which illustrate a negative effect of poten-
tial risk factors on attitude and technology adop-
tion. The more the movement of delivery drones
in urban space is expected to be stressful (0.838)
and noisy (0.678), would generate the impression of
blocked skies (0.718), the more respondents create
a negative attitude towards the technology. While i. The model reveals a significant association of atti-
tude with expected risks (H1). These results are
generally consistent with previous risk studies [50,
71, 72], which illustrate a negative effect of poten-
tial risk factors on attitude and technology adop-
tion. The more the movement of delivery drones
in urban space is expected to be stressful (0.838)
and noisy (0.678), would generate the impression of
blocked skies (0.718), the more respondents create
a negative attitude towards the technology. 4 Discussionh The aim of this study was to identify significant factors
and their direction of influence on the formation of atti-
tudes towards drone delivery. For this purpose, a SEM
was tested on a sample of 819 respondents and showed
reliable results. The Comparative Fit Index (CFI), the
Tucker-Lewis Index (TLI), and the Root Mean Square
Error of Approximation (RMSEA) indicate an accept-
able model fit of 0.936, 0.923 and 0.05, respectively. The
composition of the four latent constructs of public atti-
tude, expected risks, expected benefits, and technophilia
from the total of 17 observed variables derived from
qualitative research can be confirmed. 3.3 Structural model and hypothesis Table 3 shows the results of the relationships. The
model reveals significant associations of attitude with
expected benefits (H2) by (β = 0.439, p < 0.001) and
expected risks (H1) by (β = −0.738, p < 0.001). Hence,
H1 and H2 are confirmed. In contrast, the p-value of
H3 is 0.111, which means that the model does not sug-
gest a significant association between technophilia and
attitude. 3.1 Fitness of modelhi The fitness of the model is checked through the compara-
tive fit index (CFI), the Tucker–Lewis index (TLI), and the
root mean square error of approximation (RMSEA). As
the chi-square statistic is sensitive to the large sample size
[66], it is recommended to use the normed chi-square, that
the chi-square is divided by the degrees of freedom (χ2/df)
as a measure of model fit, with the acceptance-value of 5
or less [67]. The chi-square of this model is 458.389 with
df = 113, therefore the normed chi-square is 4.05. Kellermann et al. European Transport Research Review (2023) 15:38 Page 8 of 14 Table 2 Results of the measurements and constructs (***P ≤
0.001)
Estimate
S.E
Standardized
estimates
P
Attitude
Environment
.856
.045
.666
***
Quality of life
.991
.042
.798
***
Safety
.872
.041
.737
***
Utility
1.000
.789
Expected risks
Noise
1.000
.678
Loss of personal contact
.917
.072
.503
***
Stress
1.345
.069
.838
***
Job loss
.722
.061
.465
***
Blocked sky
1.299
.074
.718
***
Expected benefits
Time flexibility
1.000
.737
Spatial flexibility
.993
.054
.708
***
Fast delivery
.968
.050
.760
***
Reliability
.853
.051
.646
***
Environmentally friendly
.456
.043
.401
***
Technophilia
Well informed
.743
.045
.612
***
Enthusiasm
1.086
.056
.844
***
Interested
1.000
.811 Table 2 Results of the measurements and constructs (***P ≤
0.001) Finally, technophilia is measured most reliably by
interest and enthusiasm in new technologies with
standardized estimates of 0.81 and 0.844, respectively,
while the level of being informed shows a weaker yet
significant influence on technophilia (0.61). 3.2 Factor loadings While i
In the construct of expected risks and its measure-
ment, the observed variables of stress and blocked sky
view show the highest standardized estimates, which
are 0.84 and 0.72, respectively, while the variable of Job
loss has the lowest impact factor (0.46) on the latent
variable. Kellermann et al. European Transport Research Review (2023) 15:38 Page 9 of 14 Fig. 3 Structural equation model noise, in particular, has been addressed in accept-
ance research and engineering sciences [73, 74], the
above-mentioned aesthetic implications of drones
and their salient for citizens have yet to receive
little attention [31, 75]. Furthermore, our study
unveiled the significance of losing personal contact
with delivery personnel (0.503) in forming the atti-
tude towards drone delivery. This aspect was pre-
viously confirmed in the context of adopting self-
service parcel lockers [76], but—apart from general
considerations [23]—had not yet been explic-
itly analyzed in the context of drone delivery. As
another social factor, the risk perception of drone
delivery generating job losses (0.465) was shown to
have a significant negative effect on the public atti-
tude. This outcome appears to be strongly consist-
ent with related works, particularly Aydin [32]. ii. The SEM reveals a significant association of atti-
tude with expected benefits (H2). These results are
consistent with findings within technology accept-
ance research which have repeatedly demonstrated
the central relevance of beneficial factors for posi-
tively affecting attitudes towards technology adop-
tion in various contexts [15, 50, 72]. Specifically,
our study provides evidence that people form a
more positive attitude towards the technology who
expect the delivery to be faster (0.760) and more
flexible regarding delivery date (0.737) and location
of parcel drop-off (0.708). Moreover, the expecta-
tion of delivery drones providing a reliable service
(0.646) and being environmentally friendly (0.401)
were also shown to form a more positive attitude. Fig. 3 Structural equation model
Table 3 Results of the structural mode (***P ≤ 0.001)
Hypotheses
Standardized
Estimates
P
Results
H1: Attitude < –- Expected risks
− 0.520
***
Supported
H2: Attitude < –- Expected
benefits
0.424
***
Supported
H3: Attitude < –- Technophilia
0.053
0.111 Not supported the central relevance of beneficial factors for posi-
tively affecting attitudes towards technology adop-
tion in various contexts [15, 50, 72]. 3.2 Factor loadings Specifically,
our study provides evidence that people form a
more positive attitude towards the technology who
expect the delivery to be faster (0.760) and more
flexible regarding delivery date (0.737) and location
of parcel drop-off (0.708). Moreover, the expecta-
tion of delivery drones providing a reliable service
(0.646) and being environmentally friendly (0.401)
were also shown to form a more positive attitude. noise, in particular, has been addressed in accept-
ance research and engineering sciences [73, 74], the
above-mentioned aesthetic implications of drones
and their salient for citizens have yet to receive
little attention [31, 75]. Furthermore, our study
unveiled the significance of losing personal contact
with delivery personnel (0.503) in forming the atti-
tude towards drone delivery. This aspect was pre-
viously confirmed in the context of adopting self-
service parcel lockers [76], but—apart from general
considerations [23]—had not yet been explic-
itly analyzed in the context of drone delivery. As
another social factor, the risk perception of drone
delivery generating job losses (0.465) was shown to
have a significant negative effect on the public atti-
tude. This outcome appears to be strongly consist-
ent with related works, particularly Aydin [32]. Table 3 Results of the structural mode (***P ≤ 0.001)
Hypotheses
Standardized
Estimates
P
Results
H1: Attitude < –- Expected risks
− 0.520
***
Supported
H2: Attitude < –- Expected
benefits
0.424
***
Supported
H3: Attitude < –- Technophilia
0.053
0.111 Not supported ble 3 Results of the structural mode (***P ≤ 0.001) ii. The SEM reveals a significant association of atti-
tude with expected benefits (H2). These results are
consistent with findings within technology accept-
ance research which have repeatedly demonstrated Page 10 of 14 Kellermann et al. European Transport Research Review (2023) 15:38 Page 10 of 14 Kellermann et al. European Transport Research Review (2023) 15:38 These results are partly consistent with studies
focusing on drone deliveries, which also found the
relative advantages of speed and environmental
friendliness [18], respectively a “green image” [21],
to be significant predictors for a positive attitude
towards delivery drone usage. In addition, the pre-
ceding focus groups have qualitatively revealed the
factors of reliability and flexibility (time, location
as new and previously uncharted factors influenc-
ing attitudes towards drone delivery. 3.2 Factor loadings In contrast
to other studies that rather focused on examining
the factor of reliability in the context of a poten-
tial "performance risk" [18, 25], the focus groups
unveiled that participants actually considered ser-
vice reliability as a beneficial factor. This concep-
tual re-interpretation was confirmed in our model
and may contribute to a more precise definition of
acceptance factors in future studies. of drone delivery might yet appear abstract and hardly
imaginable, such as the suggested flexible drop off time
and locations for packages, or benefits might be per-
ceived as not sufficiently compelling. This, in turn, may
to some extent, be explained by the lack of familiarity and
experience with potential beneficial features. Leading
behavioral and technology adoption models have demon-
strated the relevance of familiarity and experience with a
technology [17]. This may particularly apply to drone ser-
vices, which have just been rarely implemented. Conse-
quently, experience and real-life applications with drone
deliveries may become a relevant attitudinal aspect [79]. On the other hand, individuals without real-life experi-
ence might more easily imagine obvious threats of drone
delivery (physically falling, being misused for criminal
purposes, being noisy, or violating privacy) compared to
imagining potential benefits. i
Beyond that, the comparably lower influence of
expected benefits against expected risks strengthens the
assumption that in the sample, drone delivery is not con-
sidered a prior necessity for improving the delivery sys-
tem. This may also be explained by respondents’ strongly
varying environments. Most respondents live in rural
areas or smaller towns or cities below 500,000 inhabit-
ants (79% of respondents), where congestion problems
affecting last-mile delivery traffic may not be experienced
as critical as in bigger urban areas of more than 500,000
inhabitants (21% of respondents). Moreover, respondents
may live in heterogeneous housing conditions (single
house, multi-story apartment building etc.), thus possi-
bly creating varying perceptions of drone deliveries being
practically feasible in their individual environments. iii. i. The model does not suggest a significant associa-
tion between a person’s technophilia and attitude
(H3) towards drone delivery. The result seems
trustworthy, as the latent variable was measured in
accordance with an established questionnaire [57]
by three separate items that show a good Cron-
bach alpha (0.8). Appendix 1: Socio‑demographic distribution
of the sample Appendix 1: Socio‑demographic distribution
of the sample pp
g
p
of the sample
Attribute
Value
Count %
Gender
Male
445
54.3
Female
373
45.5
Divers
1
0.1
Age
18–29 years
84
10.3
30–39 years
152
18.6
40–49 years
157
19.2
50–59 years
173
21.1
60+ years
253
30.9
Education
Without lower secondary/
vocational school leaving
certificate
3
0.4
Lower secondary/voca-
tional school leaving
certificate
62
7.6
Secondary school leaving
certificate, technical college
entrance qualification
199
24.3
Completion of polytechnic
secondary school (8th/10th
grade)
20
2.4
Advanced technical college
entrance qualification,
completion of a specialized
upper secondary school
63
7.7
High school diploma,
general or subject-linked
higher education entrance
qualification
144
17.6
Technical/college studies
317
38.7
Other school-leaving
qualification
9
1.1
No indication
2
0.2
Employment
Employed
519
63.4
Unemployed
300
36.6
Household monthly income Below 500 EUR
4
0.5 3.2 Factor loadings This suggests that even consum-
ers who have a general interest in learning about
new technologies, who consider themselves to
know a lot about new technologies and easily feel
enthusiastic about using and trying them, don’t
create significantly more positive attitudes towards
drone delivery services. This finding is in contrast
to previous studies that highlighted the relevance
of concepts such as “personal innovativeness” [18]
or “cognitively motivated consumer innovative-
ness” [21] in significantly influencing attitudes and,
thus technology adoption. Our finding of a person’s
technophilia proving to be neglectable for forming
attitudes towards drone delivery appears especially
remarkably as technophilia was found to have a sig-
nificant positive correlation with forming attitudes
towards the adoption of electric vehicles [77] or
on the attitude towards drone-enabled passenger
transport in air taxis [78]. The later may indirectly
hint at drone logistics being perceived as less spec-
tacular and rather pragmatic compared to the indi-
vidual thrill and risk of being transported as a pas-
senger in an automated drone. Irrespective of the geographical context, prior stud-
ies demonstrated that commercial drone delivery is not
considered among the most relevant use cases of drone
technology [13]. Furthermore, results from technology
assessment show that added values of delivery drones
often remain abstract and must be made more plausible
to the public in order to sustain a publicly accepted usage
of urban airspace as a new transport layer [80]. Strong
added values can be created, however when the deploy-
ment of the technology is more evidently targeted to
serve the common good, e.g. in medical or humanitarian
use cases [81]). This study faces several limitations. First, the transfer-
ability of results may be restricted as data were collected
in Germany only. However, a cross-national study on
the acceptance of drone applications in Europe shows
no serious divergences between the examined member
countries [13]. Against the background of this finding, the
model framework can be considered a relevant research
reference for future studies in the European context or
regions with comparable socio-demographic features as Concerning the relationship between expected benefits
and risks, we found the latter to have a stronger asso-
ciation with the attitude towards drone delivery (−0.52)
than the expected benefits (0.424). On the one hand, this
may be explained by the assumption that the benefits Kellermann et al. European Transport Research Review (2023) 15:38 Kellermann et al. 3.2 Factor loadings European Transport Research Review (2023) 15:38 Page 11 of 14 Page 11 of 14 used in this paper. Secondly, the survey data on drone
delivery had the limitation of asking respondents about
a technology that is not yet tangible in the daily life of
citizens. Future applications of drone delivery should
emphasize accompanying social science research to col-
lect alternative data. Third, the database for this study
consists of a survey that was conducted in January 2020,
so right before the outbreak of the Covid-19 pandemic. As various studies have highlighted that the pandemic
induced (long-term) changes in mobility and consump-
tion behaviors [82, 83], our findings may not reflect these
changed societal framework conditions. As people con-
fronted with the pandemic might have perceived a big-
ger value of contact-less delivery methods than before
the pandemic, a share of respondents might have slightly
reduced their expectation of risks and might have slightly
increased their expectation of benefits related to drone
delivery [84]. In addition, the results of this research show that techno-
philia, as we framed the individual innovativeness towards
technology, has no significant association with the attitude
towards drone delivery. This may have general implications
for the industry as drone service providers may not easily
be able to count on the innovativeness of certain consumer
subgroups, e.g., innovators or early adopters. Finally, the presented model may be of methodologi-
cal utility as both the qualitative identification of novel
acceptance factors and their quantitative transition into a
survey and the test in a model highlights the appropriate-
ness of following a mixed methods approach in technol-
ogy acceptance research. Availability of data and materials The primary survey dataset (telephone survey) is available on request to the
authors. 17. Rogers, E. M. (2003). Diffusion of innovations (5th ed.). Free Pre 18. Yoo, W., Eun, Yu., & Jung, J. (2018). Drone delivery: factors affecting the
public’s attitude and intention to adopt. Telematics and Informatics, 35(6),
1687–1700. https://doi.org/10.1016/j.tele.2018.04.014 Funding Funding
Open Access funding enabled and organized by Projekt DEAL. g
Open Access funding enabled and organized by Projekt DEAL. 16. Fishbein, M., & Ajzen, I. (1975). Belief, attitude, intention, and behavior:
An introduction to theory and research. Addison-Wesley series in social
psychology. Reading, Mass: Addison-Wesley Pub. Co. Received: 20 May 2022 Accepted: 28 August 2023 21. Mathew, A. O., Jha, A. N., Lingappa, A. K., & Sinha, P. (2021). Attitude
towards drone food delivery services—Role of innovativeness, perceived
risk, and green image. Journal of Open Innovation: Technology, Market, and
Complexity, 7(2), 144. https://doi.org/10.3390/joitmc7020144 5 Conclusionsh Delivery drones from a technology
assessment perspective. Institute for Technology Assessement Vienna (ITA). http://epub.oeaw.ac.at/ita/ita-projektberichte/2018-01.pdf. 11. Nentwich, M., & Horváth, D. M. (2018). Delivery drones from a technology
assessment perspective. Institute for Technology Assessement Vienna (ITA). http://epub.oeaw.ac.at/ita/ita-projektberichte/2018-01.pdf. Acknowledgements
h d
b
f h 12. Dannenberger, N., Schmid-Loertzer, V., Fischer, L., Schwarzbach, V.,
Kellermann, R., Biehle, T. (2020). Traffic solution or technical hype? Repre-
sentative population survey on delivery drones and air taxis in Germany. https://skylimits.info/wp-content/uploads/2020/06/Sky-Limits-_Results_
Survey_english.pdf. The data base of this article (focus groups and telephone survey) originates
from the research project “Sky Limits” that was conducted between Janu-
ary 2019 and December 2020 (funded by the German Federal Ministry for
Research and Education (BMBF), Grant Number: 16ITA216A). 13. EASA. (2021). Study on the societal acceptance of Urban Air Mobility in
Europe. https://www.easa.europa.eu/sites/default/files/dfu/uam-full-
report.pdf. 13. EASA. (2021). Study on the societal acceptance of Urban Air Mobility in
Europe. https://www.easa.europa.eu/sites/default/files/dfu/uam-full-
report.pdf. Author contributions Dr. Robin Kellermann: Conceptualization, Project Administration, Supervision,
Writing—Original Draft. Tobias Biehle: Conceptualization, Formal analysis,
Methodology, Visualization, Writing—Review & Editing. Dr. Hamid Mostofi: Data
Curation, Formal analysis, Methodology, Visualization, Validation, Writing—
Review & Editing. 14. Cesur, A., Yi̇Ğenoğlu, K., Aydin, İ, Çeli̇K, Z. (2022). A biblyometric analysis of
empirical studies on drone delivery. Yüzüncü Yıl Üniversitesi Sosyal Bilimler
Enstitüsü Dergisi. https://doi.org/10.53568/yyusbed.1172019. 14. Cesur, A., Yi̇Ğenoğlu, K., Aydin, İ, Çeli̇K, Z. (2022). A biblyometric analysis of
empirical studies on drone delivery. Yüzüncü Yıl Üniversitesi Sosyal Bilimler
Enstitüsü Dergisi. https://doi.org/10.53568/yyusbed.1172019. 15. Davis, F. D. (1989). Perceived usefulness, perceived ease of use, and user
acceptance of information technology. MIS Quarterly, 13(3), 319. https://
doi.org/10.2307/249008 Declarations 19. Hwang, J., Kim, J. J., Lee, K.-W. (2021). Investigating consumer innovative-
ness in the context of drone food delivery services: Its impact on attitude
and behavioral intentions. Technological Forecasting and Social Change
163(February). https://doi.org/10.1016/j.techfore.2020.120433. 4.
Haidari, L. A., Brown, S. T., Ferguson, M., Bancroft, E., Spiker, M., Wilcox, A.,
Ambikapathi, R., Sampath, V., Connor, D. L., Lee, B. Y. (2016). The economic 5 Conclusionsh The vision of commercial drone delivery in urban envi-
ronments incorporates not only an individual service
choice for potential customers but also entails an inher-
ently public dimension. In current acceptance research,
this comprehensive understanding appears rather under-
represented. Therefore, this study aimed to build and test
an integrated model to define and explain the public atti-
tude towards drone delivery that includes both relevant
aspects of consumer preferences as well as significant
societal and public dimensions. Using a large sample (n = 819) from a representative
survey in Germany from 2020, the results of the model
reveal insights of practical utility. We confirm that par-
ticularly the expectation of a fast and time-flexible
delivery are highly valued attributes from a customer per-
spective. On the other hand, especially the expectation
of spatial and visual implications (noise, visual distur-
bance, and rising stress levels through drone movements)
are relevant factors that negatively impact respondents’
attitudes from a citizen’s perspective. Rather than solely
focusing on individual aspects of adopting the use of
drone delivery, we, therefore, suggest that the planning
of business cases and drone-related policies should give
stronger emphasis on the consideration of public dimen-
sions of drone delivery. What is more, by highlighting the centrality of social
risk factors that outweigh individual benefits, the results
suggest that the public is currently not willing to accept
the risks of large-scale drone delivery of consumer goods. From an acceptance perspective, rather than focusing on
business-to-customer solutions, drone delivery service
providers may generate more robust business cases by
focusing on business to business applications. Kellermann et al. European Transport Research Review (2023) 15:38 Page 12 of 14 Attribute
Value
Count %
500 until below 1.000 EUR
21
2.6
1.000 until below 1.500 EUR
53
6.5
1.500 until below 2.000 EUR
58
7.1
2.000 until below 2.500 EUR
87
10.6
2.500 until below 3.000 EUR
74
9.0
3.000 until below 3.500 EUR
72
8.8
3.500 until below 4.000 EUR
76
9.3
4.000 until below 4.500 EUR
80
9.8
4.500 and more
222
27.1
No indication
72
8.8
City size
Below 5.000 EW
119
14.5
Between 5.000 and 20.000
177
21.6
Between 20.000
and 100.000
227
27.7
Between 100.000
and 500.000
123
15
More than 500.000
173
21.1 10. Doole, M., Ellerbroek, J., Hoekstra, J. (2018). Drone delivery: Urban airspace
traffic density estimation. https://www.sesarju.eu/sites/default/files/
documents/sid/2018/papers/SIDs_2018_paper_3.pdf. 11. Nentwich, M., & Horváth, D. M. (2018). Competing interests
Not applicable. Competing interests
Not applicable. Not applicable. 20. Hwang, J., & Kim, H. (2019). Consequences of a green image of drone
food delivery services: The moderating role of gender and age. Business
Strategy and the Environment, 28(5), 872–884. https://doi.org/10.1002/bse.
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BB 4312 ORGANIZATIONAL DEVELOPMENT AND CHANGE
INDIVIDUAL REPORT
“Multiple Case Studies”
Prepared By: Nurul Azzimatul Adawiyah Hj Omar
Student ID: 16B8509
Prepared For: Pg Dr Siti Rozaidah Pg Hj Idris
Submission Date: 5 May 2020 (Tuesday) Table of Contents
Abstract ................................................................................................................................................... 3
1.0 Introduction ....................................................................................................................................... 4
1.1 Problem Statement ........................................................................................................................ 4
1.1.1 BruTEL 's 'Going Paperless' Initiative ................................................................................... 4
1.1.2 Sumbiling Eco Village: Promoting the Ecotourism in the Temburong District .................... 4
1.1.3 Society for Community Outreach and Training (SCOT)’s Green Xchange Project .............. 5
2.0 Discussion ......................................................................................................................................... 6
2.1 Case Analysis ................................................................................................................................ 6
2.1.1 BruTEL's 'Going Paperless' Initiative .................................................................................... 6
2.1.2 Sumbiling Eco Village: Promoting the Ecotourism in the Temburong District .................... 7
2.1.3 Society for Community Outreach and Training (SCOT)’s Green Xchange Project .............. 8
2.2 Alternative Solutions .................................................................................................................... 9
2.2.1 BruTEL's 'Going Paperless' Initiative .................................................................................... 9
2.2.2 Sumbiling Eco Village: Promoting the Ecotourism in the Temburong District .................... 9
2.2.3 Society for Community Outreach and Training (SCOT)’s Green Xchange Project ............ 10
3.0 Recommendation ............................................................................................................................ 11
References ............................................................................................................................................. 12 Table of Contents Abstract This is a multiple case study report of three local cases: 1) BruTEL 's 'Going Paperless'
Initiative, 2) Sumbiling Eco Village: Promoting the Ecotourism in the Temburong District, and
3) Society for Community Outreach and Training (SCOT)’s Green Xchange Project. The
objective of this report is to identify and discuss the organizational development and change
strategy or strategies employed in each of the cases. The writer also discusses the key raised
issues based on some scholar’s planned change theories. A few recommended solutions are
included in this report. It is found that communication is the most optimal strategy solution
that befit into all three cases. This is because, this type of strategy is the most general and best
fit to all types of interventions when planning a change within the organization. Keywords: ODC strategies, planned change theory, Lewin’s three step model, Kotter’s eight
step model, positive model, communication 1.0 Introduction This multiple case study report consists of the analyses of three cases: 1) BruTEL 's
'Going Paperless' Initiative, 2) Sumbiling Eco Village: Promoting the Ecotourism in the
Temburong District, and 3) Society for Community Outreach and Training (SCOT)’s Green
Xchange Project. The main objective of this multiple case study report is to identify the
organizational development and change strategy or strategies employed in these three cases. The writer will first identify the key raised issues from each of these cases. These issues will
later be discussed based on the organizational change theory in the Discussion section. A
number of solutions will also be discussed. At the end of this report, the writer will recommend
the most optimal solutions to be deployed in these cases, followed by the conclusion of this
case studies. 1.1 Problem Statement In this section, it will consists of the overview of the raised issue(s) from each
of the cases (assuming the reader has read the cases mentioned). 1.1.1 BruTEL 's 'Going Paperless' Initiative It is stated that BruTel is planning to ‘go paperless’ where they wished
to introduce an absolute utilization of the technology in their business and omit
the usage of papers. The main aim of this plan is to accomplish the third goal of
BruTel i.e. to conserve the natural environment. However, some segments of its
customers are giving the adverse response – especially the elder people. It is
said that majority of the elderly experienced some difficulties on using the
technology due to the computers incompetency which caused them to insist on
the traditional system (using paper to make transactions). This therefore hinders
BruTel to achieve its third goal. Hence, the main issue for BruTel is the
reluctance or hesitance of these customer segments to the intended system
change. It is also stated that if BruTel succeed on persuading this group of
customers to switch to the new system, BruTel could smoothly develop and
execute its strategies on the ‘go paperless’ system. 1.1.2 Sumbiling Eco Village: Promoting the Ecotourism in the Temburong
District Similar to the BruTel case, this case is also facing the challenges on
implementing the green activities. It is said that Sumbiling Eco Village (SEV)
is having issue in obtaining regular sources of electricity and water. This is
because the location of SEV is far from the city where the flow of these sources
are quite limited. Besides having a scarce source of electricity and water, SEV
is also scarce in skilled workers who are well versed in the green activities. Another issue is that SEV struggles to maintain the facilities such as the benches
and chairs due to the inevitable circumstances such as fluctuations in climate
i.e. Brunei humid weather and the salinity of the wind (this is because SEV is
located next to the salt-water river bank of Sungai Temburong). These type of
nature caused the fast erosion of SEV facilities and architectures. In this regard,
SEV’s founder wanted help from the building schemes that utilized green
practices to prolong its facilities ‘endurance. Not only that, SEV also felt threatened by the existence of other ecotourism spots in the neighbouring
countries such as Sarawak and Miri; which they may have better offers in its
tourism businesses. Additionally, the local communities and the indigenous
cultures may also be ‘polluted’ by the foreign visitors and wealth. 2.1.1 BruTEL's 'Going Paperless' Initiative It is stated that BruTel is having challenges in implementing the new
change initiative. It is also stated that the status quo are from some of its
customer segments – particularly the elder people, where they are hesitant or
resistant to the change initiative i.e. go paperless project; this project is also in
line with BruTel’s third goal of corporate social responsibilities (to be
conservative to the natural environment). It is understood that the main cause
for these resistance are because the elder people are not techno-savvy and are
not familiar to the usage of technology. This indicates that BruTel’s main issue
is the difficulty in making the customer (elder people) to ‘digest’ and adapt to
the new intended change (switching from traditional transaction to electronic
billing). Therefore, it is assumed that BruTel is utilizing the Lewin’s three step
model. Lewin’s three step model functions to evaluate the organizational
change process; and to evaluate the challenges caused from the status quo on
the implementation of the change. Beforehand, the following is the brief
explanation of the three stages of Lewin’s model (Cummings & Worley, 2009): 1) Freezing – at this stage, the organization prepares the initial set of activities
of the intended change. This is to gently introduce the organization members or
other parties on the intended change. 2) Moving – this is a process when people starts to ‘digest’ the new change. At
this stage, status quo may still exist. 3) Refreezing – this is when people are accustomed and stabilized to the new
change. And when this occur, the organization is ready to consolidate its
planned change initiatives. Referring to the above model, at the first stage, BruTel has successfully
prepared the change initiative (‘go paperless’ initiative) as in line with its third
corporate social responsibilities goal i.e. being conservative to the natural
environment. At the second stage, it is where BruTel faces the status quo of its
customers (elderly), where they resist to the change of going paperless due to
not being a techno-savvy and unfamiliar with the usage of technology. According to Kotter & Schlesinger (2008), these resistance to change are due to
the misunderstanding and the lack of trust to the change. It is misunderstood of
the boon effects of the change. These people assumes that the change would
give them more bane as compared to boon. 2.1 Case Analysis 2.1 Case Analysis 1.1.1 BruTEL 's 'Going Paperless' Initiative Nevertheless,
SEV founder is persistent in the establishment of SEV and the founder wanted
to change people’s mind sets– especially the local community, about
ecotourism. It is also stated that an active tourism activities in the natural areas
tend to lead to environmental degradation. Therefore, it is crucial for SEV to set
a thorough plans and management in order to minimize the negative impact
from its activities – especially the jungle camping activities, towards the natural
environment. 1.1.3 Society for Community Outreach and Training (SCOT)’s Green Xchange
Project 1.1.3 Society for Community Outreach and Training (SCOT)’s Green Xchange
Project Last but not least, the SCOT case. It can be considered that SCOT has
run its Non-Government Organization (NGO) quite well. However, there are
several issues raised throughout its operation. The first issue is, SCOT is highly
reliant to the donations and funding from the government and private
organization. The main advantage from these donations and funding are to
realize SCOT’s next projects (after one project ends). This means that if there is
not enough money to conduct the next project, SCOT would have difficulties in
continuing the NGO to help the underprivileged people in entrepreneurship. Hence, SCOT’s mission of reducing poverty could also be hindered because the
underprivileged people is dependent on SCOT’s project e.g. building coops for
chicken rearing to earn money. In this regard, SCOT hopes that their Green
Xchange project is sufficient enough to generate their own financial source. The
Green Xchange project is to collect recyclable materials from the public and
exchange it with basic necessities such as rice. However, through this activity,
another issue arise. The price for the basic necessities are uncertain and may
fluctuate as they are highly dependent to some factors such as supplier countries’
climate and currency rate. This in result, will affect the price of exchange rate
between recyclable materials and the basic necessities. 2.0 Discussion This section will include the discussion of the root causes of the key raised issues. The
writer will discuss these issues based of the assumed theories that is implemented in each cases
based on the scholar’s planned change theory. The second subsection consists of the possible
solution(s) that can be employed in the respective cases. 2.1 Case Analysis 2.1.1 BruTEL's 'Going Paperless' Initiative Thus, resulting in the lack of trust
to the organization who implemented the change. In this case, the elder people
can be considered to misunderstand of the benefit of ‘go paperless’ initiative. At the final stage, as per stated in the case study, if these elder customers
are successfully persuade to switch to the new paperless system, then BruTel
can proceed to develop and implement its green strategies. This indicate that
BruTel’s dilemma on whether to continue or stop the green initiative to
accomplish its third goal. This dilemma is as a response to the non-techno-savvy
customer. Hence, it is concluded that BruTel failed to proceed to the final stage
of the Lewin’s three step model due to the challenges of status quo caused from
its elderly customers. It indicates that the main issue of BruTel is to get the
change implementation to be fully digested and accepted by its elderly
customers. 2.1.2 Sumbiling Eco Village: Promoting the Ecotourism in the Temburong
District In is assumed that SEV utilizes the Kotter’s eight step model. Kotter’s
eight step model consists of the of eight stages: 1) Establishing a sense of
urgency, 2) Creating the guiding coalition, 3) Developing a vision and strategy,
4) Communicating the change, 5) Empowering the broad-based action, 6)
Generating short-term wins, 7) Consolidating gains and producing more change,
and 8) Anchoring new approaches in the culture (Cummings & Worley, 2009). Below are the brief explanation of each stages (“The 8-Step Process”, n.d.),
followed by the writer’s analysis based on these eight stages: 1) Establishing a sense of urgency – in this stage, the organization are to seek
an urgent and affirmed attention from the others and telling them that change is
needed within the organization. Referring this to SEV case, SEV is aware that
its organization is lacking the assets such as irregular flow of electricity and
water, lack of skilled employees who are well versed on the green activities, as
well as lacking in maintenance capability where their architectures are easily
worn out by natural processes due ti its geographical factors (e.g. Brunei’s
humid climate and saline wind from the salt-water river beside SEV). It is also
stated that SEV feels threatened by the neighbouring ecotourism businesses
which may offer better services. 2.1.1 BruTEL's 'Going Paperless' Initiative 2) Creating the guiding coalition – this is a process where the organization
finds the most influential person or people (within or outside the organization)
to create the most effective way to telling people on the importance of the
change. In this case, it is stated that SEV is trying to get connected with green
building schemes to help them prolonging its architectures and facilities’
endurance. It is also understood that SEV founder intends to urge the local
community especially the nearby community to support SEV as it is mentioned
that the nearby community are against SEV change initiative to run the
ecotourism business. 3) Developing a vision and strategy – this phase involves the creation of the
change vision in order to provide the organization members the clear idea or the
rationale of the planned change. The development of strategies also takes place
in this phase. Referring to the case, SEV founder explained that she intends to
make its business more attractive (in architecture and services wise) in order to
attract more customers, to be competitive with the other neighbouring
competitors, as well as to change the community’s mind sets on the ecotourism. 4) Communicating the change – this is where the organization members gather
as many people in the organization to ensure all organization members are aware
of the new change and clear of its vision. As for this stage onwards, it is not
stated in the case of whether SEV has conducted the strategies based on these
stages of Kotter’s eight step model. Thus, the writer assumes that SEV has not
gone to this stage yet. Hence, the writer will discuss on this stages relating to
SEV case study in the Alternative Solution section of this report. 5) Empowering the broad-based action – this phase omits as much barriers
as possible e.g. hierarchical barriers, in order to promote freedom in
communication and it enables the members of the organization to do their task
or jobs well. 6) Generating short-term wins – this process involves the structuring of the
short term steps towards the long term goals. It is to create the sense of winning
the goals often in which this action will promote the organization members to
be motivated and can perform better. 7) Consolidating gains and producing more change – this phase involves in
the constant effort put after success in order to produce more successful changes.
This process is mainly to achieve the organization’s long term vision. 7) Consolidating gains and producing more change – this phase involves in
the constant effort put after success in order to produce more successful changes. This process is mainly to achieve the organization’s long term vision. 8) Anchoring new approaches in the culture – this final stage of the planned
change model consists of the conveyed connections between the new planned
changes. 8) Anchoring new approaches in the culture – this final stage of the planned
change model consists of the conveyed connections between the new planned
changes. 2.1.3 Society for Community Outreach and Training (SCOT)’s Green Xchange
Project 2.1.3 Society for Community Outreach and Training (SCOT)’s Green Xchange
Project As for the SCOT case, it is assumed that SCOT uses the Positive model
because it is stated in the case study that SCOT is focusing on its success
activities to develop to a much better outcome. Positive model “focuses on
positive dynamics in organizations that give rise to extraordinary outcomes”
(Cummings & Worley, 2009). It means that the organization focuses on its good
side as a driver to develop to a better outcome. Positive model has five stages:
1) Initiate the inquiry, 2) Inquire into Best Practice, 3) Discover the themes, 4)
Envision a preferred future, and 5) Design and deliver ways ti create the future. 1) Initiate the Inquiry – this is a process where the organization members
identifies the main organization issue. This is when SCOT identifies that they
are facing financial issue where they highly reliant to the government and
private organization’s funding and donations for most of its conducted projects. 2) Inquire into Best Practices – in this stage, communicated information
amongst the organization members are gathered; it involves the topic on what is
their best attributes. It is understood that SCOT has communicated to its staffs
and volunteers that their Green Xchange project is the most successful project
and that has the most potential to overcome its financial issue. 3) Discover the Themes – this is when the organization members shares ideas
or innovation of change and create the theme of change. In this phase, SCOT
founder itself belief that their Green Xchange project has benefitted them financially since it is fund-driven. This project at the same time enables them to
achieve its mission on the green initiatives (raising green awareness and
reducing poverty). financially since it is fund-driven. This project at the same time enables them to
achieve its mission on the green initiatives (raising green awareness and
reducing poverty). 4) Envision a Preferred Future – after all information are gathered, the
members identifies which theme is the most mutually agreed. The members also
envision its change initiative’s vision; it involves on the long term goal of the
intended change. At this phase, SCOT has visualized the possible goals of its
Green Xchange project where they strongly belief that this project would give
them an internal financial source. 5) Design and Deliver Ways to Create the Future – this is the final stage of
the Positive model. 2.1.3 Society for Community Outreach and Training (SCOT)’s Green Xchange
Project It involves the set of activities that are to be conducted to
form and implement on the change strategies. As stated in the case study, SCOT
has successfully innovate changes to achieve an even better outcome. They are
also able to generate its own finance that is sufficient enough to be allocated to
their next projects. Additionally, this project has also successfully promoted the
public to the green awareness which is aligned with SCOT’s mission. This
indicates that SCOT has fully utilized this change model. 2.2 Alternative Solutions In this section, the writer will suggest some solutions in order to overcome the
issues faced in each case. 2.2.1 BruTEL's 'Going Paperless' Initiative 2.2.1 BruTEL's 'Going Paperless' Initiative It is recommended that BruTel communicate to the elderly on the actual
benefit of the ‘go paperless’ change. This is to ensure that the elder
customers are aware of the green initiative – not only to the organization
itself, but to the world as a whole. BruTel should not completely implement the change of going paperless as
according to Kotter & Schlesinger (2008), some people are limited to
change that caused them to resist the change. To overcome this issue, BruTel
may provide employees to guide them on how to use the technology. However, this strategy may not be as effective thus, BruTel could hire a
specific employee to be assigned as the guider or helper whenever these
non-techno-savvy people struggles on the new change. 2.2.2 Sumbiling Eco Village: Promoting the Ecotourism in the Temburong
District Since it is assumed that the SEV has not fully executed its change strategies,
it is recommended for the SEV to continue its strategies from developing its
change vision and strategies to communicating the change. It is crucial to
communicate to the whole organization of the intended change. This is to aid
and guide all members of the organization to conduct and execute its
respective jobs. Additionally, this step is crucial in order to avoid any uncertainty experiences of the organization members. When they are
uncertain, the change strategy would have not successfully executed. Next is empowering the broad-based action; this is where the organization
members of SEV, especially the manager, should avoid any barriers. The
barriers include, allowing rumours, strict hierarchical structures during
meeting and etc. This hinders the freedom of its member to express their
feedback on the intended change which may affect the effectiveness of
change strategies. SEV also have to create appreciation more often for its employees. This is to
create motivation to the employees. Thus, the employees could be motivated
to exert its expertise in their jobs (e.g. survival skills for the jungle camping
programme). As for the seventh stage of Kotter’s eight step model, SEV should make
continuous efforts in its strategies as SEV is an ecotourism business which
most of its income are of the interaction with human. SEV should keep track
of its customers’ feedback on the business as a whole. SEV should also pay
attention to the nearby community where one of the SEV’s activity is jungle
camping. 2.2.1 BruTEL's 'Going Paperless' Initiative This activity could or may interrupt the nearby community’s
routines which eventually cause them to oppose to the establishment of SEV. Last but not least, SEV should portray the necessity and the benefit of the
implemented change. This is to help the process of adaptation and the
acceptation of the new change from the respective affected parties to the
SEV. 2.2.3 Society for Community Outreach and Training (SCOT)’s Green Xchange
Project 2.2.3 Society for Community Outreach and Training (SCOT)’s Green Xchange
Project It is recommended that SCOT would have at least, another fund-generated
project in order to generate its own self-financed green projects. This is to
avoid limited options for its financial resources. Seeing the possibilities, if
the Green Xchange project is no longer plausible to the public, then SCOT
would have again dependent of other parties’ funds and donations. SCOT should also communicate to the public in order to have an optimal
effect on raising its green awareness. This action will eventually help the
public to engage or participate to SCOT’s green activities and thus,
enabling them to accomploish their missions (i.e. raising green awareness,
promoting green behaviour and reducing poverty by teaching the
underprivileged people to do entrepreneurship though managing waste
materials). 3.0 Recommendation Through the analyses and the proposed solutions, it is concluded that communication is the
best befitting strategy to be implemented in all three cases as this the most basic type of
organizational development and change strategy. According to Kotter and Schleisinger (2008),
communication is the best strategy to use when dealing with changes, especially when there is
resistance to change. Communication provides people to give and take the rationale of changes. It also aids the organization member or any parties to have the clear view of what and why
there is the needs to be change. Elving (2005) also mentioned in his study that communication
promtes the sense of community where organization members can work in harmony. This will
eventually enhances the execution of its organizational change strategies. While there may be
several other solutions to overcome organizational change implementation, the writer believes
that communication is the most common and the best solution to these three given cases. The 8-Step Process for Leading Change | Dr. John Kotter. Kotter. Retrieved 5 May 2020, from
https://www.kotterinc.com/8-steps-process-for-leading-change/. References Cummings, T. G., Worley, C. G. (2009): Organization Development & Change (9th ed.). Cengage Learning. Elving, W. (2005). The role of communication in organisational change. Corporate
Communications: An International Journal. 10. 129-138. 10.1108/13563280510596943. Kotter, J., & Schlesinger, L. (2008). Choosing Strategies for Change. Harvard Business
Review. Retrieved 4 May 2020, from https://hbr.org/2008/07/choosing-strategies-for-
change. The 8-Step Process for Leading Change | Dr. John Kotter. Kotter. Retrieved 5 May 2020, from
https://www.kotterinc.com/8-steps-process-for-leading-change/.
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PUBLIC COMMUNICATION ON SOCIAL MEDIA: CONNECTING GOVERNMENTS AND CITIZENS ONLINE
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PUBLIC COMMUNICATION ON SOCIAL MEDIA:
CONNECTING GOVERNMENTS AND CITIZENS ONLINE
Author: Priscila Minussii PUBLIC COMMUNICATION ON SOCIAL MEDIA:
CONNECTING GOVERNMENTS AND CITIZENS ONLINE
Author: Priscila Minussii PUBLIC COMMUNICATION ON SOCIAL MEDIA:
CONNECTING GOVERNMENTS AND CITIZENS ONLINE
Author: Priscila Minussii Author: Priscila Minussii Social media use is growing worldwide. Although research shows that they are used primarily for
leisure, it has been increasingly used for political and public communication. On the one hand, many
experts have been condemning social media for undermining democracy and enhancing political
polarisation. On the other hand, these networks potentially bring positive outcomes, such as greater
transparency from public institutions and direct communication between governments and
citizens. However, the institutions have a long way to go in tailoring their communications efforts
to convey public information while appealing to the public successfully — and ultimately
strengthening people's trust and interest in politics. If entertainment seems to be the most popular content on social media, how could public agencies
compete for people's attention online? How could public communication be adapted to social
media? This public service must not simply replicate the content designed for traditional broadcast
media. Instead, it should balance different needs: the audience's demands and the states'
commitment to informing citizens while maximizing each platform's particular demographics,
features and algorithms. BACKGROUND authors found that 45% of all posts regarded symbolic and presentational exchanges, which m
that the Facebook posts had messages containing a favourable presentation of the departm
political positioning, symbolic acts and/or marketing. Further, they found that 17.5% of the p
FINDINGS This paradigm shift inevitably led to discussions about the potential democratizing power of the
Internet and how it could hinder citizen participation and engagement. Among the several
criticisms, it’s argued that the same political elite tendencies of the pre-Internet age continue
evolving in the new media age; digital communication is used predominantly as “a new technical
system for distributing old-fashioned mass communications content” according to Meyer and
Hinchman [6]. In the end of the day, the public only gets further from the political centre [7], and
the expansion of new media remains far from achieving its potential for effectively engaging
citizens. Regardless of such criticisms, governments worldwide are increasingly investing in and
professionalizing their social media communication strategies. The reasons behind this approach
include: a) reaching out to citizens and stakeholders; b) sharing information across and within
political agencies and governments; c) disseminating information about the governmental
operation; d) promoting citizen participation and government transparency; among others
[8][9][10][11]. Despite its flaws, the potential for connecting people and governments should not be
underestimated: on a global scale, people are spending more time on social media than ever,
increasing the likelihood of coming across government’s news and statements on social media first
and, then, on other media, such as TV or radio. BACKGROUND Surveys show citizens’ growing scepticism towards politics: their concerns range from
dissatisfaction with the democracies they live in [1] to utter distrust in political institutions [2]. Nevertheless, their expectations towards democratic institutions and demands for accountability
and transparency only increase [3]. "Transparency is now proposed as the solution to one of the
most intangible problems of democratic governance: citizens' increasing mistrust of government",
argues Grimmelikhuijsen and Porumbescu [4]. 1
rnment",
ucer and
ation are
f Manuel In parallel, social media flourishes, and virtually anyone can simultaneously be a producer and
consumer of information or become an influencer [5]. Thus, public and political communication are
transformed: what was once limited to mass communication becomes, in the words of Manuel
Castells, "mass self-communication" [5] — fertile soil for misinformation. 1 interagency.institute N.1 APR 2022
ISSN 2789-8040 interagency institute
This paradigm shift inevitably le
Internet and how it could hin
criticisms, it’s argued that the
evolving in the new media age;
system for distributing old-fash
Hinchman [6]. In the end of the
the expansion of new media re
citizens. Regardless of such criticisms
professionalizing their social me
include: a) reaching out to citiz
political agencies and governm
operation; d) promoting citize
[8][9][10][11]. Despite its flaws, the potent
underestimated: on a global sc
increasing the likelihood of comi
and, then, on other media, such a
Studies show that effective publi
and political activism; and that
affects the latter's perception of
Case studies on government soci
the E.U. found that these netw
gestures and information provis
Self-presentation is connected to
be used to counteract a governm
Marketing is used in communic
separate it from the informativ
promote a municipality or vacc
resorted to present government
or condolences due to an eve
governmental activities and fact
teach citizens about an importan
DePaula, Dincelli and Harrison
which they used to analyze Am
authors found that 45% of all po
that the Facebook posts had me
political positioning, symbolic ac
FINDINGS interagency.institute
N 1 AP
This paradigm shift inevitably led to discussions
Internet and how it could hinder citizen part
criticisms, it’s argued that the same political el
evolving in the new media age; digital communic
system for distributing old-fashioned mass com
Hinchman [6]. In the end of the day, the public o
the expansion of new media remains far from
citizens. BACKGROUND Despite its flaws, the potential for connecting people and governments should not be
underestimated: on a global scale, people are spending more time on social media than ever,
increasing the likelihood of coming across government’s news and statements on social media first
and, then, on other media, such as TV or radio. Studies show that effective public communication in social media can increase citizen engagement
and political activism; and that online interaction between political representatives and citizens
affects the latter's perception of government transparency, efficiency, and corruption [13]. Case studies on government social media communication at local and national levels in the U.S. and
the E.U. found that these networks are used mostly for self-presentation, marketing, symbolic
gestures and information provision purposes [8][9][10][11], which are described below. Self-presentation is connected to impression management and public relations efforts, which could
FINDINGS This paradigm shift inevitably led to discussions about the potential democratizing power of th
Internet and how it could hinder citizen participation and engagement. Among the severa
criticisms, it’s argued that the same political elite tendencies of the pre-Internet age continu
evolving in the new media age; digital communication is used predominantly as “a new technica
system for distributing old-fashioned mass communications content” according to Meyer an
Hinchman [6]. In the end of the day, the public only gets further from the political centre [7], an
the expansion of new media remains far from achieving its potential for effectively engagin
citizens. Regardless of such criticisms, governments worldwide are increasingly investing in an
professionalizing their social media communication strategies. The reasons behind this approac
include: a) reaching out to citizens and stakeholders; b) sharing information across and withi
political agencies and governments; c) disseminating information about the governmenta
operation; d) promoting citizen participation and government transparency; among other
[8][9][10][11]. Despite its flaws, the potential for connecting people and governments should not b
underestimated: on a global scale, people are spending more time on social media than eve
increasing the likelihood of coming across government’s news and statements on social media firs
and, then, on other media, such as TV or radio. FINDINGS This paradigm shift inevitably led to discussions about the potential democratizing power o
Internet and how it could hinder citizen participation and engagement. BACKGROUND Among the sev
criticisms, it’s argued that the same political elite tendencies of the pre-Internet age cont
evolving in the new media age; digital communication is used predominantly as “a new tech
system for distributing old-fashioned mass communications content” according to Meyer
Hinchman [6]. In the end of the day, the public only gets further from the political centre [7],
the expansion of new media remains far from achieving its potential for effectively enga
citizens. Regardless of such criticisms, governments worldwide are increasingly investing in
professionalizing their social media communication strategies. The reasons behind this appr
include: a) reaching out to citizens and stakeholders; b) sharing information across and w
political agencies and governments; c) disseminating information about the governme
operation; d) promoting citizen participation and government transparency; among ot
[8][9][10][11]. Despite its flaws, the potential for connecting people and governments should not
underestimated: on a global scale, people are spending more time on social media than e
increasing the likelihood of coming across government’s news and statements on social media
and, then, on other media, such as TV or radio. Studies show that effective public communication in social media can increase citizen engagem
and political activism; and that online interaction between political representatives and citi
affects the latter's perception of government transparency, efficiency, and corruption [13]. Case studies on government social media communication at local and national levels in the U.S
the E.U. found that these networks are used mostly for self-presentation, marketing, symb
gestures and information provision purposes [8][9][10][11], which are described below. Self-presentation is connected to impression management and public relations efforts, which c
be used to counteract a government's low popularity or push citizens' interest in public institut
Marketing is used in communications in many ways and for many purposes — it's often har
separate it from the informative content. In plain words, marketing is useful, for instanc
promote a municipality or vaccination campaign. Symbolic presentation is emblematic, usu
resorted to present governments taking a stance on a political issue or expressing congratulat
or condolences due to an event. Information provision regards the traditional reportin
governmental activities and factual information and educational campaigns, which are intende
teach citizens about an important issue or simply to make public service announcements [12]. DePaula, Dincelli and Harrison created a typology of government social media communica
which they used to analyze American local government departments' Facebook pages [12]. BACKGROUND Regardless of such criticisms, governments w
professionalizing their social media communicat
include: a) reaching out to citizens and stakeho
political agencies and governments; c) dissem
operation; d) promoting citizen participation
[8][9][10][11]. Despite its flaws, the potential for connecti
underestimated: on a global scale, people are s
increasing the likelihood of coming across govern
and, then, on other media, such as TV or radio. Studies show that effective public communication
and political activism; and that online interactio
affects the latter's perception of government tran
Case studies on government social media commun
the E.U. found that these networks are used m
gestures and information provision purposes [8][
Self-presentation is connected to impression man
be used to counteract a government's low popular
Marketing is used in communications in many w
separate it from the informative content. In pl
promote a municipality or vaccination campaig
resorted to present governments taking a stance
or condolences due to an event. Information
governmental activities and factual information a
teach citizens about an important issue or simply
DePaula, Dincelli and Harrison created a typolo
which they used to analyze American local gove
authors found that 45% of all posts regarded sym
that the Facebook posts had messages containin
political positioning, symbolic acts and/or marke
FINDINGS This paradigm shift inevitably led to discussions about the potential democratizing power of the
Internet and how it could hinder citizen participation and engagement. Among the several
criticisms, it’s argued that the same political elite tendencies of the pre-Internet age continue
evolving in the new media age; digital communication is used predominantly as “a new technical
system for distributing old-fashioned mass communications content” according to Meyer and
Hinchman [6]. In the end of the day, the public only gets further from the political centre [7], and
the expansion of new media remains far from achieving its potential for effectively engaging
citizens. Regardless of such criticisms, governments worldwide are increasingly investing in and
professionalizing their social media communication strategies. The reasons behind this approach
include: a) reaching out to citizens and stakeholders; b) sharing information across and within
political agencies and governments; c) disseminating information about the governmental
operation; d) promoting citizen participation and government transparency; among others
[8][9][10][11]. FINDINGS Ultimate
collaboration with citizens, promote intera
Public institutions still have a long way to
While these interactive channels have the
underused, serving as a replica of the mess
channels — that is, one-way communicatio
Changing the public communication para
Although using social media isn't exclusive
reason behind most governments' decisio
the core of transparency efforts, they need
all and, most importantly, effectively reach
Effective online communication is not onl
that interests them but also following an
certain topics and formats to the detrimen
the case mentioned above studies must b
and political communication on social me
online platforms, types of content or medi
Although social media's features, populari
useful for public agencies and government
CONCLUSIONS
RECOMMENDATIONS regard
employ
Minuss
nation
of post
operat
to the C
Sobaci
local p
i f regarded fav
employees w
Minussi used
national gove
of posts conc
operations an
to the Covid- regarded favour
employees were
Mi
i
d h regarded favourable presentation alone, in which achievements of the organization or one of its
employees were the main content of the post. Minussi used the typology mentioned earlier to analyze the Facebook posts published by the
national governments of Portugal, Spain and Denmark [10][11]. It was found that the great majority
of posts concerning information provision, that is, messages about public service announcements,
operations and events that these governments joined or organized, and policies - especially related
o the Covid-19 pandemic. Sobaci and Karkin researched Twitter's impact on transparent, participatory and citizen-oriented
local public services in Turkey. The analysis revealed that Turkish mayors use it mostly to share
information, send personal messages and share their location and activities. Promoting
transparent, participatory and citizen-oriented public service wasn't the goal. Indeed, self-
promotion and political marketing were the main purposes for using Twitter. Looking at all research findings described above, it was fairly usual to find social media posts that
overlap two or more purposes. Ultimately, the research findings agree that posts that foster
collaboration with citizens, promote interactivity, or ask for citizens' input are rare. CONCLUSIONS Public institutions still have a long way to maximize their communication efforts on social media. While these interactive channels have the potential to bridge politics and citizens, they have been
underused, serving as a replica of the messages and strategy designed for the old media, broadcast
channels — that is, one-way communication of news, marketing and symbolic acts. FINDINGS Changing the public communication paradigm presents both new challenges and opportunities. Although using social media isn't exclusively for promoting public transparency, this is the alleged
reason behind most governments' decision to join these networks. If institutions aim at making it
the core of transparency efforts, they need to start by ensuring their communications are open to
all and, most importantly, effectively reach citizens. Effective online communication is not only a matter of providing social media users with content
that interests them but also following and adapting to each platform’s algorithms, which boosts
certain topics and formats to the detriment of others. On the whole, findings and conclusions from
the case mentioned above studies must be taken with a grain of salt: not only research on public
and political communication on social media is still at an early stage, but the popularity of certain
online platforms, types of content or media formats are debatable and constantly changing. 3
could be
In short, FINDINGS Public institutions still have a long way to maximize their communication efforts on socia
While these interactive channels have the potential to bridge politics and citizens, they ha
underused, serving as a replica of the messages and strategy designed for the old media, br
channels — that is, one-way communication of news, marketing and symbolic acts. Changing the public communication paradigm presents both new challenges and oppor
Although using social media isn't exclusively for promoting public transparency, this is the
reason behind most governments' decision to join these networks. If institutions aim at m
the core of transparency efforts, they need to start by ensuring their communications are
all and, most importantly, effectively reach citizens. Effective online communication is not only a matter of providing social media users with
that interests them but also following and adapting to each platform’s algorithms, which
certain topics and formats to the detriment of others. On the whole, findings and conclusio
the case mentioned above studies must be taken with a grain of salt: not only research o
and political communication on social media is still at an early stage, but the popularity of
online platforms types of content or media formats are debatable and constantly changing
CONCLUSIONS regarded favourable presentation alone, in which achievements of the
employees were the main content of the post. Minussi used the typology mentioned earlier to analyze the Faceboo
national governments of Portugal, Spain and Denmark [10][11]. It was fo
of posts concerning information provision, that is, messages about pub
operations and events that these governments joined or organized, and
to the Covid-19 pandemic. Sobaci and Karkin researched Twitter's impact on transparent, particip
local public services in Turkey. The analysis revealed that Turkish ma
information, send personal messages and share their location a
transparent, participatory and citizen-oriented public service wasn
promotion and political marketing were the main purposes for using Tw
Looking at all research findings described above, it was fairly usual to f
overlap two or more purposes. FINDINGS Further, they found that 17.5% of the posts 2 N.1 APR 2022
ISSN 2789-8040 regarded favourable presentation alone, in which achievements of the organization or one of its
employees were the main content of the post. Minussi used the typology mentioned earlier to analyze the Facebook posts published by the
national governments of Portugal, Spain and Denmark [10][11]. It was found that the great majority
of posts concerning information provision, that is, messages about public service announcements,
operations and events that these governments joined or organized, and policies - especially related
to the Covid-19 pandemic. Sobaci and Karkin researched Twitter's impact on transparent, participatory and citizen-oriented
local public services in Turkey. The analysis revealed that Turkish mayors use it mostly to share
information, send personal messages and share their location and activities. Promoting
transparent, participatory and citizen-oriented public service wasn't the goal. Indeed, self-
promotion and political marketing were the main purposes for using Twitter. Looking at all research findings described above, it was fairly usual to find social media posts that
overlap two or more purposes. Ultimately, the research findings agree that posts that foster
collaboration with citizens, promote interactivity, or ask for citizens' input are rare. regarded favourable presentation alone, in which achievements of the organization or on
employees were the main content of the post. Minussi used the typology mentioned earlier to analyze the Facebook posts published
national governments of Portugal, Spain and Denmark [10][11]. It was found that the great m
of posts concerning information provision, that is, messages about public service announc
operations and events that these governments joined or organized, and policies - especially
to the Covid-19 pandemic. Sobaci and Karkin researched Twitter's impact on transparent, participatory and citizen-o
local public services in Turkey. The analysis revealed that Turkish mayors use it mostly t
information, send personal messages and share their location and activities. Pro
transparent, participatory and citizen-oriented public service wasn't the goal. Indee
promotion and political marketing were the main purposes for using Twitter. Looking at all research findings described above, it was fairly usual to find social media po
overlap two or more purposes. Ultimately, the research findings agree that posts tha
collaboration with citizens, promote interactivity, or ask for citizens' input are rare. FINDINGS Studies show that effective public communication in social media can increase citizen engagement
and political activism; and that online interaction between political representatives and citizens
affects the latter's perception of government transparency, efficiency, and corruption [13]. Case studies on government social media communication at local and national levels in the U.S. and
the E U found that these networks are used mostly for self presentation marketing symbolic Studies show that effective public communication in social media can increase citizen engagement
and political activism; and that online interaction between political representatives and citizens
affects the latter's perception of government transparency, efficiency, and corruption [13]. and political activism; and that online interaction between political representatives and citizens
affects the latter's perception of government transparency, efficiency, and corruption [13]. Case studies on government social media communication at local and national levels in the U.S. and
the E.U. found that these networks are used mostly for self-presentation, marketing, symbolic
gestures and information provision purposes [8][9][10][11], which are described below. Case studies on government social media communication at local and national levels in the U.S. and
the E.U. found that these networks are used mostly for self-presentation, marketing, symbolic
gestures and information provision purposes [8][9][10][11], which are described below. Self-presentation is connected to impression management and public relations efforts, which could
be used to counteract a government's low popularity or push citizens' interest in public institutions. Marketing is used in communications in many ways and for many purposes — it's often hard to
separate it from the informative content. In plain words, marketing is useful, for instance, to
promote a municipality or vaccination campaign. Symbolic presentation is emblematic, usually
resorted to present governments taking a stance on a political issue or expressing congratulations
or condolences due to an event. Information provision regards the traditional reporting of
governmental activities and factual information and educational campaigns, which are intended to
teach citizens about an important issue or simply to make public service announcements [12]. 2
DePaula, Dincelli and Harrison created a typology of government social media communication,
which they used to analyze American local government departments' Facebook pages [12]. The
authors found that 45% of all posts regarded symbolic and presentational exchanges, which means
that the Facebook posts had messages containing a favourable presentation of the departments,
political positioning, symbolic acts and/or marketing. FINDINGS Ultimately, the research findings ag
collaboration with citizens, promote interactivity, or ask for citizens' inp
Public institutions still have a long way to maximize their communicat
While these interactive channels have the potential to bridge politics a
underused, serving as a replica of the messages and strategy designed fo
channels — that is, one-way communication of news, marketing and sym
Changing the public communication paradigm presents both new cha
Although using social media isn't exclusively for promoting public trans
reason behind most governments' decision to join these networks. If in
the core of transparency efforts, they need to start by ensuring their co
all and, most importantly, effectively reach citizens. Effective online communication is not only a matter of providing socia
that interests them but also following and adapting to each platform’s
certain topics and formats to the detriment of others. On the whole, find
the case mentioned above studies must be taken with a grain of salt: n
and political communication on social media is still at an early stage, bu
online platforms, types of content or media formats are debatable and c
Although social media's features, popularity and algorithms aren't defin
CONCLUSIONS
RECOMMENDATIONS regarded favourable presentation alone, i
employees were the main content of the p
Minussi used the typology mentioned ea
national governments of Portugal, Spain an
of posts concerning information provision
operations and events that these governm
to the Covid-19 pandemic. Sobaci and Karkin researched Twitter's im
local public services in Turkey. The analy
information, send personal messages
transparent, participatory and citizen-o
promotion and political marketing were th
Looking at all research findings described
overlap two or more purposes. RECOMMENDATIONS Public institutions from local to national levels sh
strategies that would boost their reach and
governments could work on promoting citizen p
projects and meetings with political represen
governments plan on using social media prima
more effective if the posted contents concern th
see as impactful on their lives" [14]. Since local and regional governments seem
institutions could focus on wider campaigns sh
lives. Otherwise, citizens might continue to feel
have a unique opportunity to benefit from s
respective national identities and, consequently,
Governmental agencies need to use social med
ongoing communication that creates awareness
encouraging the population to get vaccinated a
with bureaucratic-tone news or strong self-prom
algorithms nor interest people. In the end,
accountable and informing: it's also about st
representatives. [1] Alliance of Democracies; Rasmussen Global;
Index 2018. [2] Ferrín-Pereira, M. (2012). What is Democr
Evaluations of Democratic Systems in Contem
University Institute, Florence, Italy). [3] Coicaud, J. M. (2019). The Paradoxical Pe
Question of its Future. Global Policy, 10(1), 110–
[4] Grimmelikhuijsen, S., & Porumbescu, G. (
REFERENCES Every country and region has its more popular platforms (Facebook, Instagram, TikTok, Twitter,
etc.), and each platform requires different communication strategies, supports certain media
formats (photos, videos, text, etc.) and has different user demographics. Thus, publishing content
tailored for each network is quintessential. Public institutions from local to national levels should collaborate and liaise digital communications
strategies that would boost their reach and efficacy online. For instance, local and regional
governments could work on promoting citizen participation, such as volunteering for community
projects and meetings with political representatives. Bonsón and Ratkai argue that “if local
governments plan on using social media primarily for citizen engagement, their efforts might be
more effective if the posted contents concern the citizens' topics of interest — that is, topics they
see as impactful on their lives" [14]. p
[
]
Since local and regional governments seem more approachable to citizens, national-level
institutions could focus on wider campaigns showing their impact on the population's everyday
lives. Otherwise, citizens might continue to feel detached from representative institutions. States
have a unique opportunity to benefit from social media communication to strengthen their
respective national identities and, consequently, citizen involvement and participation in politics. Governmental agencies need to use social media as a campaign tool and apply consistent and
ongoing communication that creates awareness and advocates for the public good. RECOMMENDATIONS For instance,
encouraging the population to get vaccinated and vote. Uncoordinated efforts to inform citizens
with bureaucratic-tone news or strong self-promotional messages won't cut through the networks'
algorithms nor interest people. In the end, public communication is not only about being
accountable and informing: it's also about strengthening citizens' trust in politics and their
representatives. Since local and regional governments seem more approachable to citizens, national-level
institutions could focus on wider campaigns showing their impact on the population's everyday
lives. Otherwise, citizens might continue to feel detached from representative institutions. States
have a unique opportunity to benefit from social media communication to strengthen their
respective national identities and, consequently, citizen involvement and participation in politics. Governmental agencies need to use social media as a campaign tool and apply consistent and
ongoing communication that creates awareness and advocates for the public good. For instance,
encouraging the population to get vaccinated and vote Uncoordinated efforts to inform citizens Since local and regional governments seem more approachable to citizens, national-level
institutions could focus on wider campaigns showing their impact on the population's everyday
lives. Otherwise, citizens might continue to feel detached from representative institutions. States
have a unique opportunity to benefit from social media communication to strengthen their
respective national identities and, consequently, citizen involvement and participation in politics. Governmental agencies need to use social media as a campaign tool and apply consistent and
ongoing communication that creates awareness and advocates for the public good. For instance,
encouraging the population to get vaccinated and vote. Uncoordinated efforts to inform citizens
with bureaucratic-tone news or strong self-promotional messages won't cut through the networks'
algorithms nor interest people. In the end, public communication is not only about being
accountable and informing: it's also about strengthening citizens' trust in politics and their
representatives. REFERENCES RECOMMENDATIONS Although social media's features, popularity and algorithms aren't definitive, the findings could be
useful for public agencies and governments reshape and improve online communications. In short, 3 interagency.institute N.1 APR 2022
ISSN 2789-8040 online communications can't be limited to providing old-fashioned mass communications news and
information. Every country and region has its more popular platforms (Facebook, Instagram, TikTok, Twitter,
etc.), and each platform requires different communication strategies, supports certain media
formats (photos, videos, text, etc.) and has different user demographics. Thus, publishing content
tailored for each network is quintessential. Public institutions from local to national levels should collaborate and liaise digital communications
strategies that would boost their reach and efficacy online. For instance, local and regional
governments could work on promoting citizen participation, such as volunteering for community
projects and meetings with political representatives. Bonsón and Ratkai argue that “if local
governments plan on using social media primarily for citizen engagement, their efforts might be
more effective if the posted contents concern the citizens' topics of interest — that is, topics they
see as impactful on their lives" [14]. Since local and regional governments seem more approachable to citizens, national-level
institutions could focus on wider campaigns showing their impact on the population's everyday
lives. Otherwise, citizens might continue to feel detached from representative institutions. States
have a unique opportunity to benefit from social media communication to strengthen their
respective national identities and, consequently, citizen involvement and participation in politics. Governmental agencies need to use social media as a campaign tool and apply consistent and
ongoing communication that creates awareness and advocates for the public good. For instance,
encouraging the population to get vaccinated and vote. Uncoordinated efforts to inform citizens
with bureaucratic-tone news or strong self-promotional messages won't cut through the networks'
algorithms nor interest people. In the end, public communication is not only about being
accountable and informing: it's also about strengthening citizens' trust in politics and their
representatives. REFERENCES online communications can't be limited to provid
information. Every country and region has its more popular
etc.), and each platform requires different co
formats (photos, videos, text, etc.) and has diffe
tailored for each network is quintessential. REFERENCES [1] Alliance of Democracies; Rasmussen Global; & Dalia Research. (2018). Democracy Perception
Index 2018. [1] Alliance of Democracies; Rasmussen Global; & Dalia Research. (2018). Democracy Perception
Index 2018. [2] Ferrín-Pereira, M. (2012). What is Democracy to Citizens? Understanding Perceptions and
Evaluations of Democratic Systems in Contemporary Europe (Doctoral dissertation, European
University Institute, Florence, Italy). [2] Ferrín-Pereira, M. (2012). What is Democracy to Citizens? Understanding Perceptions and
Evaluations of Democratic Systems in Contemporary Europe (Doctoral dissertation, European
University Institute, Florence, Italy). 4
and the
Trust in
w, 73(4),
y. [3] Coicaud, J. M. (2019). The Paradoxical Perception of Contemporary Democracy, and the
Question of its Future. Global Policy, 10(1), 110–121. y
( )
[4] Grimmelikhuijsen, S., & Porumbescu, G. (2013). The Effect of Transparency on Trust in
Government: A Cross-National Comparative Experiment. Public Administration Review, 73(4),
575–586. [5] Castells, M. (2007). Communication, Power and Counter-Power in the Network Society. International Journal of Communication, 1, 238–266 [5] Castells, M. (2007). Communication, Power and Counter-Power in the Network Society. International Journal of Communication, 1, 238–266 interagency.institute interagency.institute N.1 APR 2022
ISSN 2789-8040 N.1 APR 2022
ISSN 2789-8040 [6] Meyer, T., & Hinchman, L. (2002). Media Democracy: How the Media Colonize Politics. Malden:
Blackwell. [7] Davis, A. (2010). Political Communication and Social Theory. London: Routledge. [7] Davis, A. (2010). Political Communication and Social Theory. London: Routledge. [8] Sobaci, M. Z., & Karkin, N. (2013). The Use of Twitter by Mayors in Turkey: Tweets for Better
Public Services? Government Information Quarterly, 30(4), 417–425. [ ]
,
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)
y
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[8] Sobaci, M. Z., & Karkin, N. (2013). The Use of Twitter by Mayors in Turkey: Tweets for Better
Public Services? Government Information Quarterly, 30(4), 417–425. [9] Bellström, P., Magnusson, M., Pettersson, J. S., & Thorén, C. (2016). Facebook Usage in a Local
Government: A Content Analysis of Page Owner Posts and User Posts. Transforming Government,
10(4), 548–567. ( )
[10] Minussi, P. (2020). Political Communication on Facebook: Comparing the Regeringen and La
Moncloa Pages (Master dissertation, University of Porto, Porto, Portugal). [11] Minussi, P. (2021). Political Communication on Facebook: Comparing the República
Portuguesa and LaMoncloa Pages. Journal of Education, Innovation, and Communication, 3(2), 39–
51. [12] DePaula, N., Dincelli, E., & Harrison, T. (2018). Toward a Typology of Government Social Media
Communication: Democratic Goals, Symbolic Acts and Self-Presentation. Government Information
Quarterly, 35(1), 98–108. [13] Valle-Cruz, D., Sandoval-Almazan, R., & Gil-Garcia, R. (2016). i Master in Political Communication from the University of Porto. E-mail: priscilaminussi@gmail.com REFERENCES Citizens' perceptions of the impact
of information technology use on transparency, efficiency and corruption in local governments. Information
Polity,
21(3),
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https://content.iospress.com/articles/information-polity/ip393 https://content.iospress.com/articles/information-polity/ip393
[14] Bonsón, E., Royo, S., & Ratkai, M. (2014). Citizens' Engagement on Local Governments'
Facebook Sites. An Empirical Analysis: The Impact of Different Media and Content Types in Western https://content.iospress.com/articles/information polity/ip393
[14] Bonsón, E., Royo, S., & Ratkai, M. (2014). Citizens' Engagement on Local Governments'
Facebook Sites. An Empirical Analysis: The Impact of Different Media and Content Types in Western
Europe. Government Information Quarterly 32(1), 52–62. 5 interagency.institute interagency.institute N.1 APR 2022
ISSN 2789-8040
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Commentary: From ‘sense of number’ to ‘sense of magnitude’ – The role of continuous magnitudes in numerical cognition
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GENERAL COMMENTARY
published: 04 January 2017
doi: 10.3389/fpsyg.2016.02032 Peter Kramer* and Paola Bressan Keywords: sense of number, sense of magnitude, numerosity estimation, occupancy, statistical learning A commentary on From ‘sense of number’ to ‘sense of magnitude’ – The role of continuous magnitudes in
numerical cognition
by Leibovich, T., Katzin, N., Harel, M., and Henik, A. (2016). Behav. Brain. Sci. [Epub ahead of print]. doi: 10.1017/S0140525X16000960 From ‘sense of number’ to ‘sense of magnitude’ – The role of continuous magnitudes in
numerical cognition
by Leibovich, T., Katzin, N., Harel, M., and Henik, A. (2016). Behav. Brain. Sci. [Epub ahead of print]. doi: 10.1017/S0140525X16000960 Unlike abstract ones in mathematics, concrete sets of elements in the real world have continuous
physical properties, such as overall area and density. The dominant view has it that humans
can estimate the discrete numerosities of such sets independently of the co-varying continuous
magnitudes; i.e., that humans have a “sense of number.” It has indeed been claimed that various
animals, ranging from monkeys to tiny fish, have this sense too. A recent paper by Leibovich et al. (2016) questions all of this (see also Morgan et al., 2014; Gebuis et al., 2016) and argues convincingly
that numerosity estimation is not independent from continuous magnitudes but relies on them;
that we have not a “sense of number” but a “sense of magnitude.” Edited by:
Bert Reynvoet,
KU Leuven, Belgium
Reviewed by:
Marcus Lindskog,
Uppsala University, Sweden
*Correspondence:
Peter Kramer
peter.kramer@unipd.it Edited by:
Bert Reynvoet,
KU Leuven, Belgium
Reviewed by:
Marcus Lindskog,
Uppsala University, Sweden
*Correspondence:
Peter Kramer
peter.kramer@unipd.it Edited by:
Bert Reynvoet,
KU Leuven, Belgium Reviewed by:
Marcus Lindskog,
Uppsala University, Sweden Yet the authors fail to cite a classic article that made the very same argument 25 years ago,
and—unlike Leibovich et al.—supported it with a quantitative model (Allik and Tuulmets, 1991). Although neither density, nor overall area, nor any other single continuous magnitude can provide
reliable information about numerosity, Leibovich et al. imply that all of them together can; they
suggest that “statistical learning” will take care of extracting this information and turn numerosity
estimates out of it. How statistical learning achieves this feat and whether the resulting numerosity
estimates will fit observed ones remains, unfortunately, unclear. Allik and Tuulmets’s alternative
“occupancy” model has its limits (e.g., Kramer et al., 2011; Bertamini et al., 2016) but it is specific,
it is quantitative, and it predicts observed numerosity estimation surprisingly well with just a single
free parameter. *Correspondence:
Peter Kramer
peter.kramer@unipd.it Specialty section:
This article was submitted to
Cognition,
a section of the journal
Frontiers in Psychology To understand the occupancy model, consider a set of identical dots and imagine that each of
them is covered with a larger disk, as in Figure 1. The model posits that the total area occupied by
the disks (occupancy) will be linearly related to the estimated numerosity of the dots. As long as dot
densities remain relatively low (Durgin, 1995), the model accounts for nearly 90% of the variance
in human data (Allik and Tuulmets, 1991). The closer together the dots are and thus the more the
disks overlap, the smaller occupancy is and thus the smaller the estimated numerosity of the dots is
predicted to be—as indeed observed (e.g., DeWind et al., 2015). Notably, because large numerosities
tend to be dense, the occupancy model predicts they will tend to be underestimated. This prediction
has been corroborated repeatedly too (e.g., Izard and Dehaene, 2008). How Leibovich et al.’s
statistical learning could make the same prediction is hard to tell. Received: 31 October 2016
Accepted: 14 December 2016
Published: 04 January 2017 FIGURE 1 | Effect of item spacing on occupancy. The “occupancy” of a
dot may be represented as a larger, concentric virtual disk that covers it,
shown here in gray. (The radius of the disk is a free parameter that, during
model fitting, Allik and Tuulmets estimated to be 0.33◦of visual angle.) The
three black dots in the bottom row are closer together than those in the top
row. As a result, the virtual disks in the bottom row overlap, making for a
smaller occupancy (total gray area) and therefore a smaller predicted
numerosity estimate. For illustration purposes only a few dots are shown; note,
however, that numerosity estimation concerns numerosities outside the
“subitizing” range of one to four items. The occupancy model—even in Durgin’s (1995) variant—is
too simple to work under all conditions (Kramer et al., 2011). Still, if the degree of overlap of the virtual disks covering
the dots is interpreted as an inverse measure of the mutual
discriminability of the dots, the model makes an intuitive point. The point is that the less discriminable from one another items
are, the lower their estimated numerosity ought to be. If so,
the occupancy model could be expanded and improved (Kramer
et al., 2011) by taking into account anything that affects the
items’ (1) discriminability (their distance from one another and
from fixation, for example), (2) retention across saccades (if
presentation times allow eye movements), and (3) retention in
memory (if the stimulus is presented or inspected sequentially or
compared to another one). The occupancy model deserves at the very least discussion—
if not revival and development. It has the potential to challenge
not only “sense of number” models but also Leibovich et al.’s
statistical-learning alternative to them. Unlike the latter, the
occupancy model is a “sense of magnitude” model that does not
require learning, is deterministic, quantitative, perception- rather
than cognition-based, and not inconsistent with the idea that
numerosity estimation might be innate and so simple that even
fish could do it. AUTHOR CONTRIBUTIONS Although people are under the impression they are blessed
with a high-resolution image of the world around them, careful
experimentation has revealed that this is largely an illusion
(e.g., Durgin, 1995). In numerosity-estimation experiments the PK wrote the first draft of the manuscript; both authors discussed
it, critically revised it, and agreed on the final version. dispersion. Atten. Percept. Psychophys. 78, 1460–1471. doi: 10.3758/s13414-01
6-1100-0
DeWind, N. K., Adams, G. K., Platt, M. L., and Brannon, E. M. (2015). Modeling
the approximate number system to quantify the contribution of visual stimulus
features. Cognition 142, 247–265. doi: 10.1016/j.cognition.2015.05.016
Durgin, F. H. (1995). Texture density adaptation and the perceived numerosity and
distribution of texture. J. Exp. Psychol. Hum. Percept. Perform. 21, 149–169.
Gebuis, T., Cohen Kadosh, R., and Gevers, W. (2016). Sensory-integration system
rather than approximate number system underlies numerosity processing: Citation: Kramer P and Bressan P (2017)
Commentary: From ‘sense of number’
to ‘sense of magnitude’ – The role of
continuous magnitudes in numerical
cognition. Front. Psychol. 7:2032. doi: 10.3389/fpsyg.2016.02032 January 2017 | Volume 7 | Article 2032 Frontiers in Psychology | www.frontiersin.org Item Discriminability and Numerosity Estimation Kramer and Bressan stimuli tend to be randomly placed items, usually dots, and
some of them end up clumped together. These items may be
impossible to discriminate from one another, even when they can
all be perceptually detected. This crowding effect (Whitney and
Levi, 2011) is a serious concern, because the opportunities for
crowding necessarily grow with numerosity and have been shown
to affect its estimation (Valsecchi et al., 2013; Anobile et al., 2015). So far, studies that claim that numerosity estimation does not
fully rely on continuous magnitudes, or that it is more cognitive
than perceptual in nature, have not been very specific about how
the perceptual system extracts numerosity from the stimulus (see
also Morgan et al., 2014). These studies have considered a wide
range of perceptual factors such as density, overall area, and
item size. None of these factors, however, has ever been claimed
to be crucial in numerosity estimation. The one factor that has
for as long as 25 years (i.e., occupancy) is instead consistently
ignored, including now by Leibovich et al. (2016). Izard and
Dehaene (2008), for example, do cite Allik and Tuulmets (1991)
and control for various factors including “occupied area,” but
they use the term to mean overall area (which nobody considers
critical to numerosity estimation) and not occupancy (which
Allik and Tuulmets themselves consider critical). Izard and
Dehaene dismiss occupancy as “a complex combination” of two
parameters (density and occupied area). Occupancy does not
strike us as complex, but even if it were, the fact that it reflects
well-established perceptual discriminability limitations that have
been demonstrated to affect numerosity estimation—rather than
hypothetical cognitive ones that continue to be debated—should
give the occupancy model an edge over any “sense of number”
model. In defense of their own “sense of magnitude” model,
which Leibovich et al. fail to cite too, Morgan et al. (2014) make a
similar point. dispersion. Atten. Percept. Psychophys. 78, 1460–1471. doi: 10.3758/s13414-01
6-1100-0 DeWind, N. K., Adams, G. K., Platt, M. L., and Brannon, E. M. (2015). Modeling
the approximate number system to quantify the contribution of visual stimulus
features. Cognition 142, 247–265. doi: 10.1016/j.cognition.2015.05.016 Frontiers in Psychology | www.frontiersin.org January 2017 | Volume 7 | Article 2032 REFERENCES Allik, J., and Tuulmets, T. (1991). Occupancy model of perceived numerosity. Percept. Psychophys. 49, 303–314. doi: 10.3758/BF03205986 Anobile, G., Turi, M., Cicchini, G. M., and Burr, D. C. (2015). Mechanisms for
perception of numerosity or texture-density are governed by crowding-like
effects. J. Vis. 15, 1–12. doi: 10.1167/15.5.4 Bertamini, M., Zito, M., Scott-Samuel, N. E., and Hulleman, J. (2016). Spatial clustering and its effect on perceived clustering, numerosity, and January 2017 | Volume 7 | Article 2032 Frontiers in Psychology | www.frontiersin.org 2 Item Discriminability and Numerosity Estimation Kramer and Bressan Whitney,
D.,
and
Levi,
D. M. (2011). Visual
crowding:
a
fundamental
limit
on
conscious
perception
and
object
recognition. Trends
Cogn. Sci. 15,
160–168. doi:
10.1016/j.tics.2011. 02.005 a critical review. Acta Psychol. 171, 17–35. doi: 10.1016/j.actpsy.2016. 09.003 Izard, V., and Dehaene, S. (2008). Calibrating the mental number line. Cognition
106, 1221–1247. doi: 10.1016/j.cognition.2007.06.004 Kramer, P., Di Bono, M. G., and Zorzi, M. (2011). Numerosity estimation in
visual stimuli in the absence of luminance-based cues. PLoS ONE 6:e17378. doi: 10.1371/journal.pone.0017378 Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Leibovich, T., Katzin, N., Harel, M., and Henik, A. (2016). From ‘sense of number’
to ‘sense of magnitude’ - the role of continuous magnitudes in numerical
cognition. Behav. Brain. Sci. doi: 10.1017/S0140525X16000960. [Epub ahead of
print]. Copyright © 2017 Kramer and Bressan. This is an open-access article distributed
under the terms of the Creative Commons Attribution License (CC BY). The
use, distribution or reproduction in other forums is permitted, provided the
original author(s) or licensor are credited and that the original publication in
this journal is cited, in accordance with accepted academic practice. No use,
distribution or reproduction is permitted which does not comply with these
terms. Morgan, M. J., Raphael, S., Tibber, M. S., and Dakin, S. C. (2014). A texture-
processing model of the ‘visual sense of number’. Proc. Biol. Sci. 281:20141137. doi: 10.1098/rspb.2014.1137 Valsecchi, M., Toscani, M., and Gegenfurtner, K. R. (2013). Perceived numerosity
is reduced in peripheral vision. J. Vis. 13, 1–16. doi: 10.1167/13.13.7 January 2017 | Volume 7 | Article 2032 Frontiers in Psychology | www.frontiersin.org 3
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Pertussis Post-Exposure Prophylaxis among Household Contacts: A Cost-Utility Analysis
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RESEARCH ARTICLE Nisha Thampi1,2*, Ipek Gurol-Urganci3, Natasha S. Crowcroft4,5, Beate Sander5,6,7,8 1 Division of Infectious Diseases, Children’s Hospital of Eastern Ontario, Ottawa, ON, Canada, 2 University
of Ottawa, Ottawa, ON, Canada, 3 Department of Health Services Research and Policy, London School of
Hygiene and Tropical Medicine, London, United Kingdom, 4 Laboratory Medicine and Pathobiology,
University of Toronto, Toronto, ON, Canada, 5 Public Health Ontario, Toronto, ON, Canada, 6 Institute of
Health Policy, Management & Evaluation, University of Toronto, Toronto, ON, Canada, 7 Institute for Clinical
Evaluative Sciences, Toronto, ON, Canada, 8 Toronto Health Economics and Technology Assessment
Collaborative, Toronto, ON, Canada * nthampi@cheo.on.ca OPEN ACCESS OPEN ACCESS
Citation: Thampi N, Gurol-Urganci I, Crowcroft NS,
Sander B (2015) Pertussis Post-Exposure
Prophylaxis among Household Contacts: A Cost-
Utility Analysis. PLoS ONE 10(3): e0119271. doi:10.1371/journal.pone.0119271 OPEN ACCESS
Citation: Thampi N, Gurol-Urganci I, Crowcroft NS,
Sander B (2015) Pertussis Post-Exposure
Prophylaxis among Household Contacts: A Cost-
Utility Analysis. PLoS ONE 10(3): e0119271. doi:10.1371/journal.pone.0119271 Methods A Markov model was constructed to examine 4 mutually exclusive strategies: erythromycin,
azithromycin, clarithromycin, or no intervention, stratified by age group of contacts (“infant”,
“child”, and “adult”). Transition probabilities, costs and quality-adjusted life years (QALYs)
were derived from the literature. Chronic neurologic sequelae were modeled over a lifetime,
with costs and QALYs discounted at 5%. Associated health outcomes and costs were com-
pared, and incremental cost-effectiveness ratios (ICER) were calculated in 2012 Canadian
dollars. Deterministic and probabilistic sensitivity analyses were performed to evaluate the
degree of uncertainty in the results. Academic Editor: T. Mark Doherty, Glaxo Smith
Kline, DENMARK Copyright: © 2015 Thampi 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. * nthampi@cheo.on.ca Background Recent pertussis outbreaks have prompted re-examination of post-exposure prophylaxis
(PEP) strategies, when immunization is not immediately protective. Chemoprophylaxis is
recommended to household contacts; however there are concerns of clinical failure and
significant adverse events, especially with erythromycin among infants who have the high-
est disease burden. Newer macrolides offer fewer side effects at higher drug costs. We
sought to determine the cost-effectiveness of PEP strategies from the health care payer
perspective. Pertussis Post-Exposure Prophylaxis among
Household Contacts: A Cost-Utility Analysis Nisha Thampi1,2*, Ipek Gurol-Urganci3, Natasha S. Crowcroft4,5, Beate Sander5,6,7,8 Findings Azithromycin offered the highest QALYs in all scenarios. While this was the dominant strate-
gy among infants, it produced an ICER of $16,963 per QALY among children and $2,415
per QALY among adults. Total QALYs with azithromycin were 19.7 for a 5-kg infant, 19.4 for
a 10-year-old child, and 18.8 for a 30-year-old adult. The costs of azithromycin PEP among
infants, children and adults were $1,976, $132 and $90, respectively. While results were
sensitive to changes in PEP effectiveness (11% to 87%), disease transmission (variable Funding: These authors have no support or funding
to report. Competing Interests: The authors have declared
that no competing interests exist. 1 / 17 PLOS ONE | DOI:10.1371/journal.pone.0119271
March 6, 2015 Pertussis Post-Exposure Prophylaxis: A Decision Model among age groups) and hospitalization costs ($379 to $59,644), the choice of strategy
remained unchanged. among age groups) and hospitalization costs ($379 to $59,644), the choice of strategy
remained unchanged. Interpretation Pertussis PEP is a cost-effective strategy compared with no intervention and plays an im-
portant role in contact management, potentially in outbreak situations. From a healthcare
payer perspective, azithromycin is the optimal strategy among all contact groups. Introduction In the pre-vaccine era, pertussis was a major childhood illness affecting children primarily
under 10 years of age, and a major cause of death among infants under 1 year [1]. Caused by
Bordetella pertussis, the disease was pandemic throughout the 20th century, with cyclical epi-
demic peaks every 2 to 5 years [1]. Widespread immunization of children has not lengthened
the epidemic cycle as much as expected [2], suggesting that adults serve as a reservoir for dis-
ease in young children [3]; pertussis is common and endemic among this group [4,5]. Prolonged pertussis outbreaks have recently been reported across North America [6–9],
prompting re-examination of control and prevention strategies [10]. In 2010, 101 cases of pertus-
sis were reported in Ontario (0.8 cases per 100,000 population) [6]. Following a 2011–2012 out-
break, 792 cases were reported across both years, or 5.9 per 100,000 population. Immunization
coverage in Ontario among 7- and 17-year olds in 2012 was 76% and 68%, respectively [6]. The resurgence of pertussis has been attributed to waning immunity in older children and
poor vaccine efficacy against other Bordetella species associated with pertussis-like illnesses
[7,11,12]. A randomized, controlled trial (RCT) of post-exposure prophylaxis (PEP) with
erythromycin compared with placebo found PEP to be effective in preventing secondary cases
in 67.5% of adult and pediatric household contacts [13]. Practice guidelines in Canada, the
United States and the United Kingdom recommend chemoprophylaxis with a macrolide for
household and close contacts of pertussis cases of all ages, irrespective of immunization status
[14–16]. While studies have examined erythromycin as a treatment and PEP agent, the newer
macrolides, azithromycin and clarithromycin, have also been shown to be active against B. per-
tussis in vitro [17], and have been recommended as better-tolerated, equally effective [18,19]
but more costly treatment options [15,16]. Economic evaluations can provide “value-for-money” information to support health care
decision-making [20]. A cost-utility analysis was performed to compare the additional cost per
QALY gained across the 4 strategies of erythromycin, azithromycin, clarithromycin and no
prophylaxis, with an aim to estimate the effects, costs and cost-effectiveness of alternative strat-
egies for the prevention of pertussis among household contacts. This analysis is especially time-
ly in informing clinical practice and health care resource allocation given the recent outbreaks
among under-immunized populations in countries that have well-established immunization
programs. Cost-utility analysis In accordance with established guidelines for economic evaluations [20,21], a cost-utility analy-
sis was performed to determine whether PEP with a macrolide was cost-effective compared to 2 / 17 PLOS ONE | DOI:10.1371/journal.pone.0119271
March 6, 2015 Pertussis Post-Exposure Prophylaxis: A Decision Model no PEP (i.e. treatment of cases as they arise) in household contacts exposed to pertussis. Specif-
ic PEP regimens considered were erythromycin and azithromycin in all age groups (infants,
children and adults) and clarithromycin in children and adults only. The primary outcome was
the incremental cost-effectiveness ratio (ICER), derived from estimated quality-adjusted life
years (QALYs) and direct healthcare cost over a lifetime time horizon. The analysis was con-
ducted from the healthcare payer perspective (i.e. Ontario’s Ministry of Health and Long-Term
Care), with a commonly-used cost-effectiveness threshold of $50,000 per QALY gained [22]. Healthcare costs for PEP and the treatment of cases are included. All costs were expressed in
2012 Canadian dollars (1 US dollar was 0.99958 Canadian dollars) [23–26]. Costs and health
outcomes were discounted at 5% per year as recommended for health economic evaluations in
Canada [20]. Data Probabilities of disease-specific outcomes and medication-related gastrointestinal effects were
drawn from RCTs and observational cohort studies. All data are shown in Table 1. Secondary
attack rates of pertussis are highest among young infants at 65% [28], with a 10-fold decrease
among children and adults [13]. Similarly, more than two-thirds of infants with pertussis are
managed in hospital [29], compared to only 7% of children [30] and 3.5% of adult contacts
[29]. Encephalitis is an infrequent but serious complication of pertussis, affecting 0.5% infants
[31], 0.08% children [30] and 0.05% adults who are hospitalized [30]. Early economic evalua-
tions and population studies reported residual effects in one-third of survivors of pertussis en-
cephalitis [1,32,33]. Death is least likely to occur among adult patients with pertussis, at 0.01%,
followed by children at 0.06%. The probability of death is highest among infants, at 0.6% [29]. Model and assumptions A Markov model was constructed to evaluate the lifetime health outcomes and costs of devel-
oping pertussis among household contacts following expectant management or PEP. Most
events occurred in the first year. The Markov model had a cycle length of 1 year and incorpo-
rated 2 health states: survival and death. The duration of acute events in the first year was taken
into account. Household contacts were stratified into 3 age groups: less than 1 year of age
(“infant”), 1 to 12 years old (“child”) and greater than 12 years old (“adult”). The model structure was the same for all contacts (Fig. 1A and B). Infant contacts had a
higher frequency of hospitalization for severe disease, including significant respiratory or neu-
rologic complications. Children and adult contacts with respiratory symptoms were managed
in the outpatient setting depending on severity, although all patients with neurologic presenta-
tions required hospitalization. Household contacts that developed mild symptoms went to
their general practitioner (GP) rather than an emergency department. Mild-to-moderate
cough resulted in an additional GP visit and antibiotics, and those with severe cough or pneu-
monia had 2 additional GP visits, antibiotics and a chest x-ray. Survivors of respiratory disease
requiring hospitalization did not experience long-term sequelae. In all groups, pertussis-related
deaths were assumed to occur during hospitalization. Age-specific mortality was obtained
from Statistics Canada [27]. Given that the total duration of illness is 6–8 weeks [1], all costs at-
tributable to pertussis were assumed to have been incurred in the first year unless patients ex-
perienced neurological sequelae, which were assumed to require lifetime healthcare services. Intervention An RCT of erythromycin prophylaxis against pertussis demonstrated 67.5% effectiveness (95%
CI 7.6% to 88.7%) in preventing culture-positive pertussis among household contacts [13]. 3 / 17 PLOS ONE | DOI:10.1371/journal.pone.0119271
March 6, 2015 Pertussis Post-Exposure Prophylaxis: A Decision Model Fig 1. Markov model for post-exposure prophylaxis strategy. The square represents a decision node, and circle represents a chance node. The triangle
represents the final outcome for that event pathway. Consequences associated with the chance node are mutually exclusive. PEP: post-exposure
prophylaxis; GI: gastrointestinal. Fig 1. Markov model for post-exposure prophylaxis strategy. The square represents a decision node, and circle represents a chance node. The triangle
represents the final outcome for that event pathway. Consequences associated with the chance node are mutually exclusive. PEP: post-exposure
prophylaxis; GI: gastrointestinal. Fig 1. Markov model for post-exposure prophylaxis strategy. The square represents a decision node, and circle represents a chance node. The triangle
represents the final outcome for that event pathway. Consequences associated with the chance node are mutually exclusive. PEP: post-exposure
prophylaxis; GI: gastrointestinal. doi:10.1371/journal.pone.0119271.g001 doi:10.1371/journal.pone.0119271.g001 With no subsequent studies re-examining this intervention, this point estimate of effectiveness
was extrapolated to azithromycin and clarithromycin based on their comparatively similar effi-
cacy with erythromycin in treatment studies [18,19] which is in keeping with current pertussis
PEP guidelines [15,16]. PEP was assumed to have been given within 21 days of onset of cough
in the index case and before a secondary case had occurred [28]. Clarithromycin has not been
studied for those under 1 month of age and so was not included as an option for the infant
group [19]. g
p
All contacts who receive an antibiotic have a likelihood of developing a gastrointestinal ad-
verse event. These probabilities were derived from case series and clinical trials involving eryth-
romycin, clarithromycin and/or azithromycin. Neonates may be more than 10 times at risk for
infantile hypertrophic pyloric stenosis (IHPS) in the month following orally administered
erythromycin, compared to unexposed neonates, with a peak incidence of 1.28% in under
3 month olds [34,35]. As the risk of IHPS with erythromycin has had an impact on recommen-
dations for infant PEP, this was explicitly examined in the model. There were 2 case reports of
azithromycin-associated IHPS [36]. PLOS ONE | DOI:10.1371/journal.pone.0119271
March 6, 2015 Intervention With over 4,000 cases noted over a 10-year period in the
US [29] and azithromycin having been adopted as the preferred agent for young infants [16], a PLOS ONE | DOI:10.1371/journal.pone.0119271
March 6, 2015 4 / 17 PLOS ONE | DOI:10.1371/journal.pone.0119271
March 6, 2015 Pertussis Post-Exposure Prophylaxis: A Decision Model Table 2. Utility values and duration of relevant health states. Health State
Utility value for base case (SD)
Days in first year
Reference (utility; duration)
INFANT
Mild illness, outpatient
0.67 (0.30)
76
[40,41]
Hospitalization
0.58 (0.37)
8
[38,39]
Respiratory complications
0.58 (0.37)
8
[38]
Encephalitis without sequelae
0.51 (0.38)
14
[38]
Chronic neurologic sequelae1
0.77 (0.25)2
365
[38]
IHPS
0.51 (0.38)3
6
Assumption; [34,43]
CHILD
Mild cough, outpatient
0.85 (0.26)4
76
[38,41]
Moderate-severe cough, outpatient
0.81 (0.30)
76
[38,41]
Hospitalization with recovery
0.67 (0.33)
3
[38,42]
Encephalitis without sequelae
0.51 (0.38)
14
[38]; Assumption
Chronic neurologic sequelae
0.77 (0.25)
365
[38]; Assumption
GI adverse event
0.70 (0.15)5
7
[84]; Assumption
ADULT
Mild cough
0.85 (0.26)
87
[38,40]
Moderate cough
0.81 (0.30)
87
[38,40]
Hospitalization for respiratory complications
0.62 (0.40)
3
[38,42]
Encephalitis without sequelae
0.51 (0.38)
14
[38]; Assumption
Chronic neurologic sequelae
0.77 (0.25)
365
[38]; Assumption
GI adverse event
0.70 (0.15)5
7
[84]; Assumption
1 Utility value and duration in health state assumed to be the same for all age groups
2 Interpreted as willing to give up 84 days of life to prevent 1 year of neurologic sequelae
3 Assumed to be same as for encephalitis
4 Interpreted as willing to give up 8 days of life to prevent 8 weeks of mild cough that does not require hospitalization
5 Standard error Table 2. Utility values and duration of relevant health states. Utility value for base case (SD)
Days in first year
Reference (utility; duration) Utility value for base case (SD)
Days in first year 1 Utility value and duration in health state assumed to be the same for all age groups 1 Utility value and duration in health state assumed to be the same for all age groups
2 Interpreted as willing to give up 84 days of life to prevent 1 year of neurologic sequelae
3 Assumed to be same as for encephalitis
4 Interpreted as willing to give up 8 days of life to prevent 8 weeks of mild cough that does not require hospitalization
5 Standard error
GI: gastrointestinal
IHPS: infantile hypertrophic pyloric stenosis
PEP: post-exposure prophylaxis
doi:10.1371/journal.pone.0119271.t002 IHPS: infantile hypertrophic pyloric stenosis Health outcomes. QALYs combine quality of life and duration of life, or life years, into a
single measure. Health states were valued with preferences (Table 2) drawn from individuals
with pertussis [38]. Pertussis Post-Exposure Prophylaxis: A Decision Model Table 1. Event probabilities for decision model. Contact group
Parameter
Base case value
Range examined
References
ALL
Prophylaxis effectiveness
0.675
0.076–0.887
[13]
INFANT
Intervention
IHPS following erythromycin
0.0128
0.0026–0.0275
[35]
IHPS following azithromycin
0.0005
0.0001–0.0010
Assumption1
Pertussis
Develops pertussis post-exposure
0.65
0.25–0.81
[28,70]
Hospitalization for severe disease
0.69
0.59–0.82
[29,30]
Develops complication in hospital
0.14
0.10–0.19
[42,66]
Death
0.006
0.001–0.009
[29,66,82]
Develops encephalitis
0.005
0.002–0.009
[29,31,42]
Develops chronic neurologic sequelae
0.33
0.25–0.50
[32,33]
CHILD
Intervention
GI adverse event with erythromycin
0.34
0.27–0.44
[13,19,28]
GI adverse event with azithromycin
0.19
0.12–0.20
[18]
GI adverse event with clarithromycin
0.32
0.20–0.402
[19]
Pertussis
Acquires pertussis post-exposure
0.061
0.048–0.440
[13,28]
Develops moderate to severe respiratory pertussis
0.25
0.10–0.32
[42,83]
Hospitalization for severe disease
0.07
0.04–0.08
[29,30,58]
Death
0.0006
0.0004–0.0010
[29,30,70]
Develops encephalitis/ encephalopathy
0.0008
0.0005–0.0040
[29,30,32]
Develops chronic neurologic sequelae
0.33
0.25–0.50
[1,32,33]
ADULT
Intervention
GI adverse event with erythromycin
0.34
0.27–0.44
[13,19,28]
GI adverse event with azithromycin
0.19
0.12–0.20
[18]
GI adverse event with clarithromycin
0.32
0.2–0.402
[19]
Pertussis
Acquires pertussis post-exposure
0.061
0.048–0.200
[13,68]
Develops moderate to severe respiratory pertussis
0.035
0.021–0.040
[29,30]
Hospitalization for severe disease
0.027
0.008–0.060
[28,30,73]
Death
0.0001
0–0.0010
[29,30,75]
Develops encephalitis/ encephalopathy
0.0005
0.0002–0.0040
[29,30,32]
Develops chronic neurologic sequelae
0.33
0.25–0.50
[32,33]
1 A
ti
b
d
2
t
f
ith
i
i t d IHPS [36] Table 1. Event probabilities for decision model. 1 Assumption based on 2 case reports of azithromycin-associated IHPS [36]
2 Assumption of range
GI: gastrointestinal
IHPS: infantile hypertrophic pyloric stenosis
PEP: post-exposure prophylaxis
doi:10.1371/journal.pone.0119271.t001 point estimate of 0.05%, or 2 cases among 4,000 infants treated with azithromycin, was
assumed. If no PEP was received, or no gastrointestinal event occurred on chemoprophylaxis, then
erythromycin was used for treatment of pertussis as the least expensive option [37]. If IHPS oc-
curred during erythromycin PEP, infants who subsequently developed pertussis received azi-
thromycin for treatment [16]. However, if IHPS followed azithromycin PEP, then infants with
subsequent pertussis were treated with erythromycin. As azithromycin and clarithromycin
have fewer side effects than erythromycin, child and adult contacts that developed pertussis
after experiencing PEP-related side effects were treated with either of these agents. PLOS ONE | DOI:10.1371/journal.pone.0119271
March 6, 2015 5 / 17 PLOS ONE | DOI:10.1371/journal.pone.0119271
March 6, 2015 doi:10.1371/journal.pone.0119271.t002 In this study, time trade-off and contingent valuation methods were used
to determine utilities of short-term health states among adult patients and parents of adoles-
cent patients diagnosed with pertussis [38]. Infant utilities were derived from the adult respon-
dents, who were presented with the scenario of a 1-month old who developed pertussis, and
asked to value the prevention of short-term health states, that is respiratory or neurologic com-
plications lasting 8 weeks’ duration, as compared to long-term health states, which reflect neu-
rologic sequelae [38]. The time spent in each health state was derived from studies examining the natural course
of pertussis in under-immunized populations [39–43], and was multiplied by the correspond-
ing preference value to calculate the QALYs. The literature search did not identify a study re-
porting a preference value for IHPS, and so it was assumed to be the same as for encephalitis
without sequelae, given the need for hospitalization and short-term disability. 6 / 17 PLOS ONE | DOI:10.1371/journal.pone.0119271
March 6, 2015 Pertussis Post-Exposure Prophylaxis: A Decision Model Costs. The direct medical costs were expressed in 2012 Canadian dollars, and were
associated with exposure to and treatment of pertussis. These costs include contact tracing by a
public health nurse to review the need for PEP within households, medications, GP visits, diag-
nostic testing, hospitalization and long-term medical costs associated with neurologic sequelae. While there is no universal drug coverage in Ontario, medication costs were included in this
analysis as Canadian public health policy makers have recommended that prophylaxis, where
appropriate, should be supplied by public health [15]. Cost estimates are summarized in
Table 3. Costs of health care visits and procedures were obtained from the Ontario Health In-
surance Plan Schedule of Benefits [44], and prices for generic formulations from the Ontario
Drug Benefit Formulary [37,45]. The doses and dispensing costs for each macrolide are outlined in Table 4. Given the infant
contact is at highest risk for complications with pertussis in the first 4 months of life [9,46], the
model assumed a 6-week old infant whose weight was at the 50th percentile-for-age, or 5 kg, as
per the CDC Growth Charts for the United States [47]. Thus, the cost of erythromycin was
$2 and azithromycin $8.90. Similarly, a child was assumed to have completed the primary im-
munization series and prefer taking liquid preparations of antibiotics, so that a 10-year old
child whose weight was at the 50th percentile-for-age at 34 kg had erythromycin at a cost of
$13.58, azithromycin at $42.87 and clarithromycin at $8.16. An adult was assumed to be
30 years old and weigh 70 kg, so that erythromycin cost $10.24, azithromycin $7.84 and clari-
thromycin $11.54, respectively. The duration of prophylaxis and treatment of pertussis was as
per Health Canada guidelines [15]. Medications, GP visits and diagnostic testing are set by the Ministry for all practitioners and
pharmacies. Older children and adults with PEP-related gastrointestinal symptoms were as-
sumed to visit their GP to discuss discontinuation of therapy. Acquiring the disease incurred
further costs. Hospitalization costs for pertussis and encephalitis were drawn from the Ontario Case Cost-
ing Initiative (OCCI), which collects case costing data for acute inpatient, day surgery and am-
bulatory care cases, complex continuing care and community care cases [39]. The mean cost
per hospitalized case includes costs associated with ward and intensive care, and diagnostic
and therapeutic resources. During the fiscal year 2010–2011, there were 10 pertussis-related
hospitalizations and fewer than 5 associated encephalitis cases. Fifty-one cases of encephalitis
associated with other infectious etiologies were reported to the OCCI and those costs were re-
viewed. Physician costs are not part of the OCCI, but an estimate of 5% of hospitalization costs
was used based on previous studies of influenza analyzing health administrative data (expert
opinion, based on [48,49]). There are no data on long-term outcomes of patients with pertussis encephalitis. Direct
medical costs associated with the long-term neurologic sequelae of pertussis encephalitis were
extrapolated from a review of direct medical costs for children with complex medical condi-
tions in Ontario. Excluding initial hospitalization costs, the average costs of care for a child
with neurological impairment and technology assistance (including cerebrospinal fluid ventric-
ular shunt, gastrostomy, or tracheostomy) was $34,574 (interquartile range: $10,178-$97,063)
over a 2-year period [50]. This population was comparable with respect to burden of disease
and resource utilization patterns. Pertussis Post-Exposure Prophylaxis: A Decision Model Table 3. Estimated direct medical costs per contact. Costs in Canadian dollars, 2012 valuation. Parameter
Base case value
Standard Error
Reference
GENERAL
Contact tracing (public health)
37.74
-
Assumption1
Visit to GP
33.70
-
[44]
Chest X-ray (professional and
technical cost)
32.65
-
[44]
No pertussis
0
-
Assumption
MEDICATIONS
Erythromycin2
Infant
2.00
-
[45]
Child
13.58
-
[37]
Adult
10.24
-
[37]
Azithromycin3
Infant
8.90
-
[37]
Child
42.87
-
[37]
Adult
7.84
-
[37]
Clarithromycin4
Child
8.16
-
[37]
Adult
11.54
-
[37]
PERTUSSIS
GI symptoms in child or adult contact
33.705
[44]
Outpatient with mild illness
GP + treatment
Assumption
Outpatient with moderate-severe
illness
2 GP visits + 1 chest x-ray + treatment
Assumption
Hospitalization6
12,160
5,689
[39]
IHPS
10,340
550
[43]
COMPLICATIONS
Encephalitis
27,643
8370
[39]
Chronic neurologic sequelae7
103,652
148,867
[50] Table 3. Estimated direct medical costs per contact. Costs in Canadian dollars, 2012 valuation. 1 Public health nurse with 4 years of seniority paid an hourly rate of CAD 37.74 (personal communication, Public Health Ontario, July 2012)
2 4 clarithromycin at 15 mg/kg/day divided twice daily, maximum 1000g/day, dispensed for a child at $0.5712 per 250mg/5mL; and an adult at $0.4122 per
250mg tablet p
p
6 Hospitalization includes the infant with an uncomplicated admission; infant with respiratory complications; child and adult hospitalizations; death in
all groups 6 Hospitalization includes the infant with an uncomplicated admission; infant with respiratory complications; child a
all groups 7 Mean cost and standard deviation over 2-year period; includes initial hospitalization
GI: gastrointestinal GP: general practitioner IHPS: infantile hypertrophic pyloric stenosis PEP: post-exposure prophylaxis effectiveness. The ranges examined in the deterministic sensitivity analysis were derived either
from the confidence intervals of the point estimate used for the base case; or, if not available,
minimum and maximum values in the model were derived from point estimates in other ob-
servational studies (Table 1). Probabilistic sensitivity analysis was performed to address uncer-
tainty involving parameters with an underlying probability distribution, varying all selected effectiveness. The ranges examined in the deterministic sensitivity analysis were derived either
from the confidence intervals of the point estimate used for the base case; or, if not available,
minimum and maximum values in the model were derived from point estimates in other ob-
servational studies (Table 1). Analysis Base case analysis was conducted for a previously healthy 6-week old infant at 5 kg; 10-year old
child at 34 kg; and 30-year old adult [51,52]. Deterministic sensitivity analysis was performed
to explore uncertainty in the point estimates, and a threshold analysis was performed for PEP 7 / 17 PLOS ONE | DOI:10.1371/journal.pone.0119271
March 6, 2015 doi:10.1371/journal.pone.0119271.t003 Pertussis Post-Exposure Prophylaxis: A Decision Model Table 4. Macrolide strategies for pertussis PEP among household contacts. Strategy
Dose (oral)
Cost
Duration
(days)
Erythromycin
40 mg/kg/day divided three times daily (maximum 2,000 mg/day)
$0.0713 per 50 mg/mL and $0.1828 per 250
mg tablet
7
Azithromycin
10 mg/kg (maximum 500 mg) on day 1 followed by 5 mg/kg (maximum
250 mg) once daily
$5.9347 per 100mg/5mL and $1.3070 per
250mg tablet
5
Clarithromycin1
15 mg/kg/day divided twice daily (maximum 1,000 mg/day)
$0.5712 per 250mg/5mL and $0.4122 per
250mg tablet
7
1 Not included as strategy among infants Table 4. Macrolide strategies for pertussis PEP among household contacts. variables simultaneously [53]. In this assessment, beta distributions for probabilities and utili-
ties and gamma distributions for costs were derived, based on mean and standard error [54]. The model was run 10,000 times so that each event of interest may occur at least once in the
simulation. Results were summarized as cost-effectiveness acceptability curves. Probabilistic sensitivity analysis was performed to address uncer-
tainty involving parameters with an underlying probability distribution, varying all selected PLOS ONE | DOI:10.1371/journal.pone.0119271
March 6, 2015 8 / 17 Results From the model, PEP with azithromycin prevents 439 secondary cases per 1,000 infant con-
tacts, and 41 secondary cases per 1,000 child and adult contacts. The results for the base case
analysis are shown in Table 5. A previously-healthy 5-kg infant contact who did not receive
PEP experienced 19.22 QALYs, whereas a macrolide resulted in at least 0.45 more expected
QALYs, or 165 quality-adjusted life days. Between the macrolides, azithromycin dominated
erythromycin with lower expected costs ($1,976 versus $2,096) and outcomes (19.6661 versus
19.6660 QALYs). These findings were sensitive to costs of hospitalization, costs of encephalitis
and probability of hospitalization, death, PEP effectiveness and acquiring disease following ex-
posure, and not sensitive to the probability of complications, death, annual costs of neurologic
sequelae, probability of an IHPS event with either macrolide, and changes in utility on PEP or
with any pertussis health state. However, in all scenarios in univariate and probabilistic sensi-
tivity analyses, azithromycin dominated erythromycin and no PEP. Table 5. Costs, effects and cost-effectiveness of prophylaxis with erythromycin, azithromycin or clarithromycin compared with no intervention
for household contacts of cases of pertussis, stratified by age group, discounted at 5%. Age
group
Strategy
Average expected cost
($)
Average expected
QALY
Difference in cost
($)
Difference in
QALY
ICER ($ per
QALY)
Infant
azithromycin
1,975.87
19.66612
-
-
erythromycin
2,095.70
19.66602
-
-
dominated
none
5,815.10
19.21593
-
-
dominated
Child
none
35.13
19.37751
-
-
-
clarithromycin
101.98
19.40780
66.86
0.0303
2,207
erythromycin
107.85
19.40753
-
-
dominated
azithromycin
132.04
19.40957
30.06
0.0018
16,963
Adult
none
14.22
18.72856
-
-
-
azithromycin
90.25
18.76004
76.02
0.0315
2,415
erythromycin
97.74
18.75800
-
-
dominated
clarithromycin
98.37
18.75827
-
-
dominated
doi:10 1371/journal pone 0119271 t005 Table 5. Costs, effects and cost-effectiveness of prophylaxis with erythromycin, azithromycin or clarithromycin compared with no intervention
for household contacts of cases of pertussis, stratified by age group, discounted at 5%. 5. Costs, effects and cost-effectiveness of prophylaxis with erythromycin, azithromycin or clarithromycin c
usehold contacts of cases of pertussis, stratified by age group, discounted at 5%. ess of prophylaxis with erythromycin, azithromycin or clarithromycin compared with no intervention
sis, stratified by age group, discounted at 5%. Results PLOS ONE | DOI:10.1371/journal.pone.0119271
March 6, 2015 9 / 17 Pertussis Post-Exposure Prophylaxis: A Decision Model The lifetime expected health outcomes for a previously healthy 10-year-old contact were
19.38 QALYs without intervention and increased by 12 days (0.032 QALYs) with azithromycin
PEP, with a $97 cost increase for an ICER of $16,963 per QALY when compared with clarithro-
mycin. While clarithromycin was less expensive than azithromycin, there were fewer QALYs
gained, thus making it the non-preferred option in the base case scenario and sensitivity analy-
ses. In the probabilistic sensitivity analysis, the median ICER of azithromycin compared to
clarithromycin was $16,709 per QALY (mean $16,966 per QALY, 95% CI: $16,900-$16,972 per
QALY), and was sensitive to the probability of a GI adverse event with clarithromycin and azi-
thromycin, the probability of acquiring disease, and PEP effectiveness, but not sensitive to the
complications and costs of pertussis, including probability and cost of hospitalization, the
probability and costs of encephalitis and its sequelae, probability of death, and changes in utili-
ty on PEP or with any pertussis health state (Fig. 2A). No PEP had a 95% probability of being
cost-effective when the willingness-to-pay threshold was below $2,000 per QALY, whereas azi-
thromycin had a 95% probability of being cost-effective at the threshold of $50,000 per QALY
(Fig. 3A). Among adult contacts, the quality-adjusted life expectancy was 18.73 years without PEP. Among adult contacts, the quality-adjusted life expectancy was 18.73 years without PEP. Azithromycin was the dominant strategy compared to the other macrolides, with more QALYs
and lower costs. Compared to no intervention, which was the least costly option, PEP with azi-
thromycin resulted in an additional 0.031 QALYs, or 11 quality-adjusted life days, and $76 in
incremental costs, for an ICER of $2,415 per QALY. The median ICER was $2,574 per QALY
(mean $2,426 per QALY, 95% CI $2,425-$2,426 per QALY) and was sensitive to PEP effective-
ness, probability of acquiring disease and probability of encephalitis, and not sensitive to the
probability of an adverse GI event with azithromycin, sequelae and death, costs of hospitaliza-
tion and encephalitis, and changes in utility on PEP or with any pertussis health state; however,
azithromycin remained the preferred strategy (Fig. 2B). Among adult contacts, azithromycin
was the preferred strategy, with a 95% probability of being cost-effective at $4,000 per QALY
(Fig. 3B). Results A threshold analysis for the effectiveness of PEP in preventing a secondary case of pertussis
is shown in Table 6. Among infants, when the PEP effect was 0.2% or greater, azithromycin of-
fered the highest net benefit of all strategies. Among children and adults, this threshold proba-
bility was 10% and 9%, respectively. Below these values, none of the macrolides offered a
higher net benefit than no post-exposure prophylaxis. PLOS ONE | DOI:10.1371/journal.pone.0119271
March 6, 2015 Fig 2. Tornado diagram of the univariate sensitivity analysis for the azithromycin PEP strategy for child (A) and adult (B) contacts. Azithromycin
remained the dominant strategy among infants. The bars represent the variation in cost-effectiveness ratios from the base case scenario in response to
sequential changes in model parameters, with the vertical axis reflecting the base case ICER. The maximal and minimal values were tested according to
ranges outlined in Table 1. (A) Axis at $16,963 per QALY. (C) Axis at $2,415 per QALY. Fig 2. Tornado diagram of the univariate sensitivity analysis for the azithromycin PEP strategy for child (A) and adult (B) contacts. Azithromycin
remained the dominant strategy among infants. The bars represent the variation in cost-effectiveness ratios from the base case scenario in response to
sequential changes in model parameters, with the vertical axis reflecting the base case ICER. The maximal and minimal values were tested according to
ranges outlined in Table 1. (A) Axis at $16,963 per QALY. (C) Axis at $2,415 per QALY. Fig 2. Tornado diagram of the univariate sensitivity analysis for the azithromycin PEP strategy for child (A) and adult (B) contacts. Azithromycin
remained the dominant strategy among infants. The bars represent the variation in cost-effectiveness ratios from the base case scenario in response to
sequential changes in model parameters, with the vertical axis reflecting the base case ICER. The maximal and minimal values were tested according to
ranges outlined in Table 1. (A) Axis at $16,963 per QALY. (C) Axis at $2,415 per QALY. doi:10.1371/journal.pone.0119271.g002 doi:10.1371/journal.pone.0119271.g002 A recent systematic review found only 2 RCTs related to pertussis PEP [13,61], and insuffi-
cient evidence to support erythromycin for all household contacts [62]; although, they may not
have been sufficiently powered to detect a significant difference. An argument against PEP is
that widespread immunization has been associated with a milder clinical course among sec-
ondary cases [41,63–65]. Most chemoprophylaxis studies occurred in the context of an under-
immunized adult population [3,5,66,67] and large cohort studies have demonstrated some ben-
efit in offering PEP to household contacts [27,28,68,69]. However, a recent study among a
highly-immunized population of health care professionals found no difference in rates of
symptomatic pertussis between those who received PEP compared to daily symptom monitor-
ing [64]. Discussion A cost-utility analysis was undertaken to examine 4 strategies against pertussis transmission to
household contacts. The decision model synthesized evidence from a broad range of published
literature and used QALYs as a preference-based outcome measure. Azithromycin offered the
most QALYs in all age groups. Among infants, PEP with azithromycin had the lowest expected
cost and most QALYs. This strategy was cost-effective among children and adult contacts at
$16,963 and $2,415 per QALY gained, respectively, compared to no PEP, which was well under
the cost-effectiveness threshold of $50,000 per QALY gained. The findings were robust in sen-
sitivity analyses. In particular, when the probability of disease acquisition and disease severity
were minimized (reflecting an immune population), disease-related costs were still significant,
especially among infants, and azithromycin remained the preferred strategy. Pertussis transmission has been mitigated historically through immunization and chemo-
prophylaxis. While numerous studies have analyzed the impact of varying immunization strat-
egies [32,38,40,55–60], this is the first study to comparatively evaluate the benefits, risks and
costs associated with antibiotic PEP options among household contacts. PLOS ONE | DOI:10.1371/journal.pone.0119271
March 6, 2015 10 / 17 PLOS ONE | DOI:10.1371/journal.pone.0119271
March 6, 2015 Pertussis Post-Exposure Prophylaxis: A Decision Model Pertussis Post-Exposure Prophylaxis: A Decision Model doi:10.1371/journal.pone.0119271.g002 Pertussis Post-Exposure Prophylaxis: A Decision Model Fig 3. Cost-effectiveness acceptability curves for child (A) and adult (B) contacts. These curves reflect the proportion of times each intervention is likely
to be cost-effective for a given cost-effectiveness threshold, up to $50,000 per additional QALY. Clarithromycin post-exposure prophylaxis was never a
preferred strategy for adult contacts, and so does not feature for clarity. Fig 3. Cost-effectiveness acceptability curves for child (A) and adult (B) contacts. These curves reflect the proportion of times each intervention is likely
to be cost-effective for a given cost-effectiveness threshold, up to $50,000 per additional QALY. Clarithromycin post-exposure prophylaxis was never a
preferred strategy for adult contacts, and so does not feature for clarity. doi:10.1371/journal.pone.0119271.g003 associated with contact tracing, evaluation and counseling for PEP. However, all participants
had been immunized against pertussis within the previous 2 years, suggesting higher rates of
immunity than what may be found in the general community [67]. Thus, the marginal benefits
of PEP in this study may have been smaller compared to what may be found in the general
population, particularly among infants and young children who are at highest risk for severe
disease [29,42,66]. The analysis has several limitations. The model does not account for changes in disease inci-
dence as it is not an infectious disease transmission model; however the probability of acquir-
ing pertussis and the impact of prophylaxis were varied in sensitivity analyses. The healthcare
payer perspective includes only direct medical costs related to health care resource utilization,
such as medication, laboratory, procedures and personnel, hospitalizations and physician visits
[55]. Numerous studies have highlighted the significant indirect medical and non-medical
costs of pertussis [32,40,59,60,70–75]. Given that the antibiotic strategies had similar effective-
ness, the differences in non-medical costs between each regimen may be minimal. However,
the indirect economic benefit of PEP in avoiding pertussis and its complications may have
been underestimated, as the model did not account for QALY loss of caregivers looking after
contacts with pertussis or with neurologic sequelae following pertussis encephalitis. Table 6. Threshold analysis of post-exposure prophylaxis effectiveness. PEP effect
Contact Group
0
0.001
0.002
0.005
0.01
0.08
0.09
0.1
0.9
1
Infant
no
no
A
A
A
A
A
A
A
A
Child
no
no
no
no
no
no
no
A
A
A
Adult
no
no
no
no
no
no
A
A
A
A
“no” indicates no PEP as the preferred option. The authors questioned whether antibiotic PEP could be eliminated in such popula-
tions, given the potential for reduced prescriptions, rates of adverse events, and labor costs PLOS ONE | DOI:10.1371/journal.pone.0119271
March 6, 2015 11 / 17 “A” indicates azithromycin. When PEP has zero effect, the net benefit of no prophylaxis exceeds that of azithromycin and other macrolides. At or above
10% effectiveness, PEP with azithromycin offers a higher net benefit than no intervention. Table 6. Threshold analysis of post-exposure prophylaxis effectiveness. “no” indicates no PEP as the preferred option. p
p
“A” indicates azithromycin. When PEP has zero effect, the net benefit of no prophylaxis exceeds that of azithromycin and other macrolides. At or above
10% effectiveness, PEP with azithromycin offers a higher net benefit than no intervention. PLOS ONE | DOI:10.1371/journal.pone.0119271
March 6, 2015 12 / 17 Pertussis Post-Exposure Prophylaxis: A Decision Model The costs incurred during lifeyears gained from the intervention were not included in this
model, in keeping with Canadian guidelines on economic evaluations [20]. Whether to include
future unrelated healthcare costs, that is costs solely due to changes in survival, has been con-
troversial. Theoretical arguments for [76] and against inclusion [77] of unrelated costs have
been advanced and debated [78].To date no consensus has been reached, and to the best of our
knowledge, no current guideline on economic evaluation calls for inclusion of unrelated cost as
a requirement. The Canadian guideline recommends including unrelated costs only in sensitiv-
ity analysis, if at all [20]. While substantial survival benefits may have a major impact on
spending, in the case of pertussis, where the highest risk of mortality is among infants and at
0.6%, offering chemoprophylaxis arguably does not have substantial effects on survival. Since
these costs were excluded, the cost-effectiveness of the intervention was likely overestimated. Existing clinical and cost data were applied to each age group used in this model, and so nu-
merous assumptions were made regarding the calculation and application of costs and proba-
bilities where published data were not available. It is arguable that the populations from which
secondary attack rates are estimated may differ from the best available data, which is from Que-
bec during the 1990s. Despite subsequent changes in the immunization schedules, including a
booster dose during adolescence and among adults in contact with children, and in vaccine
product, from an adsorbed whole cell vaccine to acellular vaccine, there is no prospective sur-
veillance on immunization rates or household secondary attack rates. PLOS ONE | DOI:10.1371/journal.pone.0119271
March 6, 2015 References 1. Cherry JD. The epidemiology of pertussis and pertussis immunization in the United Kingdom and the
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[11]. In this economic analysis, to explore the uncertainty in secondary attack rates, the proba-
bility of acquiring disease was varied broadly, and did not result in a change in the preferred
strategy for contacts in any of the age groups. The assumption that respiratory and neurologic diseases are mutually exclusive does not
necessarily reflect clinical experience; however the associated costs and QALYs of both compli-
cations are likely to overlap, and thus be potentially overestimated in this analysis. The utility
scores for infants were derived from adolescent and adult respondents, which may have im-
pacted the validity of the QALYs measured. Direct elicitation of preferences can be especially
challenging in children due to evolving developmental maturity and their cognitive abilities at
various ages to value health; parents may also not reliably report on subjective outcomes of
clinical conditions, all of which potentially impact the validity and generalizability of the
QALYs measured.[80,81] Furthermore, only respiratory and neurologic complications were in-
cluded, while other serious health states in the disease spectrum were not assessed, including
apneic spells with spontaneous and complete recovery, generalized illness with outpatient care,
and long-term neurologic sequelae following encephalitis. All cases reported to the OCCI were less than 18 years of age, and the costs of encephalitis
and long-term neurologic sequelae have been extrapolated from other disease processes. The
cost of death was the same as for hospitalization in this model; however end-of-life care is asso-
ciated with higher costs [46,49]. Uncertainty in the parameter estimates was tested through
univariate and probabilistic sensitivity analyses, and did not shift the preferred strategy from
the base case scenario. Alongside policy recommendations for immunization of infants, children, adolescents and
adults, there remains an important role for chemoprophylaxis in the prevention of pertussis,
especially in outbreaks, when immunization is not immediately protective, and among infants,
who are at highest risk for complicated disease. Our analysis suggests azithromycin PEP to be
cost-effective when compared with other macrolides and no intervention, protecting house-
hold contacts from acquiring infection at acceptable costs. 13 / 17 PLOS ONE | DOI:10.1371/journal.pone.0119271
March 6, 2015 Pertussis Post-Exposure Prophylaxis: A Decision Model Disclaimer The opinions, results and conclusions reported in this paper are those of the authors. No en-
dorsement by Public Health Ontario is intended or should be inferred. Acknowledgments The authors wish to thank Dr. Chaim Bell for his critical review of the manuscript Author Contributions Conceived and designed the experiments: NT IGU NC BS. Performed the experiments: NT BS. Analyzed the data: NT BS. Wrote the paper: NT IGU NC BS. PLOS ONE | DOI:10.1371/journal.pone.0119271
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8851461 17 / 17
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English
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Comprehensive analysis of the ionospheric response to the largest geomagnetic storms from solar cycle 24 over Europe
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Frontiers in astronomy and space sciences
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cc-by
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TYPE Original Research
PUBLISHED 24 April 2023
DOI 10.3389/fspas.2023.1092850 TYPE Original Research
PUBLISHED 24 April 2023
DOI 10.3389/fspas.2023.1092850 TYPE Original Research
PUBLISHED 24 April 2023
DOI 10.3389/fspas.2023.1092850 geomagnetic storms, space weather, ionosphere, ionospheric storm, midlatitude
ionospheric trough, GNSS TEC, ionosphere-plasmasphere coupling, Swarm observations geomagnetic storms, space weather, ionosphere, ionospheric storm, midlatitude
ionospheric trough, GNSS TEC, ionosphere-plasmasphere coupling, Swarm observations Comprehensive analysis of the
ionospheric response to the
largest geomagnetic storms from
solar cycle 24 over Europe OPEN ACCESS
EDITED BY
Joseph Huba,
Syntek Technologies, United States
REVIEWED BY
Shun-Rong Zhang,
Haystack Observatory, United States
Bapan Paul,
Vivekananda B.Ed and D.El.Ed
College, India
*CORRESPONDENCE
K. A. Berényi,
berenyi.kitti@epss.hu
RECEIVED 08 November 2022
ACCEPTED 11 April 2023
PUBLISHED 24 April 2023
CITATION
Berényi KA, Heilig B, Urbář J, Kouba D,
Kis Á and Barta V (2023), Comprehensive
analysis of the ionospheric response to
the largest geomagnetic storms from
solar cycle 24 over Europe. Front. Astron. Space Sci. 10:1092850. doi: 10.3389/fspas.2023.1092850 OPEN ACCESS doi: 10.3389/fspas.2023.1092850 COPYRIGHT
© 2023 Berényi, Heilig, Urbář, Kouba, Kis
and Barta. This is an open-access article
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which does not comply with these terms. Frontiers in Astronomy and Space Sciences OPEN ACCESS EDITED BY
Joseph Huba,
Syntek Technologies, United States REVIEWED BY
Shun-Rong Zhang,
Haystack Observatory, United States
Bapan Paul,
Vivekananda B.Ed and D.El.Ed
College, India
*CORRESPONDENCE
K. A. Berényi,
berenyi.kitti@epss.hu
RECEIVED 08 November 2022
ACCEPTED 11 April 2023
PUBLISHED 24 April 2023 REVIEWED BY
Shun-Rong Zhang,
Haystack Observatory, United States
Bapan Paul,
Vivekananda B.Ed and D.El.Ed
College, India K. A. Berényi1,2,3*, B. Heilig3,4, J. Urbář5, D. Kouba5, Á. Kis3 and
V. Barta3 1ELKH-ELTE Space Research Group, Budapest, Hungary, 2Doctoral School of Environmental Sciences,
ELTE Eötvös Loránd University, Budapest, Hungary, 3Institute of Earth Physics and Space Science, Sopron,
Hungary, 4Space Research Group, Eötvös Loránd University, Budapest, Hungary, 5Institute of Atmospheric
Physics CAS, Prague, Czechia CITATION
Berényi KA, Heilig B, Urbář J, Kouba D,
Kis Á and Barta V (2023), Comprehensive
analysis of the ionospheric response to
the largest geomagnetic storms from
solar cycle 24 over Europe. Front. Astron. Space Sci. 10:1092850. doi: 10.3389/fspas.2023.1092850 A multi-instrumental analysis of the meridional ionospheric response is presented
over Europe during the two largest ICME-driven geomagnetic storms of solar
cycle #24 maximum. Data from 5 European digisonde stations, ground-based
Global Navigation Satellite System, Total Electron Content (GNSS TEC), the ratio of
the TEC difference (rTEC), as well as Swarm and Thermosphere, Ionosphere,
Mesosphere, Energetics and Dynamics (TIMED) satellite observations have been
used for the investigation of selected intervals (11–17 November, 2012, and
16–25
March,
2015). The
storm
evolution
is
monitored
by
digisonde
foF2 critical frequency (related to the maximum electron density of F2-layer)
and GNSS TEC data. Moreover, Global Ultraviolet Imager (GUVI) measurements
from the TIMED satellite are used to investigate the changes in the thermospheric
O/N2 ratio. Our main focus was on the main phase of the geomagnetic storms,
when during the nighttime hours extremely depleted plasma was detected. The
extreme depletion is observed in foF2, TEC and rTEC, which is found to be directly
connected to the equatorward motion of the midlatitude ionospheric trough (MIT)
on the nightside. We demonstrate a method (beside the existing ones) which
allows the monitoring of the storm-time evolution of the disturbances (e.g., MIT,
SAPS, SED) in the thermosphere-ionosphere-plasmasphere system by the
combined analysis of the worldwide digisonde system data (with the drift
measurements and the ionospheric layer parameters with 5–15 min cadence),
with rTEC and GNSS TEC data, and with the satellite data like Swarm, TIMED/GUVI. Front. Astron. Space Sci. 10:1092850. doi: 10.3389/fspas.2023.1092850 Front. Astron. Space Sci. 10:1092850. Frontiers in Astronomy and Space Sciences frontiersin.org 1 Introduction In addition, several other influencing factors must be taken
into consideration, such as: geomagnetic storm size, local time (LT)
of the sudden storm commencement (SSC), time of the day, season,
geomagnetic latitude and longitude (Immel and Mannucci, 2013;
Mendillo and Narvaez, 2010; Mendillo and Narvaez, 2009). Within
the
Earth’s
plasma
environment
(ionosphere,
plasmasphere,
outer magnetosphere), all regions are closely
related to each other. In the presence of an external forcing (e.g.,
ICME or CIR/HSSWS), perturbations can be observed in each
plasma layer. The F-layer of the ionosphere has the highest
electron density, so electron density changes are the most
pronounced
there. The
electric
fields
generated
during
geomagnetically disturbed periods (e.g., prompt penetration of
magnetospheric convection electric field-PPEF and disturbance
dynamo electric field-DDEF) map along geomagnetic field lines
through all these regions and can interact with all of them (see e.g.,
Nava et al., 2016). The Earth’s plasma environment is a very
complex, tightly coupled system, and the effects cannot be
studied and explained in their entirety if we do not consider
them as part of the system. Geomagnetic storms generate so-called ionospheric storms,
which have similar evolution and phases to those of geomagnetic
storms, but with a faster procession. Ionospheric storms have
already been studied using several types of observations of the
F2-layer: ionosonde data of the maximum electron density
(NmF2); measurements of the total electron content; incoherent
scatter
radar
measurements
of
electron
and
ion
densities,
temperatures and plasma dynamics; satellite measurements of
ionospheric/thermospheric parameters along their orbits (see e.g.,
Kane and Makarevich, 2010). Comprehensive reviews of storm
effects in NmF2 and TEC have been given by Prölss (1995) and
Mendillo (2006), for incoherent scatter radar results by Buonsanto
(1999), and for in-situ satellite data by Prölss and Zahn (1974). Long-duration
positive
ionospheric
storm
phase
can be
generated
by
the
enhanced
storm-induced
equatorward
meridional winds because they cause the downwelling of the
neutral atomic oxygen and the uplifting of the F-layer along the
magnetic field lines due to wind induced vertical ExB drift (Prölss,
1995; Danilov, 2013). A similar drift can also be caused by an
increase in E-fields of other origin (e.g., from the magnetosphere)
(Danilov, 2013). As the loss-rate decreases with altitude, the density
increases. 1 Introduction Previous studies concluded that at high and middle latitudes
this negative phase type occurs more often, and has much more
dangerous effect on HF propagation (Danilov, 2013). the geomagnetic activity. For intense storms, it could be found
below L = 2.5 (Mendillo and Narvaez, 2009). There are two
major consequences of high-latitude heating: composition
changes (specifically, decrease in the O/N2 ratio) and the
intensification of equatorward winds that can carry the
composition
change
toward
lower
latitudes
(Buonsanto,
1999). Both processes contribute to the formation of long-
duration negative storm phases. The electron density near
the
F2-layer
maximum
is,
approximately,
directly
proportional to the O/N2 ratio (Rishbeth and Barron, 1960;
Pirog, 2006). This means, if other conditions are unchanged
(constant pressure), we should detect depletion in electron
density (negative phase) in all regions where O/N2 ratio has
been decreased at F2-layer heights (Danilov, 2013). Under
geomagnetic storm conditions this so-called compositional
disturbance zone at F2-layer heights reaches lower latitudes
because of the intensified heating induced equatorward
meridional winds. Negative phase is most common in the
summer hemisphere (both day and night) due to the
seasonal variation of the background thermospheric wind
circulation, but during intense geomagnetic storms it can be
detected even in winter months. It favors the postmidnight and
morning sectors, because then the background and the storm-
induced circulation coincide and add up and therefore can reach
much lower latitudes (Prölss, 1995; Buonsanto, 1999; Danilov,
2013). This equatorward expansion of the negative phase has
about 50–300 m/s velocity (Danilov and Belik, 1991). Besides
the O/N2 ratio depletion, the increased temperature of the
heated thermospheric gas itself is an important factor in
forming a negative storm phase in the ionosphere (Mikhailov
and Foster, 1997). The increase in temperature leads to an
increase in the recombination coefficient causing a further
decrease in electron density (see Mikhailov et al., 1995). Previous studies concluded that at high and middle latitudes
this negative phase type occurs more often, and has much more
dangerous effect on HF propagation (Danilov, 2013). perturbations. Besides there are several processes that have to be
taken into consideration during the examination of the mid- and low
latitude ionosphere, as follows: photo-production, chemical loss,
and transport by thermal expansion, neutral winds, waves, tides and
electric fields of internal and external origin (Mendillo and Narvaez,
2009). Frontiers in Astronomy and Space Sciences frontiersin.org 1 Introduction The physical processes of the Earth’s plasma environment responsible for the
perturbations of the system and the exact mechanisms of action of solar events affecting
the near-Earth space have been of interest to researchers for decades. The most remarkable
disturbing solar activity events are interplanetary coronal mass ejections (ICMEs) and high-
speed solar wind streams (HSSWS)/corotating interaction regions (CIRs). When they collide
with the Earth’s magnetic field, they trigger geomagnetic storms. The two types of
geomagnetic storms have different time courses and result in different magnitudes of Frontiers in Astronomy and Space Sciences 01 frontiersin.org frontiersin.org 10.3389/fspas.2023.1092850 Berényi et al. the geomagnetic activity. For intense storms, it could be found
below L = 2.5 (Mendillo and Narvaez, 2009). There are two
major consequences of high-latitude heating: composition
changes (specifically, decrease in the O/N2 ratio) and the
intensification of equatorward winds that can carry the
composition
change
toward
lower
latitudes
(Buonsanto,
1999). Both processes contribute to the formation of long-
duration negative storm phases. The electron density near
the
F2-layer
maximum
is,
approximately,
directly
proportional to the O/N2 ratio (Rishbeth and Barron, 1960;
Pirog, 2006). This means, if other conditions are unchanged
(constant pressure), we should detect depletion in electron
density (negative phase) in all regions where O/N2 ratio has
been decreased at F2-layer heights (Danilov, 2013). Under
geomagnetic storm conditions this so-called compositional
disturbance zone at F2-layer heights reaches lower latitudes
because of the intensified heating induced equatorward
meridional winds. Negative phase is most common in the
summer hemisphere (both day and night) due to the
seasonal variation of the background thermospheric wind
circulation, but during intense geomagnetic storms it can be
detected even in winter months. It favors the postmidnight and
morning sectors, because then the background and the storm-
induced circulation coincide and add up and therefore can reach
much lower latitudes (Prölss, 1995; Buonsanto, 1999; Danilov,
2013). This equatorward expansion of the negative phase has
about 50–300 m/s velocity (Danilov and Belik, 1991). Besides
the O/N2 ratio depletion, the increased temperature of the
heated thermospheric gas itself is an important factor in
forming a negative storm phase in the ionosphere (Mikhailov
and Foster, 1997). The increase in temperature leads to an
increase in the recombination coefficient causing a further
decrease in electron density (see Mikhailov et al., 1995). 1 Introduction In early studies, two different phases of ionospheric storms were
distinguished: positive ionospheric storm phase, when the electron
density is increased, and negative ionospheric storm phase, when the
electron density is decreased with respect to its expected value. Earlier studies during the past few decades have already found clear
and unambiguous patterns in the ionosphere during geomagnetic
storms, see for example, the great reviews of Sato (1957), Matsushita
(1959), Prölss (1995), Danilov (2013) (for ionosonde data) and
Mendillo (2006) (for TEC data). (2) The ionospheric F- region plasma is the base of the
plasmasphere (Mendillo et al., 1974), i.e., the plasmasphere is
filled from the dayside ionosphere and empties into the
nightside F-region through diffusion along the geomagnetic
field lines. Under quiet conditions the midlatitude region
maps into the plasmasphere corotating with the Earth. During storms the footprint of the plasmapause (PP) often
moves to midlatitudes, especially after sunset (Mendillo and
Narvaez, 2009) as the plasmapause moves inward due to the
increased geomagnetic activity, while the plasmasphere can still
remain conjugated with the midlatitude ionosphere during The various perturbations in the midlatitude ionosphere during
geomagnetic storms can be linked to various processes detailed
below. For case studies of ionospheric storm effects, it is necessary to
determine the key drivers of the actual events. (1) During geomagnetic storms, the Joule dissipation of currents
and the absorption of precipitating particles in the auroral
region of the lower thermosphere (100–140 km) are the
cause of the so called auroral heating of the thermosphere
(Prölss, 1995; Danilov, 2013). The location of both the
precipitation boundary and the auroral currents depends on 02 frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 10.3389/fspas.2023.1092850 daytime hours. Thus, from local noon to dusk the dynamical
plasmaspheric processes may contribute to daytime positive-
phase ionospheric storms (Lanzerotti et al., 1975), which is
followed after sunset by a sharp transition to a depleted state
beyond the footprint of the plasmapause (Mendillo et al., 1974). After sunset, both the above discussed dynamics and chemistry
cause the onset of a negative phase. Furthermore, multi-instrumental comprehensive analysis of the
thermosphere-ionosphere-plasmasphere
coupling
processes
during individual geomagnetic storms are rare in the literature. Most studies are based on one or two types of observational data,
however the effects cannot be studied separately since the Earth’s
plasma environment is a very complex, multiplied coupled system. 1 Introduction In this study our aim is to determine the exact connections
between the thermosphere-ionosphere-plasmasphere system during
two intense ICME-caused geomagnetic storm events. We analyzed
the effects through a meridional chain of digisondes (foF2, h’F2 and
drift data) across Europe (Northern Hemisphere) complemented by
GNSS TEC, Swarm satellite observations of the topside electron
density, electron temperature and PP location data, as well as O/N2
ratio observations from TIMED satellite. Another aim is to identify
the processes responsible for the extreme decrease during the night
in the main phase of the geomagnetic storm. The rest of the paper is
organized as follows. First, the utilized data and the applied methods
are introduced. In Section 3 the observations are presented, followed
by the discussion in Section 4. Finally, main conclusions are
summarized in Section 5. (3) During intense geomagnetic storm events, mainly in the main
phase, at night, a drastic “peeling off” of the magnetospheric
plasma can be seen, and the ionospheric projection of the PP can
penetrate into the midlatitude region also on the dayside. In
such a case, an all-day negative phase storm is expected. Based
on earlier studies like Heilig et al. (2022), it is stated that the
ionospheric footprint of the PP is tightly coupled to the
minimum of the midlatitude (main) ionospheric trough
(MIT). The poleward edge of the MIT in the evening sector
has been observed to coincide with the equatorward boundary
of the soft electron precipitation, and the precipitation has
therefore been proposed as a major source of electrons
building up the poleward wall of the MIT in the evening
sector (Turunen and Liszka, 1972; Rodger et al., 1986;
Voiculescu et al., 2010). During the night the ionospheric
F-layer on the equatorward side of the plasmapause is
maintained by the plasmasphere, but not on the global
convection dominated poleward side (Schunk and Banks,
1975). Consequently, an MIT minimum could be bounded
by
a
plasmasphere-maintained
and
the
precipitation-
maintained ionosphere. The minimum could be sharpened
by storm-time developed subauroral polarization streams
(SAPS)
due
to
SAPS
E-field,
and
by
plasma
heating
(frictional
heating
by
the
drifting
plasma)
and
its
consequences (Rodger et al., 1992; Horváth and Lovell,
2016), and by enhancing recombination due to molecular
composition changes by neutral winds or diffusion from the
aurora oval = composition disturbance zone (1) (Schunk and
Banks, 1975). 2 Data and method Its
typical latitudinal extent (width) is 5°–10° (Voiculescu et al., 2010),
10°–15° (Liu and Xiong, 2020), but it can be as wide as 20°. MIT is
typically found somewhere between 55° and 75° geographic latitude
depending on the geomagnetic activity and MLT (e.g., Whalen,
1989; Werner and Prölss, 1997; Voiculescu et al., 2006; Deminov and
Shubin, 2018; Karpachev et al., 2019; Aa et al., 2020; Liu and Xiong,
2020). However, as we will show, in severely disturbed cases it can
move even further equatorward. MIT typically occurs in the dark
hemisphere, thus, it is most regularly observed during winter
months and equinoxes, while in summer, it is mainly restricted
to the midnight sector (Rodger et al., 1992; Voiculescu et al., 2006). We analyzed individual events to see in detail the processes and
to determine the key drivers of the geomagnetic storm generated
ionospheric perturbations. Using the meridional station chain, the
latitudinal evolution of the effects can be followed. With the
digisonde drift measurements the directions of the associated
plasma drifts can be determined. Using also satellite data, the
thermospheric
(with
TIMED,
GUVI
measurements)
and
plasmaspheric (with Swarm, Langmuir probe measurements)
processes can be linked to the perturbations observed in the
ionosphere. The measurements and data utilized are listed below: Over the years, the general behavior of the ionosphere during
storms has become well studied. However, each space weather event
and the involved processes are unique, and in many cases the
magnitude of the effects cannot yet be predicted. Consequently,
case studies of extraordinary/high magnitude events are still
important as they can deepen/refine our understanding of the
extent of change that can be expected in the individual layers of
the
Earth’s
plasma
in
response
to
a
geomagnetic
storm. Frontiers in Astronomy and Space Sciences 2 Data and method In the present study our main focus is on the analysis of the
effects
in
the
ionospheric
F2-layer
during
two
intense
(Dstmin > −100 nT) ICME related geomagnetic storms. The
storms considered are from the winter/equinox of 2012 and 2015
(the maximum of #24 solar cycle), respectively, with the following
characteristics: 11–17/11/2012 (Dstmin = −108 nT, Kpmax = 6.33)
and 16–25/03/2015 (Dstmin = −223 nT, Kpmax = 7.67). For the investigation, intervals covering all the pre-storm (24 h
before the SSC), initial, main and recovery phase were selected. We
used the geomagnetic Dst-index to identify the storm phases. A
sudden sharp increase in the data is observed when an SSC occurs,
this is followed by a major decrease in the data (main phase), and
then a slower recovery lasting for several days. The magnitude of the
storm itself is given by the minimum value of Dst. The AE-index
(auroral electrojet index - mainly characterizing the polar region)
increases as the substorm activity intensifies. MIT is a longitudinally elongated and latitudinally restricted
region of depleted plasma in the ionosphere (He et al., 2011). Its
typical latitudinal extent (width) is 5°–10° (Voiculescu et al., 2010),
10°–15° (Liu and Xiong, 2020), but it can be as wide as 20°. MIT is
typically found somewhere between 55° and 75° geographic latitude
depending on the geomagnetic activity and MLT (e.g., Whalen,
1989; Werner and Prölss, 1997; Voiculescu et al., 2006; Deminov and
Shubin, 2018; Karpachev et al., 2019; Aa et al., 2020; Liu and Xiong,
2020). However, as we will show, in severely disturbed cases it can
move even further equatorward. MIT typically occurs in the dark
hemisphere, thus, it is most regularly observed during winter
months and equinoxes, while in summer, it is mainly restricted
to the midnight sector (Rodger et al., 1992; Voiculescu et al., 2006). Over the years, the general behavior of the ionosphere during
storms has become well studied. However, each space weather event
and the involved processes are unique, and in many cases the
magnitude of the effects cannot yet be predicted. Consequently,
case studies of extraordinary/high magnitude events are still
important as they can deepen/refine our understanding of the
extent of change that can be expected in the individual layers of
the
Earth’s
plasma
in
response
to
a
geomagnetic
storm. MIT is a longitudinally elongated and latitudinally restricted
region of depleted plasma in the ionosphere (He et al., 2011). 2.2 Ionosonde data For comparison, the National Institute of Information and
Communications Technology (NICT) Ratio of the TEC difference
(rTEC) global maps constructed using the RINEX files obtained
from thousands of GNSS receivers all over the world were applied
for the European region with a grid of 0.5 × 0.5° and smoothed with a
5 × 5 boxcar. The rTEC value was defined as the difference between the
observed TEC and the monthly average quiet TEC value (mean of
10 geomagnetically quietest days), normalized by the average TEC. A meridional ionosonde station chain across Europe was selected to
represent the latitudinal changes. The following 5 stations were chosen
for the analysis (from North to South): Juliusruh (JR), Pruhonice (PQ),
Sopron (SO), Rome (RO), and Athens (AT). In Table 1, information on
the ionosonde stations is given. LT = UT +1. Most of the stations are
operating with a DPS-4D type of ionosonde, a digisonde. Only SO is an
exception where a VISRC-2 type ionosonde operated until 2018, when
the same DPS-4D type digisonde was installed also here (Bór et al.,
2020). Old
ionosonde/digisonde
data
has
typically
30/15 min
resolution, respectively, however, in some cases (like at Athens
station) data are available at 5-min cadence. Frontiers in Astronomy and Space Sciences 2.3 TEC data The Dst index shows the intensity of the ring current and as we
mentioned above are generally used to separate the geomagnetic
storm’s phases case to case. The AE index describe the evolution
of the auroral electrojets, so it inform us about the processes at
high latitudes. GNSS TEC IONEX (IONosphere Map EXchange) maps data
(http://ftp.aiub.unibe.ch/ionex/draft/ionex11.pdf) International
GPS Service for Geodynamics (IGSG) with 2-hourly cadence
were used to determine vertical TEC (vTEC) development in
areas surrounding the respective digisondes, each map area of
size 2.5 (latitude) x 5 (longitude) degrees. 2.4 TIMED satellite, global ultraviolet imager
(GUVI) O/N2 measurements During this investigation, two ionospheric parameters of the F2-
layer were examined, namely, foF2, h’F2. The foF2 parameter represent
the F2 layer critical frequency associated with the maximum plasma
(electron) density of the F2 layer, while the h’F2 parameter reflects the
changes in the virtual height of the layer. All digisonde data were
manually checked and corrected with SAO Explorer. Besides, modern
digital ionosondes provide also routine ionospheric drift measurements
in addition to classical vertical ionospheric sounding (for more see the
Supplementary Data section and Kouba et al., 2008; Kouba and Koucka
Knizova, 2012; Kouba and Koucka Knizova, 2016). The ionospheric
drift data for the selected storms of this study were manually processed
for the PQ station. Nowadays, tens of digisondes worldwide measure
ionospheric drifts routinely and store their data in Global Ionosphere
Radio Observatory (GIRO). The TIMED satellite has been operating at 625 km altitude
since December of 2001 and focuses its measurements to the
appr. 60–180 km altitude range (neutral thermosphere and
ionosphere) below the satellite (Christensen et al., 2003). TIMED
is
orbiting
on
a
circular
polar
orbit
with
an
inclination of 74.1° corresponding to 97.8 min period, which
means that a global map of the measurements can be derived
from 14.9 daily orbits. GUVI measurements provide dayside O/N2 composition
(note that this is a column integrated value for an altitude
range) and temperature profile of the Mesosphere and Lower
Thermosphere/Ionosphere (MLTI) region, as well as the auroral
energy inputs (Christensen et al., 2003; Crowley et al., 2006;
http://guvitimed.jhuapl.edu/home_background). Reference values were needed to determine the magnitude of the
storm-time deviations from the nominal state. For this, we chose the
3 closest geomagnetically quiet days (QDs) preceding the storms
investigated (8, 9, 10 November 2012 and 10, 13, 14 March 2015,
respecti721vely) based on the International Q-days (QD) list, and
averaged them. These reference values appear as green lines in
Figure 3; Figure 4; Figure 5; Figure 6. 2.1 Solar and geomagnetic indices During case studies we typically identify and characterize the
storm events considering the magnitude of geomagnetic indices
such as Kp, Dst and AE. The Kp index represents the geomagnetic
activity of the midlatitudal regions, and it has logarithmic scale. 03 frontiersin.org 10.3389/fspas.2023.1092850 Berényi et al. TABLE 1 In this table, we can see information about the used 5 digisonde stations. For the geomagnetic coordinates, International Geomagnetic Reference Field
(IGRF-13)-model (for year 2015) based calculator was used. Name of the
station
Station
ID
Geomagnetic
latitude (deg)
Geomagnetic
longitude (deg)
Geographic
latitude (deg)
Geographic
longitude (deg)
High mid-
latitude
Juliusruh
JR055
53.95° N
99.48°E
54.6° N
13.4° E
Middle
latitude
Pruhonice
PQ052
49.32° N
98.61° E
50° N
14.6° E
Sopron
SO148
46.67° N
99.75° E
47.63° N
16.72° E
Low mid-
latitude
Rome
RO041
41.7° N
93.76° E
41.8° N
12.5° E
Athens
AT138
36.17° N
103.33° E
38° N
23.5° E 2.5 Swarm satellite measurements The three Swarm satellites were launched into a polar low-
Earth orbit (LEO) in November 2013. The altitude of Swarm A Frontiers in Astronomy and Space Sciences 04 frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 C (flying side-by-side with ca 1 5° longitudinal difference)
3 Observations
GURE 1
he geomagnetic Dst, Kp and AE indices are plotted for the 2012 November storm (A), and for the 2015 March storm (B). In the upper diagrams the
aily variation of the Dst index is presented, in the middle panel the Kp index, while in the bottom the daily variation of the AE index is shown. The UT of the
C was at 23:12 for the 2012 storm marked with a red dotted line. In the 2015 storm, the first storm commenced at UT 04:45, while the second SSC was
20:54 (red dotted lines). FIGURE 1
The geomagnetic Dst, Kp and AE indices are plotted for the 2012 November storm (A), and for the 2015 March storm (B). In the upper diagrams the
daily variation of the Dst index is presented, in the middle panel the Kp index, while in the bottom the daily variation of the AE index is shown. The UT of the
SSC was at 23:12 for the 2012 storm marked with a red dotted line. In the 2015 storm, the first storm commenced at UT 04:45, while the second SSC was
at 20:54 (red dotted lines). Frontiers in Astronomy and Space Sciences 3 Observations and C (flying side-by-side with ca. 1.5° longitudinal difference)
was around 460 km in March 2015, while Swarm B was operating
at around 520 km altitude, with a 91 min orbiting period. Swarm
satellites provide in-situ measurements of the electron density
and temperature observed by Langmuir probes, as well as the
location of the MIT from which the footprint of the nightside
plasmapause can be derived. and C (flying side-by-side with ca. 1.5° longitudinal difference)
was around 460 km in March 2015, while Swarm B was operating
at around 520 km altitude, with a 91 min orbiting period. Swarm
satellites provide in-situ measurements of the electron density
and temperature observed by Langmuir probes, as well as the
location of the MIT from which the footprint of the nightside
plasmapause can be derived. In our previous article (Berényi et al., 2018), the characteristics
of the 2012 November and 2015 March storm were described in
detail, the course of the storms was investigated in several aspects
using data from the Sopron ionosonde station. In this article, we
discuss the meridional evolution and characteristics of the two Frontiers in Astronomy and Space Sciences 05 frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 FIGURE 2
The interplanetary magnetic field (IMF) Bz component and the solar wind speed data can be seen for the two investigated storm intervals. (A) Is for
the 2012 November, (B) is for the 2015 March storm. The SSC times are with red dotted lines (Supplementary Figure S1 is the same, except that it is with 1-
min resolution). FIGURE 2 FIGURE 2
The interplanetary magnetic field (IMF) Bz component and the solar wind speed data can be seen for the two investigated storm intervals. (A) Is for
the 2012 November, (B) is for the 2015 March storm. The SSC times are with red dotted lines (Supplementary Figure S1 is the same, except that it is with 1-
min resolution). investigated storms using several types of measurements, detailed in
Section 2. Right after that a negative polarity coronal hole associated high
speed solar wind stream (HSSWS) arrived. The main phase of the
geomagnetic storm started on 14 November (Dstmin = −108 nT,
Kpmax = 6.33), this can be distinctly seen in Figure 1A in the
evolution of the geomagnetic Dst, Kp and AE indices. Frontiers in Astronomy and Space Sciences 3 Observations According to the ACE satellite data, in the case of storm from
2012 November, the geomagnetic disturbance started with two
ICMEs shock arrivals at 22:16 UT on 12 November (source:
SWPC PRF 1942, 19 November, 2012; https://izw1.caltech.edu/
ACE/ASC/DATA/level3/icmetable2.htm). The storm started with
a Sudden Storm Commencement (SSC) at 23:12 UT (00:12 LT) on
12 November On Figure 2A the evolution of the interplanetary
magnetic field (IMF) Bz component and the solar wind (SW) speed
data are plotted (1-min resolution is on Supplementary Figure S1A). During the SSC time, short southward turning of the Bz appeared
which 1 h later turned northward and lasted until ca. 13 h. In the
meanwhile the SW speed from 300 km/s after the SSC went up to
420 km/s and it oscillated around this value through the whole storm
interval. Early on 14 November the storm reached its maximum
magnitude (major storm), which was caused by a prolonged period
of negative Bz (Figure 2A) from 8 h to 18 h, attributed to a
combination of lingering ICME effects and a solar sector
boundary crossing that occurred at approximately 02:45 UT. The 2015 March geomagnetic storm started with an SSC at
04:45 UT (05:45 LT) on 17 March, around equinox. The
magnitude
of
this
storm
was
(on
day
17
March):
Dstmin = −234 nT, Kpmax = 7.67 (see also Figure 1B). More
information about this ICME generated geomagnetic storm can
be found in the article of Wu et al. (2016). On Figure 2B the IMF
Bz and SW speed values were displayed (1-min resolution is on
Supplementary Figure S1B). The most significant episodes: right
after the SSC the northward Bz turned southward around 7 UT,
this followed by a northward turning around 10, then from noon
until midnight a prolonged southward Bz can be observed. The
SW speed values increased from an initial 400 km/s to 600 km/s
around noon on17 March, and peaked at 700 km/s on the night of
18 March. On the days after the ICME the Earth was inside the
flow of HSSWS (Nava et al., 2016). A subsequent geomagnetic 06 frontiersin.org Berényi et al. 3 Observations 10.3389/fspas.2023.1092850 FIGURE 3
On the upper diagrams the Dst-index values and on the lower diagrams the storm-time foF2 (with red line, A)) and h’F2 (with light blue line, B)) with
the reference foF2, h’F2 values of selected 3 quiet days [with dotted green (A) and pink line (B)] are portrayed for storm 2012 over the meridional station
chain. FIGURE 3
On the upper diagrams the Dst-index values and on the lower diagrams the storm-time foF2 (with red line, A)) and h’F2 (with light blue line, B)) with
the reference foF2, h’F2 values of selected 3 quiet days [with dotted green (A) and pink line (B)] are portrayed for storm 2012 over the meridional station
chain. frontiersin.org Frontiers in Astronomy and Space Sciences 3.1.2 Geomagnetic storm 16-25/03/2015 The evolution of the 2015 March geomagnetic storm started on
day 17th. The local time of the SSC was at 05:45 on 17 March (these
are marked with a red dotted line in Figure 4). After the SSC, during
the main phase of the ionospheric and geomagnetic storm, a positive
phase of the ionospheric storm started at all stations. These kinds of
storms are called Regular Positive Phase (RPP) storms because in the
main phase of the storm, the electron density (foF2) increases, with
no delay (Mendillo and Narvaez, 2010). At the same time, there is
also a significant increase in height of F2-layer (h’F2) during daylight
hours at all stations, but much less pronounced at RO and AT (see
the right panel of Figure 4). During the daytime hours in the main phase of the geomagnetic
storm (14 November) a negative ionospheric storm phase was
observed at all stations. In the meantime, the F2 layer was
extremely uplifted, up to 680 km at stations SO, PQ and JR
(Figure 3, right panel). In this case, we observe a negative
ionospheric storm phase during a relatively weak (i.e., closer to
moderate) geomagnetic storm. This event can be considered as an
atypical storm, since the main phase of the geomagnetic storm is
delayed by 1 day. In a regular storm, the main phase typically starts
with a significant positive or negative ionospheric storm phase
within a few hours after the SSC. The most significant increase in the virtual height of the F2-layer
occurred from around 20:00 UT (21 LT) on 17 March. The highest
peak of the h’F2 parameter was observed at Pruhonice at 587.6 km at
22:45 UT. This main phase pattern in h’F2 parameter lasts until
21 March, and appears significant at SO, PQ and JR stations (see
Figure 4). Note. during this storm the ionosonde at Sopron provided
observations only up to 8 MHz, but the negative ionospheric
response during the main phase of the geomagnetic storm clearly
shows up. Besides, during the evening/night on 17/18 March, a sharp
decrease in the electron density was observed, the start of the
negative phase began at noon at JR and it occurred later and
later with decreasing latitude. The depletion was the most
pronounced at PQ, SO and RO (see Figure 4 and Supplementary
Table S2). 3.1.1 Geomagnetic storm 11-17/11/2012 The first phenomenon known from previous studies, which can
appear in foF2 even 24 h before the SSC during the daytime hours, is
called the pre-storm enhancement (Kane, 2005; Burešová and
Laštovička, 2007; Danilov, 2013). This can be also observed in all
the 5 digisonde stations, though it is not very well pronounced (see
Figure 3). On the contrary, during the night on 11/12 November all
station data show negative phase, except for AT where no change
can be detected. From 15 November, during the early recovery phase, a so-called
early recovery phase enhancement effect was detected in foF2 at
stations AT, RO, SO and PQ (see Figure 3). This effect with
increased electron density around noon lasts 3-day long, most
significantly at Rome and Athens station, but observable at the
other stations, too (see Figure 3). On the other hand, during the
nighttime hours of 15 November a negative phase appears at all
stations, while on 16 November only JR and PQ are negative, SO
does not show any clear trend, and RO and AT are positive. On the right panel of Figure 3, the virtual height of the F2-layer
(h’F2) is presented, not any significant daytime change at any station
can be seen on 12 November, however a quite significant increase
can be observed during the night starting at 21:00 UT, just before
the SSC. During the recovery phase, no deviation from the reference quiet
time h’F2 was seen during daytime, however there was a quite
significant increase during the night, first of all at the PQ station, but
less pronounced also at SO and JR. Right after the SSC at 23:12 UT, an increase in electron density
(foF2 parameter) can be seen during that night and then during the
day on 13 November at all stations. This is the main phase of the
ionospheric storm, but the geomagnetic storm main phase starts
only later on the following day. At the beginning of the following
night, still positive phase at all stations can be seen, but around
midnight it turns negative at JR, PQ and SO. Besides, a significant
increase in h’F2 appears already around 20:00 UT which lasts even
throughout the next day (14 November). 3.1 Digisonde data over Europe In Supplementary
Table S1 the exact time and duration of the fade-outs, the start time
of the decrease/increase and the minimum/maximum values are
tabulated (the foF2 value in MHz). ionospheric storm phase can be observed, while over lower latitudes
(closer to the equator) this turns into a positive phase (the exact
latitude of the phase reversal depends e.g., on the season, LT, the
strength of the geomagnetic storm, meridional winds, etc.). First, we
present ionosonde observations for the extended 2012 and
2015 storm periods. ionograms) at these stations. Along with this electron density
drop, the virtual height of the F2-layer increased up to 400 km
(from the 250–280 km QD value) before the observed fade-out (see
Figures 1, 3, as well as the Supplementary Table S1). On the contrary,
at RO and AT stations, a significant increase of the foF2 parameter
(electron density) with respect to the reference days could be seen
during the night. Furthermore, the h’F2 was not as increased at RO
and AT during this period as at the other stations. In Supplementary
Table S1 the exact time and duration of the fade-outs, the start time
of the decrease/increase and the minimum/maximum values are
tabulated (the foF2 value in MHz). Frontiers in Astronomy and Space Sciences 3.1 Digisonde data over Europe disturbance which is connected to a HSSWS (see Figure 1B)
started at 20:54 UT on 21 March, which does not appear in the
value of the Dst index, but is clearly visible in the Kp and AE
index (see Figure 1B) (Nava et al., 2016). Several articles have
been published in recent years about this storm, also known as St. Patrick’s Day storm, which is the largest storm of the solar cycle
24 (e.g., Astafyeva et al., 2015; Cherniak and Zakharenkova, 2015;
Zhang J.J. et al., 2015; Zhang S.R. et al., 2015; Li et al., 2016; Wu
et al., 2016; Nayak et al., 2016; Nava et al., 2016; Kalita et al., 2016;
Tulasi et al., 2016; Hairston et al., 2016; Jin et al., 2017; Polekh
et al., 2017; Zhang S.R. et al., 2017a; Zhang S.R. et al., 2017b and
references therein; Berényi et al., 2018; Huang et al., 2018;
Habarulema et al., 2018; Kumar and Kumar, 2019; Ratovsky
et al., 2019; Lu et al., 2020). The main results presented in the following were derived from
ionosonde and digisonde measurements across a meridional station
chain introduced in Section 2. In this section, the evolution of the
ionospheric foF2, h’F2 is shown. Using data of European ionosonde
stations for comparison, our primary goal is to check whether the
ionosonde recorded effects at Sopron station (Berényi et al., 2018)
were local or regional/global. Furthermore, we would like to
illustrate how an ionospheric storm develops from North to
South,
along
a
geomagnetic
meridian. Through
this
demonstration, the turning point of the ionospheric phase
development can be determined (see Buonsanto, 1999; Kane,
2005). In a general case, over the auroral region a negative Frontiers in Astronomy and Space Sciences 07 frontiersin.org frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 10.3389/fspas.2023.1092850 10.3389/fspas.2023.1092850 ionograms) at these stations. Along with this electron density
drop, the virtual height of the F2-layer increased up to 400 km
(from the 250–280 km QD value) before the observed fade-out (see
Figures 1, 3, as well as the Supplementary Table S1). On the contrary,
at RO and AT stations, a significant increase of the foF2 parameter
(electron density) with respect to the reference days could be seen
during the night. Furthermore, the h’F2 was not as increased at RO
and AT during this period as at the other stations. frontiersin.org 3.1.2 Geomagnetic storm 16-25/03/2015 The electron density decreased below the detectability
level (disappeared from the ionograms) for a short period at RO and
SO station. In Supplementary Table S2 the exact time and duration
of the fade-outs, the start time of the negative phase and the time of
the observed minimum value are tabulated. Along with this electron
density drop, the virtual height of the F2-layer increased up to
483 km (from the 250–280 km QD value) before the fade-out at
Sopron (see Figure 4). This storm can be identified as a Regular Positive Phase (RPP)
storm type following the nomenclature introduced by Mendillo and
Narvaez (2010) according to the meridional evolution of foF2. Their
classification is based on the local time of the SSC, which was in this
case around midnight (00:12 LT) on 13 November The significant
increase of foF2 (electron density) lasted from around 10–11:00 to
20:00 LT at all stations, and this positive phase was followed by a
negative phase on 14 November. In Figure 3 during the main phase of the geomagnetic storm at
night hours (18:00–02:30 UT), a negative ionospheric storm phase
can be observed at the SO, PQ and JR stations. The electron density
decreased below the detectability level (disappeared from the Depletions in the foF2 parameter value (negative phase)
compared to the main phase value can be observed on the next 08 frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 FIGURE 4
In the upper diagrams the Dst index values and in the other diagrams (A) the storm-time foF2 (red line) and (B) the storm-time h’F2 (light blue line)
and the corresponding reference foF2 and h’F2 values (green (A) and pink (B), respectively) are portrayed for the 2015 storm along the meridional chain. FIGURE 4
In the upper diagrams the Dst index values and in the other diagrams (A) the storm-time foF2 (red line) and (B) the storm-time h’F2 (light blue line)
and the corresponding reference foF2 and h’F2 values (green (A) and pink (B), respectively) are portrayed for the 2015 storm along the meridional chain. days (during the early recovery phase) at SO, PQ and JR stations
(middle - and high midlatitude), during both days and nights. Even
at AT and RO, for a short period positive phase occurred around
9 UT, then it is turned into a significant negative phase around
11 UT. 3.2 GNSS TEC data To provide a wider context for the ionosonde observations,
changes in the total electron content (TEC) data was compared with
the foF2 values measured by the ionosondes. TEC data are presented
similarly to Figures 3, 4 for a better comparison. The TEC represents
the total electron content as an integrated value between the receiver
on the ground and the GNSS satellite (MEO orbit, at around
20,000 km altitude), therefore it contains not just the whole
ionosphere, but also most of the plasmasphere (Hofmann-
Wellenhof and Lichtenegger, 2001). g
g
y
A subsequent geomagnetic disturbance, associated with a
HSSWS (see Figures 1, 4) started at 20:54 UT on 21 March,
occurred during the recovery phase of the first storm and
generated
a
positive
ionospheric
storm. Decreased
electron
density during the daytime and nighttime of 21th can be
observed at all stations, but most significantly at RO. In the
h’F2 parameter just a slight increase can be seen right after the
SSC during the night, mostly at AT and RO stations. Meanwhile on
22 March no change was observed at JR, and only a slight increase in
foF2 at PQ, SO, RO, AT. During the night the storm turns negative
at all stations, except for RO and AT, where no change was observed. During the remaining days in the recovery phase: on 23 March
no changes at JR and PQ were detected, but a slight increase in
foF2 at SO, RO, AT can be seen around noon. During the night just
JR showed no changes, the others were positive. On 24 March no
changes were observed in foF2 at all stations during the day, but at
night just JR did not show changes, the other stations had positive A subsequent geomagnetic disturbance, associated with a
HSSWS (see Figures 1, 4) started at 20:54 UT on 21 March,
occurred during the recovery phase of the first storm and
generated
a
positive
ionospheric
storm. Decreased
electron
density during the daytime and nighttime of 21th can be
observed at all stations, but most significantly at RO. In the
h’F2 parameter just a slight increase can be seen right after the
SSC during the night, mostly at AT and RO stations. Meanwhile on
22 March no change was observed at JR, and only a slight increase in
foF2 at PQ, SO, RO, AT. 3.1.2 Geomagnetic storm 16-25/03/2015 At RO and AT stations (low mid-latitude) the depletion
became really significant (see Figure 4) only on 20 March. It can be
stated that in the early recovery phase the low mid-latitude and the
high mid-latitude regions behave differently. phase. On 25 March the situation was the same as on day 23. The
h’F2 parameter did not show any deviation from the QD curve
during these days. Frontiers in Astronomy and Space Sciences frontiersin.org 3.2.1 rTEC maps of the storms storm (14 November) all stations turned negative except for RO,
where there until 12 UT the TEC variation followed the mean
variation. Later a slight decrease started and lasted until 18 UT. Also
AT behaved differently, because here a TEC enhancement took place
during the whole day. In the recovery phase (15–17 November)
during the day positive phase was detected. On the contrary, during
the nighttime hours on 15 November negative phase, and on
16 November no change was observed at all stations. In Figure 7 the Ratio of the TEC difference (rTEC) maps are
shown for (geomagnetic) main phase of the two storms during the
nighttime hours (18–04:00 UT). These maps show the relative
deviation of TEC from its QD value (10 quietest days of the
month). These rTEC maps clearly present the evolution of the
nighttime negative phase along the examined stations in Europe. In the case of the 2012 storm as shown in Figure 7A, the reduced
electron density region moves from higher to lower latitudes,
reaching its minimum latitude around midnight near the Athens-
Rome line. Meanwhile, it is also nicely seen that the low-midlatitude
Athens-Rome region remains in a positive phase throughout the
night. In the 00:00 and 02:00 UT maps the depleted electron density
zone is nicely drawn out in blue, and it can be seen that JR, PQ, SO
stations are all located within it. In Figure 6 the 2015 March storm can be seen in the TEC data. On the pre-storm day and night (16 March) no deviation from the
reference values can be seen at any stations. In the main phase of the
storm (17 March) significant positive phase is detected during the
day at all stations, but during the night the storm phase is turned
negative at all stations, except at JR where first a positive phase and
then after midnight no deviation can be seen. During the early
recovery phase between 18 and 20 March, a negative effect in TEC is
detected during both day and night at all stations. Interestingly at
RO and AT on 18 March between around 06–12 UT the TEC is
increased with respect to the QD value, then rapidly decreases and
stays decreased during the following 3 days. Frontiers in Astronomy and Space Sciences 3.2 GNSS TEC data During the night the storm turns negative
at all stations, except for RO and AT, where no change was observed. In Figure 5 the TEC values (red) along with their corresponding
references (green) for the 2012 November storm are plotted. Before
the SSC time, on 11 and 12 November a small positive deviation can
be seen during the day at all stations, while during the night on
11 November no significant deviation was observed. On the
following night (12 November) a slight negative phase appeared
at RO, SO and PQ. On 13 November, the day after the SSC, very
impressive positive ionospheric storm phase developed at all stations
during both day and night. In the main phase of the geomagnetic During the remaining days in the recovery phase: on 23 March
no changes at JR and PQ were detected, but a slight increase in
foF2 at SO, RO, AT can be seen around noon. During the night just
JR showed no changes, the others were positive. On 24 March no
changes were observed in foF2 at all stations during the day, but at
night just JR did not show changes, the other stations had positive 09 frontiersin.org frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 FIGURE 5
The meridional evolution of the storm-time GNSS TEC (with red line) with the reference TEC derived from selected 3 quiet days (with green line) are
portrayed for storm 2012. SSC time is marked with a red dashed line. FIGURE 5 FIGURE 5
The meridional evolution of the storm-time GNSS TEC (with red line) with the reference TEC derived from selected 3 quiet days (with green line) are
portrayed for storm 2012. SSC time is marked with a red dashed line. Frontiers in Astronomy and Space Sciences 3.2.1 rTEC maps of the storms On 21 March the same
effect was repeated during the day, but at night JR station showed no
deviation, while all the other station data was negative. On 22 March
(the day after the 2nd SSC) a positive ionospheric storm phase
appeared in TEC during the day at all stations, but during the
nighttime hours there was no effect. Between 23 and 25 March a
slight positive phase can be seen during the day and night at all
stations, except at JR where there is no effect during the night of
23 March. For the 2015 storm, a very strong positive phase (red color in
Figure 7B) is visible at 18 UT on 17 March spreading in a NW-SE
direction, its boundary shows up in yellow along the line connecting
the Black See and North-France (see Figure 7B). SO, PQ to the North
is green, indicating that there is no deviation from the expected
value, while JR is in negative phase. Interestingly, there is a narrow
but
strong
positive-phase
region
at
high
latitudes
(over
Scandinavia). As we move into the night, at 22:00 UT the
negative phase moves towards the equator and the positive phase
region also decreases in strength in the Athens-Rome line. At 00:00,
the depleted plasma has gone even lower, and this is when both
Rome and Athens go into negative phase at night, so the low mid-
latitude region is also under the influence of this negative storm
effect, which lasts until 04:00 UT. The more specific connections are explained in the Discussion
Section 4 below. 10 frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 FIGURE 6
The meridional evolution of the storm-time GNSS TEC (with red line) with the reference TEC of selected 3 quiet days (with green line) are portrayed
for storm 2015. SSC time is marked with red dashed line. FIGURE 6 FIGURE 6
The meridional evolution of the storm-time GNSS TEC (with red line) with the reference TEC of selected 3 quiet days (with green line) are portrayed
for storm 2015. SSC time is marked with red dashed line. 3.3 GUVI measurements most significantly over the JR station. This effect also happens on the
two following days just the same way, with one difference. On
20 March a slight depletion (light blue) showed up also at RO and
AT stations (see Supplementary Figure S3). On 21 March, when the
second SSC happened, another slight decrease follows in the GUVI
data over JR, PQ, SO. During the next day, on 22 March, right over
the JR station a slight decrease can be observed. On 23–25 March
SO, PQ and JR stations are in the slightly depleted O/N2 region (light
blue) during the day. The TIMED satellite GUVI measurements were used to examine
the variation of the daytime thermospheric O/N2 composition
during the evolution of the considered ionospheric storms. In
Figures 8, 9 the three QDs as a reference are shown (Figure 8A;
Figure 9A) and also 3 days from the storm (Figure 8B; Figure 9B) to
present the main features. The whole storm intervals are plotted in
Supplementary Figure S2 and Supplementary Figure S3 (note that
time runs from right to left). For the 2012 November storm the GUVI data presented in
Figure 8 do not show any deviation in the O/N2 ratio from the QD
level over the examined stations during the daytime hours in the pre-
storm phase (12 November). On 13 November (the day after the
SSC) around noon (in LT) a slight increase in the O/N2 (yellow) can
be seen over Europe. In the main phase (when the Dst minimum
value occurred), on 14 November around 12 LT, a significant
decrease (blue) in the GUVI measured O/N2 ratio can be
observed all over Europe. On 15 November there is no deviation
(green), but on 16 and 17 November a slight increase appears in
GUVI data marked with yellow (see Supplementary Figure S2). 3.4 Swarm satellite measurements While ground ionosondes are good at monitoring the
temporal
variation
of
ionospheric
parameters
at
fixed
locations,
LEO
observations
can
provide
pole-to-pole
latitudinal profiles of the considered parameters recorded
during around 45 min. The location of the plasmapause (PP),
more
precisely
the
location
of
ionospheric
phenomena
conjugated with PP, such as the MIT can also be monitored
by LEO satellites (e.g., Heilig and Lühr, 2013; 2018; Heilig et al.,
2022). Swarm observations are only available for the 2015 storm
event. Figure 10 shows the change in the topside electron density
(top panel) for three orbits of Swarm B, each separated by 9.5 h
(in UT). All measurements were taken at around 21 h MLT but
along different meridians. Thus this plot represents primarily
the temporal evolution of the topside electron density at
21 h MLT, but the profiles are also influenced by longitudinal
effects. St. Patrick’s Day storm 2015 (Figure 9): during the pre-storm
phase was on 16 March, the O/N2 ratio shows a decrease (blue) in
the subauroral region, e.g., over JR station. After the SSC, during the
main phase of the geomagnetic and ionospheric storm, on 17 March
in the daytime GUVI data do not show any significant departure
from the reference values. On the contrary, during the following day, on 18 March, a quite
deep depletion was observed in the O/N2 ratio at JR, PQ and SO, but 11 frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 FIGURE 7
The rTEC values are plotted for the nighttime hours (18:00–04:00 UT) on 14 November, 2012 (A), and on 17 March, 2015 (B). FIGURE 7
The rTEC values are plotted for the nighttime hours (18:00–04:00 UT) on 14 November, 2012 (A), and on 17 March, 2015 (B). A deepening density minima (marked by vertical dashed lines
on the Northern Hemisphere) can be observed in both
hemispheres,
moving
progressively
equatorward
as
the
geomagnetic
storm
main
phase
develops. These
are
the
minima of the MIT that is typically well observed in the
nighttime ionosphere during equinoxes. Poleward of MIT, the
erosion by the increased ExB drift continued along the whole day,
while at lower latitudes at the altitude of Swarm B (505–525 km) a
general increase was observed. The presented profiles were all
taken
following
the
SSC
(04:45
UT). The
latest
profile
corresponds to the time of the maximum of the plasmasphere
erosion (most equatorward MIT). Frontiers in Astronomy and Space Sciences 3.4 Swarm satellite measurements In the upper plot (A) reference data for 3 quiet days are shown; in the lower plots (B)
data of 3 selected days from the storm interval are presented, namely, the day of SSC (12 November), the main phase of the ionospheric storm
(13 November), and the main phase of the geomagnetic storm (14 November) (Note: a plot of GUVI data for the whole storm can be found in
Supplementary Figure S1). FIGURE 9
The GUVI measured O/N2 ratio for the 2015 March storm. In the upper plot (A) reference data for 3 quiet days are shown; in the lower plots (B) data of
3 selected days from the storm interval are presented, namely, the day of pre-storm phase (16 March), the main phase of the ionospheric and
geomagnetic storm (17 March), and 1 day from the recovery phase (18 March) (Note: a plot of GUVI data for the whole storm can be found in
Supplementary Figure S2). The GUVI measured O/N2 ratio for the 2015 March storm. In the upper plot (A) reference data for 3 quiet days are shown; in the lower plots (B) data of
3 selected days from the storm interval are presented, namely, the day of pre-storm phase (16 March), the main phase of the ionospheric and
geomagnetic storm (17 March), and 1 day from the recovery phase (18 March) (Note: a plot of GUVI data for the whole storm can be found in
Supplementary Figure S2). As shown in the lower panel, the MLT difference between
Swarm B (red) and C (blue) observations was fairly stable,
around 1.5 h on the average. The MIT position is well-known to
have a strong MLT dependence (e.g., Heilig et al., 2022). The MIT
latitude decreases during afternoon and also during nighttime until
4–6 h MLT. Here this dependence is reflected in the latitude
difference between Swarm B and C observations, Swarm B
observations
are
consequently
equatorward
of
Swarm
C
observations by 2.3° on the average. The time evolution of the MIT minimum position based on
Swarm in-situ observations made on the Northern Hemisphere is
presented in Figure 11. The two colors depict observations of Swarm
B and C satellites. Right after the SSC, MIT started to shift
equatorward. This decrease lasted the whole day, and by the end
of the day MIT approached 45° magnetic latitude. Frontiers in Astronomy and Space Sciences 3.4 Swarm satellite measurements Sharp MIT minima are accompanied by an enhanced electron
temperature (bottom panel). This is a well-known feature of sub-
auroral dynamics and appears mainly as a result of frictional heating
by the intense westward drift that in turn are driven by an intense
substorm (precipitation) associated poleward electric field (e.g.,
Heilig et al., 2022). MIT minimum in this MLT sector is tightly coupled to the
footprint of the plasmapause (Heilig et al., 2022). Thus the
equatorward movement of MIT reflects the shrinking of the
plasmasphere due to the increased magnetospheric convection
and intensified substorm-related sub-auroral electric field (see
also in Supplementary Figure S4). 12 frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 The time evolution of the MIT minimum position based on
As shown in the lower panel, the MLT difference between
FIGURE 8
The GUVI measured O/N2 ratio for the 2012 November storm. In the upper plot (A) reference data for 3 quiet days are shown; in the lower plots (B)
data of 3 selected days from the storm interval are presented, namely, the day of SSC (12 November), the main phase of the ionospheric storm
(13 November), and the main phase of the geomagnetic storm (14 November) (Note: a plot of GUVI data for the whole storm can be found in
Supplementary Figure S1). FIGURE 9
The GUVI measured O/N2 ratio for the 2015 March storm. In the upper plot (A) reference data for 3 quiet days are shown; in the lower plots (B) data of
3 selected days from the storm interval are presented, namely, the day of pre-storm phase (16 March), the main phase of the ionospheric and
geomagnetic storm (17 March), and 1 day from the recovery phase (18 March) (Note: a plot of GUVI data for the whole storm can be found in
Supplementary Figure S2). Berényi et al. 10.3389/fspas.2023.1092850 FIGURE 8
The GUVI measured O/N2 ratio for the 2012 November storm. In the upper plot (A) reference data for 3 quiet days are shown; in the lower plots (B)
data of 3 selected days from the storm interval are presented, namely, the day of SSC (12 November), the main phase of the ionospheric storm
(13 November), and the main phase of the geomagnetic storm (14 November) (Note: a plot of GUVI data for the whole storm can be found in
Supplementary Figure S1). FIGURE 8
The GUVI measured O/N2 ratio for the 2012 November storm. 3.4 Swarm satellite measurements Using these adjustments the MIT at Sopron could have moved
down as low as to magnetic latitude 44.6°, maybe even below 42°. The width of the most equatorward MIT was 3.6° as observed by
Swarm B. This width is shared between the equatorward wall (1.9°)
and the poleward wall (1.7°). This means that on 17 March during
the night MIT (but at least its equatorward wall) should have
reached
the
Sopron
station. Thus,
the
Sopron
ionosonde
observed the depleted plasma associated with the MIT structure. It is also clear that the MIT could not approach Athens, located at
36.1° magnetic latitude. The MIT position is also known to depend on the geographic
longitude (e.g., Heilig et al., 2022). In the American sector MIT
appears a few degrees higher than in the European or Asian
sector. The Equation 5 of Deminov and Shubin (2018) estimates
this difference to 0.98°, while according to the statistical results of
Karpachev et al. (2019), this difference could easily be 2°–3°. Using these adjustments the MIT at Sopron could have moved
down as low as to magnetic latitude 44.6°, maybe even below 42°. The quality of the skymap is very good and corresponds to the
assumption of one drift velocity vector in the area above the station. Estimated vectors are determined reliably. The width of the most equatorward MIT was 3.6° as observed by
Swarm B. This width is shared between the equatorward wall (1.9°)
and the poleward wall (1.7°). This means that on 17 March during
the night MIT (but at least its equatorward wall) should have
reached
the
Sopron
station. Thus,
the
Sopron
ionosonde
observed the depleted plasma associated with the MIT structure. It is also clear that the MIT could not approach Athens, located at
36.1° magnetic latitude. On 14 November around 00:00 UT, a significantly increased
value of the vertical drift velocity component (to the tens of m/s) and
its very rapid changes is added to the above-described effect. The
drift measurement cadence was 15 min. The dramatic changes are
visible in every consequent measurement, it is obvious that the
period of the vertical drift velocity variation is significantly smaller
than 15 min and cannot be studied (described) in more detail due to
the limitations of the drift measurements. During
the
day-time
on
14
November
both
described
manifestations practically disappeared. 3.4 Swarm satellite measurements The value of the vertical
velocity component no longer fluctuates quickly, and the value of the
horizontal (westward) component drops to small values. Here it is
necessary to note that the quality of measured skymaps during the Frontiers in Astronomy and Space Sciences frontiersin.org 3.4 Swarm satellite measurements The lowest
latitude of the MIT position actually observed (by Swarm B) was
46.3°. This observation took place on 18 March at 00:15 UT (21.9 h
MLT) at −43.2° longitude. 13 frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 FIGURE 10
Meridional profiles of the topside electron density (top panel) and electron temperature (bottom panel) observed by Swarm B on 17 and 18 March,
2015. Vertical dashed lines indicate the location of MIT on the Northern hemisphere. FIGURE 10
Meridional profiles of the topside electron density (top panel) and electron temperature (bottom panel) observed by Swarm B on 17 and 18 March,
2015. Vertical dashed lines indicate the location of MIT on the Northern hemisphere. At the time of the minimum observation, MLT at Sopron was
0.72 h. Estimating from the MLT difference between Sopron and
Swarm B (1.1 h) and the rate of decrease (2.3°/1.5 h = 1.53°/h), the
MIT minimum at 0.72 h MLT was located around 45.6°. The
formula (their Equation 4) of Deminov and Shubin (2018) for the
MLT dependence yields a somewhat higher MIT latitude (45.9°). However, these values are already lower than the latitude of
Pruhonice and very close to that of Sopron. vertical motion of the F-layer during the two storm intervals. For the
detailed analysis method of the manual correction see the
Supplementary
section. Now
we
are
going
to
detail
the
phenomena found in the manual drift data for each storm
separately. The first significant manifestations of the 2012 November storm
event can be seen on the drifts in the night from 13 to 14 November
(Figure 12A). On 13 November around 20:30 UT a pronounced
episode of westward drift begins. Roughly until 14 November 10:
00 UT we observe dominantly horizontal westward drifts. The speed
of the observed drifts is significantly higher than the horizontal
speeds observed under quiet conditions (<100 m/s, according to
Kouba and Koucka Knizova, 2012). The detected maximum speeds
exceed even 350 m/s. The MIT position is also known to depend on the geographic
longitude (e.g., Heilig et al., 2022). In the American sector MIT
appears a few degrees higher than in the European or Asian
sector. The Equation 5 of Deminov and Shubin (2018) estimates
this difference to 0.98°, while according to the statistical results of
Karpachev et al. (2019), this difference could easily be 2°–3°. 3.5 Digisonde drift measurements Digisonde drift measurements from Pruhonice station were
analyzed manually in order to determine the true horizontal and 14 frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 FIGURE 11
Evolution of the MIT minimum position (quasi dipole magnetic latitude) on the course of the 2015 event (A); magnetic longitude (B) and MLT (C) of
the in-situ Swarm observations. Red and blue colors depict Swarm B and C observations, respectively. FIGURE 11
Evolution of the MIT minimum position (quasi dipole magnetic latitude) on the course of the 2015 event (A); magnetic longitude (B) and MLT (C) of
the in-situ Swarm observations. Red and blue colors depict Swarm B and C observations, respectively. During day-time 18 March no significant manifestations of the
storm are observed in the drift measurements. During day-time 18 March no significant manifestations of the
storm are observed in the drift measurements. day-time is significantly lower than during the night-time. Therefore, information about the value of westward drift should
be taken with a large margin. In most cases, the daytime skymap
quality is not sufficient to determine the horizontal drift components
with good reliability. The next episode is coming at night 18/19 March. Rapid changes
occur in the vertical component between 23:00 UT and 06:00 UT, and
the amplitude of the vertical drift is significantly greater than values
under normal conditions. In the horizontal component, we observe a
pronounced westward peak between about 18:00 UT and 06:00 UT. The
maximum detected values significantly exceed 200 m/s. The second significant episode is observed during the night of
14/15 November This episode has similar characteristics, again
between approx. 21:00 UT and 03:00 UT there is increased
activity of the vertical component – larger values and rapid
variations. In the horizontal components, a significant increase in
the westward component can be seen again in the time interval
between 23:00 – 08:00 UT. In this episode, the maximal measured
values of the westward component exceed 250 m/s. It can be seen
that the observed manifestations in the case of the second episode
are smaller than in the case of the first. The last significant episode is observed the following night. On
20 March at around 00:00 – 04:00 UT the features described for the
previous episode are repeated. It is interesting that this episode is
significantly shorter than the previous one (roughly half) but the
manifestations are more pronounced (on both the westward and the
vertical components). Frontiers in Astronomy and Space Sciences 3.5 Digisonde drift measurements Drift activity affected by the storm is apparently observable also the
following night (21 March, approx. 00:00 – 05:00 UT) on the westward
component. The maximum detected velocity value is close to 200 m/s. In this case, however, the insufficient number of high-quality
measurements does not allow proving a clear connection to the storm. In the drift measurements of 2015 March storm (Figure 12B),
the first significant manifestations of the storm are observed on
17 March around 20:00 UT. The measurements show very quick
variations of all observed parameters. The variations are
significantly faster than the cadence of measurements and
therefore the fluctuation period cannot be resolved. The value
of the vertical drift velocity component exceeds the extreme
values of +/- 50 m/s. frontiersin.org 4.1 Pre-storm phase In the pre-storm phase of the 2012 storm (11–12 November -
Dstmin = 0 nT; Kpmax = 1,33; AEmax = 134 nT) foF2 and TEC show a
slight positive deviation at all stations, while by the 2015 storm
(16 March - Dstmin = 0 nT; Kpmax = 3,77; AEmax = 300 nT) no
deviation from the reference value is detected. However, the O/N2
data for this storm shows a decrease in the subauroral region already in
this phase (16 March). Earlier studies (e.g., Kane, 1973a; Araujo-Pradere
and Fuller-Rowell, 2002; Kane, 2005; Burešová and Laštovička, 2007)
found a pre-storm feature (positive effect) in foF2 24 h before the SSC,
however other authors doubt its presence (Mikhailov and Perrone, 4 Discussion In
this
section,
we
attempt
to
define
the
cause-effect
relationships in order to gain a more detailed understanding of
the response of the terrestrial ionosphere-plasmasphere system to
geomagnetic storms. Our main focus was on the main phase of the
geomagnetic storm, when during the night extremely depleted Unfortunately, in this phase of the storm, the actual extreme
ionospheric situation (the values of foF2 and h’F2) does not allow
successful realization of drift measurement in most cases. Between 22:00 UT and 01:00 UT successful measurements
are rare. 15 frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 FIGURE 12
The manually corrected digisonde drift measurement of F-region for the 2012 November (A) and 2015 March (B) storm at Pruhonice station. FIGURE 12
The manually corrected digisonde drift measurement of F-region for the 2012 November (A) and 2015 March (B) storm at Pruhonice station. FIGURE 12
The manually corrected digisonde drift measurement of F-region for the 2012 November (A) and 2015 March (B) storm at Pruhonice station. 2009). Burešová
and
Laštovička
(2007)
examined
65
strong
geomagnetic storms from the period of 1995–2005 and found that
about 20%–25% of the storms had a significantly strong pre-storm
effect. Similarly to the finding of Liu et al. (2008), we found a slight pre-
storm enhancement in both TEC and foF2 in the 2012 storm case. Other authors suggested that this effect could be related to some
different
channel
of
penetration
of
the
energy
from
the
interplanetary environment and magnetosphere to the ionosphere
(Danilov and Belik, 1991; 1992; Blagoveshchensky and Kalishin,
2009), but this needs verification. plasma was detected. Case studies are still important to shed light on
deeper relationships that cannot be easily extracted from multi-
event-based statistical investigations. Frontiers in Astronomy and Space Sciences 4.2 The main phase of ionospheric and
geomagnetic storm The upward ion drift could also contribute to the daytime positive
phase (Zhang S.R. et al., 2017a). The diurnal evolution of the positive
phase was different, dual peak can be seen at all stations. The first
peak was at noon with maximum half hour delay, the second was
around 18 h (UT). The second peak was observed (station-hour):
JR-17 h, PQ and SO-17:30, RO and AT-18 h (UT). This could
connected to the propagation of storm-induced electric field from
north to south (e.g., PPEFs). After Kumar and Kumar (2022),
eastward/westward PPEFs are associated with the southward/
northward IMF Bz. The southward turnings of the IMF Bz
component (induce eastward PPEF) can cause electron density
increase during the daytime hours (Kumar and Kumar, 2022). There were two significant southward Bz episodes: around 8 and
14 (UT) during this time, which could support the above detailed
assumption, but this needs verification. g
g
p
y
y
y
In the main phase of the geomagnetic storm in 2012
(14 November) during the daytime extremely depleted electron
density was observed in foF2 at all stations. A similar decrease
was observed also in TEC at all stations but with lower magnitude,
except for AT and RO where it turned into negative only after 12 UT
(see Figures 3, 5). Kane (2005) also found a deep decrease in electron
density over the surrounding stations at midlatitude during the main
phase of a similar storm (Dstmin = −589 nT). He also found that the
magnitude of the Dst is not proportional to the magnitude of the
negative effect in the ionosphere at middle and high-latitude. Pirog
et al. (2006) strengthened it, where the analyzed geographic latitude
range was similar to our case. The decrease of foF2 during the main
phase of a geomagnetic storm is a known response of the ionosphere
in winter, but usually during intense storms (Dstmin<= −100 nT) can
reach lower latitudes (Danilov, 2013). According to the GUVI
measurements around 12 LT a significant decrease of O/N2 ratio
was observed over Europe by GUVI (marked by blue in Figure 8). 4.2 The main phase of ionospheric and
geomagnetic storm Generally, the main cause of a negative ionospheric storm is related
to the depletion in the O/N2 ratio, which is generated by the storm-
time Joule-heating of the auroral thermospheric neutral gas leading
to the formation of composition disturbance zone (with decreased
O/N2 rate) this zone is transported by the enhanced thermospheric
meridional winds (TADs) equatorward (Seaton, 1956; Prölss and
von Zahn, 1974; Mikhailov et al., 1989; Mikhailov et al., 1995; Prölss
et al., 1995; Bunonsanto, 1999; Astafyeva et al., 2015). The third peak
in AE-index with 1,000 nT happened at ~01:00 UT. This energy
input could generate also Joule-heating of the thermosphere and this
strengthen the possibility of our hypothesis. This is strengthening
our earlier assumption that the main reason for the negative phase is
due to the decreased O/N2 ratio transported to lower latitudes (up to
Sopron station) by meridional neutral winds (Berényi et al., 2018). 4.2 The main phase of ionospheric and
geomagnetic storm On the day right after the SSC (on 13 November 2012 -
Dstmin = −29nT; Kpmax = 4,77; AEmax = 674 nT and on 16 frontiersin.org frontiersin.org 10.3389/fspas.2023.1092850 Berényi et al. 10.3389/fspas.2023.1092850 17 March, 2015 - Dstmin = −234nT; Kpmax = 7,7; AEmax = 1570 nT)
we observed a positive ionospheric storm phase at all station in both
foF2 and TEC in both storms. This behavior is consistent with the
average winter ionospheric response (in foF2 and TEC) to
geomagnetic storms observed in previous studies (e.g., Matsuhita,
1959; Prölss, 1995; Buonsanto, 1999; Mendillo, 2006; Danilov, 2013). Astafyeva et al. (2015) examined the St. Patrick’s Day storm’s main
phase (17–18 March) globally with multi-instrument measurements
(e.g., Swarm VTEC, GUVI, GNSS TEC), and they found that the
daytime positive phase during the day of 17 March at lower latitudes
was due to increased O/N2 ratio. This finding was also investigated
and supported by Nayak et al. (2016); Kalita et al. (2016). The AE-
index mostly exceeded 500 nT and increased often up to 1,500 nT
during the day, which indicates the possibility of intense Joule
heating of the thermosphere, which can cause positive storm
phase by two ways: 1) the uplift of the layer through enhanced
meridional winds, and then lift the ionization to greater heights
(Prölss and Zahn, 1974). 2) the downwelling of atomic [O] causing
the increase of O/N2 ratio (see also in Introduction). Nava et al. (2016); Polekh et al. (2017) found also that the positive ionospheric
effects at middle and low latitudes are related to different inputs of
energy (as we see in AE-index; Figure 1B), and as they described the
high latitude heating of the thermosphere travelled toward the
equator with the velocity of 460 m/s during 17 March (along
120°E, see Polekh et al., 2017). Besides, Zhang S.R. et al. (2017a)
found PPEF signatures on dayside over American sector, appearing
as poleward/upward ion drift, and the observations showed
meridional wind equatorward surges during also daytime hours. The upward ion drift could also contribute to the daytime positive
phase (Zhang S.R. et al., 2017a). The diurnal evolution of the positive
phase was different, dual peak can be seen at all stations. The first
peak was at noon with maximum half hour delay, the second was
around 18 h (UT). The second peak was observed (station-hour):
JR-17 h, PQ and SO-17:30, RO and AT-18 h (UT). Frontiers in Astronomy and Space Sciences frontiersin.org 4.2 The main phase of ionospheric and
geomagnetic storm This could
connected to the propagation of storm-induced electric field from
north to south (e.g., PPEFs). After Kumar and Kumar (2022),
eastward/westward PPEFs are associated with the southward/
northward IMF Bz. The southward turnings of the IMF Bz
component (induce eastward PPEF) can cause electron density
increase during the daytime hours (Kumar and Kumar, 2022). There were two significant southward Bz episodes: around 8 and
14 (UT) during this time, which could support the above detailed
assumption, but this needs verification. main phase of the geomagnetic storm, but by the 2012 storm, the
geomagnetic storm’s main phase was delayed by 1 day. 17 March, 2015 - Dstmin = −234nT; Kpmax = 7,7; AEmax = 1570 nT)
we observed a positive ionospheric storm phase at all station in both
foF2 and TEC in both storms. This behavior is consistent with the
average winter ionospheric response (in foF2 and TEC) to
geomagnetic storms observed in previous studies (e.g., Matsuhita,
1959; Prölss, 1995; Buonsanto, 1999; Mendillo, 2006; Danilov, 2013). Astafyeva et al. (2015) examined the St. Patrick’s Day storm’s main
phase (17–18 March) globally with multi-instrument measurements
(e.g., Swarm VTEC, GUVI, GNSS TEC), and they found that the
daytime positive phase during the day of 17 March at lower latitudes
was due to increased O/N2 ratio. This finding was also investigated
and supported by Nayak et al. (2016); Kalita et al. (2016). The AE-
index mostly exceeded 500 nT and increased often up to 1,500 nT
during the day, which indicates the possibility of intense Joule
heating of the thermosphere, which can cause positive storm
phase by two ways: 1) the uplift of the layer through enhanced
meridional winds, and then lift the ionization to greater heights
(Prölss and Zahn, 1974). 2) the downwelling of atomic [O] causing
the increase of O/N2 ratio (see also in Introduction). Nava et al. (2016); Polekh et al. (2017) found also that the positive ionospheric
effects at middle and low latitudes are related to different inputs of
energy (as we see in AE-index; Figure 1B), and as they described the
high latitude heating of the thermosphere travelled toward the
equator with the velocity of 460 m/s during 17 March (along
120°E, see Polekh et al., 2017). Besides, Zhang S.R. et al. (2017a)
found PPEF signatures on dayside over American sector, appearing
as poleward/upward ion drift, and the observations showed
meridional wind equatorward surges during also daytime hours. 4.3 Deep electron density depletion during
the night of 14 November and 17 March Our findings are in good agreement
with the results of previous articles like Nayak et al. (2016), who
examined the equatorward motion of the MIT in the American
sector (reached up to 40° N latitude) and Huang et al. (2018) also
observed obvious westward disturbances in the zonal wind over
central China (Asia, 38.7°N, 111.6°E). From 22:00 -02:00 UT strong
westward neutral wind appeared, and at 03:00 UT a poleward wind
surge of ~100 m/s appeared due to the poleward Coriolis force
arising from the significant westward wind amplitudes (see Zhang
S.R. et al., 2015; Tulasi et al., 2016; Huang et al., 2018). These were
found to be driven by strong SAPS westward ion drift of >500 m/s
peaking in the MIT and were accompanied by > 50 m/s upward ion
drift in the E−and F-layers during SAPS periods at 22:43 UT at
subauroral and midlatitudes (Zhang S.R. et al., 2015; Zhang S.R. et al., 2017a). But at lower latitudes poleward winds might not be
driven by SAPS, most probably these wind changes are associated
with travelling atmospheric disturbances (TADs) originated from
the disturbance source region in the southern hemisphere (Huang
et al., 2018). of MIT lasts until 04:00 UT. Our findings are in good agreement
with the results of previous articles like Nayak et al. (2016), who
examined the equatorward motion of the MIT in the American
sector (reached up to 40° N latitude) and Huang et al. (2018) also
observed obvious westward disturbances in the zonal wind over
central China (Asia, 38.7°N, 111.6°E). From 22:00 -02:00 UT strong
westward neutral wind appeared, and at 03:00 UT a poleward wind
surge of ~100 m/s appeared due to the poleward Coriolis force
arising from the significant westward wind amplitudes (see Zhang
S.R. et al., 2015; Tulasi et al., 2016; Huang et al., 2018). These were
found to be driven by strong SAPS westward ion drift of >500 m/s
peaking in the MIT and were accompanied by > 50 m/s upward ion
drift in the E−and F-layers during SAPS periods at 22:43 UT at
subauroral and midlatitudes (Zhang S.R. et al., 2015; Zhang S.R. et al., 2017a). But at lower latitudes poleward winds might not be
driven by SAPS, most probably these wind changes are associated
with travelling atmospheric disturbances (TADs) originated from
the disturbance source region in the southern hemisphere (Huang
et al., 2018). 4.3 Deep electron density depletion during
the night of 14 November and 17 March According to the observed rTEC values, as we move into the
night at 22:00 UT (note that in reality most possibly this happened
~01:00 UT based on the aforementioned 3 h delay in rTEC), the
zone with depleted electron density moves towards the equator, and
even the positive phase region has been pushed back in the Athens-
Rome line. By 00:00, the depleted zone moved even lower, at this
time both Rome and Athens go into negative phase based on the
foF2 data, indicating that the low mid-latitude region is also under
the influence of MIT. The location of the MIT reached the 44.6°
magnetic latitude (or maybe even below 42°) at 0.72 h MLT based on
Swarm electron density and temperature measurements (Figures 10,
11) and the method described in Heilig et al. (2022). This sharp MIT
minima was also accompanied by increased electron temperature
with more than 5000 K (Figure 10), which was also observed by Liu
et al. (2016), Zhang S.R. et al. (2015, Zhang S.R. et al. (2017a) during
16–19:30 UT on 17 March, which was presumably caused by strong
frictional heating due to large plasma drifts. The equatorward
motion
of
the
minima
of
MIT
is
indicating
that
the
plasmasphere was shrinking (see also Supplementary Figure S4),
and the stations (JR, PQ, SO) went outside the plasmasphere where
the ionosphere was not filled any more from the plasmasphere
leading to extremely decreased electron density (detailed in Section
2). The severely depleted ionosphere was conjugated to the depleted
outer magnetosphere (plasma trough) (see also Heilig et al., 2022). The F-layer drift measurement (at PQ) detected an intense ~500 m/s
speed westward drift from appr. 20–02:00 UT (see Figure 12B)
which is associated with the MIT formation mechanism. The effect After the observation of Zhang S.R. et al. (2017a) the SED plume
over America (Millstone Hill) 20–24 UT can be linked to the positive
phase over Southern Europe (AT and RO) from 18–22 UT: the
westward winds could bring the plasma from Europe to North
America leading to the detected SED plume over there (see Figure 4;
Figure 7B) within a few hours, however, PPEF may also play some
role in its formation (Liu et al., 2016). Investigation of Liu et al. (2016) and model results of Lu et al. (2020) confirm this possibility. 4.3 Deep electron density depletion during
the night of 14 November and 17 March caused by plasmasphere-ionosphere coupling due to the enhanced
downward ExB drift (leading to a maintaining effect by the
plasmasphere), which in the meantime led to a strong erosion of
the plasmasphere. For the 2012 storm the drift measurements from
PQ during that night shows the presence of a strong westward
motion of F-layer plasma (v ~ 350 m/s) indicating the presence of
the ionospheric trough (MIT) (see also Heilig et al., 2022). Thus we
can conclude based on foF2 and drift data that the equatorward
motion of the MIT region (so the PP footprint) was the main cause
of the detected sharp electron density decrease in foF2, GNSS TEC
and rTEC data during the night of the geomagnetic storm main
phase at SO, PQ and JR stations. Another contributor was possibly
also the depleted O/N2 ratio, GUVI measurements show significant
depletion above Europe during daytime, which could corotate with
Earth (by westward winds) into the night sector, causing long-lasting
negative phase (see Figure 8). During the 2015 St. Patrick’s Day storm both foF2 and TEC
decreased at all stations during the night. In the rTEC maps, a very
strong positive phase is visible at 18 UT on 17 March at lower
latitudes, its boundary located along the line connecting the Black
See and N-France (shown by yellow in Figure 7B). SO, PQ are in the
green area, indicating that there is no deviation from the quiet level,
as confirmed by the foF2 value (Figure 4). At the same time, JR is in
negative phase, the foF2 shows significantly decreased electron
density. It should be noted that the start time of the negative
phase in TEC (and rTEC) was delayed by 3 h comparing it with
the foF2 parameter at the same location. Based on the Swarm data
(Figure 11) the MIT has already reached that latitude and it moved
further to lower latitudes later, so it possibly caused the negative
effect at JR. Previous studies indicated that the afternoon appearance
of MIT may be linked to a plasmaspheric plume (see Heilig et al.,
2022). 4.3 Deep electron density depletion during
the night of 14 November and 17 March Interestingly, a strong positive-phase region appeared at high
latitudes (over Scandinavia), which can represent the poleward
boundary of the MIT. The data show positive deviation, partly
because the electron density was quite low in that region during
QDs, while during the subsequent storm a quite significant increase
took place in association with the equatorward movement of
the MIT. To speak about the lower latitudes, previous studies found the
cumulative effect of enhanced meridional wind and changes in the
electric fields, during night of the main phase at the 30–40°N zone. At low latitudes (like RO and AT) the detected variations in the
ionosphere were governed by the superposition of disturbed electric
fields (DDEFs), and the PPEF, which triggered the drop of foF2 in
the evening on 17 March and the morning on 18 March (see
Astafyeva et al., 2016; Jin et al., 2017; Polekh et al., 2017). For
the St. Patrick‘s Day storm, the ionospheric effects of PPEF to low
latitudes and the ionospheric disturbance dynamo (DDEF) were
examined and discussed by several other papers: Le Huy and
Amory-Mazauider, (2008); Lu et al. (2012); Abdu et al. (2013);
Nava et al. (2016); Nayak et al. (2016); Ramsingh et al. (2015); Ram
et al. (2016); Kalita et al. (2016). This could contribute to the
nighttime electron density decrease at lower latitudes (AT, RO). Frontiers in Astronomy and Space Sciences 4.3 Deep electron density depletion during
the night of 14 November and 17 March Significantly eroded ionospheric plasma was detected during the
nighttime hours in the main phase of the geomagnetic storms (from
18:00 UT on 14 November 2012 and from 20:43 on 17 March 2015)
with short duration fade-out of the layers in the ionograms at some
stations (detailed in section 3.1). The foF2 and TEC data show a clear
negative phase at JR, PQ and SO stations in the 2012 November
storm. On the contrary, a strong positive phase mainly in foF2 but
slightly also in TEC at AT and RO stations was detected during the
whole night of 14 November The rTEC maps of storm 2012
(Figure 7A) show the variation during the nighttime hours (18–4:
00 UT) on 14 November These rTEC maps show clearly how the
reduced electron density region moves from the higher latitudes
equatorward, reaching its minimum latitude around midnight along
the Athens-Rome line. Between 00:00 and 02:00 UT, the depleted
electron density region is very nicely drawn out, and it can be seen
that JR, PQ and SO stations are fully located within it. In the
meantime, the low-midlatitude Athens-Rome region remains in the
positive phase throughout the night. Based on our observations and
the findings of previous studies like Yizengaw et al. (2005), we can
assume, that this nighttime positive phase at AT and RO region, is For the 2012 November storm, the GUVI data show a slight
increase in the O/N2 over Europe on 13 November around LT noon
(Figure 8), which could contribute to the positive phase in foF2
(Figure 3). There was a second peak (after the SSC) in AE-index with
~600 nT, which here indicates also the Joule-heating of the
thermosphere. The generated processes here also could contribute
to the positive phase similarly to 2015 storm by two ways. Unfortunately, there is no digisonde drift episode, which could
strengthen the presence of uplifting of the layer or the enhanced
equatorward meridional winds. This assumption needs verification
also with Horizontal Wind Model 2007 (HWM07, Huang et al.,
2018). Kumar and Kumar (2022) found that the PPEFs were
contributors in the electron density increase during the day of
13 November, 2012. Interestingly, this day is the main phase of
the ionospheric storm, and normally it develops on the course of the Frontiers in Astronomy and Space Sciences 17 frontiersin.org 10.3389/fspas.2023.1092850 Berényi et al. of MIT lasts until 04:00 UT. frontiersin.org 4.4 Recovery phase During the recovery phase (on 15–17 November 2012 -
Dstmin = −40;-27;-13nT; Kpmax = 1,33; 2,77; 3; AEmax = 119; 310;
328 nT and 18–20 March 2015 - Dstmin = −200;-99;-81 nT; Kpmax =
6; 5;4,77; AEmax = 1,043; 1,134; 611 nT) all station’s TEC and
foF2 data showed a positive phase in the 2012 storm case, while 18 frontiersin.org frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 they showed a significant negative phase in the 2015 St. Patrick
storm case. Yizengaw et al. (2005) examined a geomagnetic storm
from 31 March, 2001 (Dstmin = −380 nT) and found also an
extended, long-lasting negative phase during the recovery phase. The reason for the long-lasting negative phase during storm
2015 can be the great extension of composition disturbance zone
to low latitudes as suggested by Danilov (2013 and references
therein). These statements are also confirmed by our analysis of
GUVI data. On 18 March, 2015, a quite deep depletion was observed
in
O/N2
ratio
over
Juliusruh
(Figure
9)
accompanied
by
foF2 decrease (Figure 4). The effect also re-occurs on 19 March,
moreover the foF2 parameter data show a slight depletion also at
PQ, SO and RO on 20 March. Nava et al. (2016), Huang et al. (2018)
and Chernigovskaya et al. (2021) also found this depletion in
electron density (18–20 March). They found that, during the
recovery phase the important role in dynamics of the midlatitude
ionosphere may belong to the wave-like thermospheric disturbances
of molecular gas, propagating westward for several days causing
electron density decrease along the trajectories of propagation. Besides, after the investigation of Kumar and Kumar (2019)
another contributor to formation of the negative phase can be
related to DDEFs. As for the 2012 storm, a negative phase
changed to a positive one, which is believed to be caused by
travelling atmospheric disturbances (TADs) (Danilov, 2013). These are generally brought by the same storm-time equatorward
meridional wind discussed above, and have velocity of about 440 m/s
(Mansilla, 2003). These processes uplift the layer to greater altitude,
where the loss rate is lower, beside there is downwelling in atomic [O],
resulting in an increase in electron density. 4.4 Recovery phase On 16–17 November a
slightly increased O/N2 was observed by GUVI (marked with yellow
in Figure 8), which could contribute to the daytime positive effect in
foF2 around 10–15 UT at all investigated stations (Figure 3; see also
article of Illes-Almar et al., 1987; Pirog et al., 2006). they showed a significant negative phase in the 2015 St. Patrick
storm case. Yizengaw et al. (2005) examined a geomagnetic storm
from 31 March, 2001 (Dstmin = −380 nT) and found also an
extended, long-lasting negative phase during the recovery phase. The reason for the long-lasting negative phase during storm
2015 can be the great extension of composition disturbance zone
to low latitudes as suggested by Danilov (2013 and references
therein). These statements are also confirmed by our analysis of
GUVI data. On 18 March, 2015, a quite deep depletion was observed
in
O/N2
ratio
over
Juliusruh
(Figure
9)
accompanied
by
foF2 decrease (Figure 4). The effect also re-occurs on 19 March,
moreover the foF2 parameter data show a slight depletion also at
PQ, SO and RO on 20 March. Nava et al. (2016), Huang et al. (2018)
and Chernigovskaya et al. (2021) also found this depletion in
electron density (18–20 March). They found that, during the
recovery phase the important role in dynamics of the midlatitude
ionosphere may belong to the wave-like thermospheric disturbances
of molecular gas, propagating westward for several days causing
electron density decrease along the trajectories of propagation. Besides, after the investigation of Kumar and Kumar (2019)
another contributor to formation of the negative phase can be
related to DDEFs. As for the 2012 storm, a negative phase
changed to a positive one, which is believed to be caused by
travelling atmospheric disturbances (TADs) (Danilov, 2013). These are generally brought by the same storm-time equatorward
meridional wind discussed above, and have velocity of about 440 m/s
(Mansilla, 2003). These processes uplift the layer to greater altitude,
where the loss rate is lower, beside there is downwelling in atomic [O],
resulting in an increase in electron density. On 16–17 November a
slightly increased O/N2 was observed by GUVI (marked with yellow
in Figure 8), which could contribute to the daytime positive effect in
foF2 around 10–15 UT at all investigated stations (Figure 3; see also
article of Illes-Almar et al., 1987; Pirog et al., 2006). 4.4 Recovery phase is the best for portraying, assessing and understanding the overall
behavior of the near-Earth thermal plasma (ionosphere and
plasmasphere) (Mendillo, 2006). It has to be mentioned here that
TEC plots used for this study (on Figures 5, 6) are based on a TEC
grid map (not from individual GNSS stations). The plotted values
are taken at a grid point (closest to each ionosonde station) of these
TEC maps, so it is a smoothed interpolated value that can also be a
reason for that it is less sensitive than the foF2 measured at
individual stations. In future studies, we found important to compare these results
with some existing models/simulations in order to analyze and test
the aforementioned processes: with NRL ionosphere/plasmasphere
model SAMI3 (Huba et al., 2017), the thermosphere-ionosphere-
electrodynamic general circulation model (TIEGCM, see the article
of Lu et al., 2020), Horizontal Wind Model 2007 (HWM07, Huang
et al., 2018), Global Self-consistent Model of the Thermosphere,
Ionosphere, and Protonosphere (GSM TIP, Ratovsky et al., 2019)
and also with the simulations of Fuller-Rowell et al. (1994) and
Forbes and Roble et al. (1990). It is important mainly for the
2012
storm
case
where
we
do
not
have
Swarm
satellite
measurement,
and
Fabry–Perot
interferometer
neutral
wind
observations to confirm our assumptions about the possible
ionospheric effect drivers. Frontiers in Astronomy and Space Sciences 5 Conclusion During the evolution of two ICME-related geomagnetic
storms, we investigated the extent of disturbances in the
ionosphere. Our supreme aim was to find connections between
geomagnetic storm induced processes in the ionosphere and to
determine the evolution of the effects over Europe through a
meridional chain of digisondes. We used various measurements
(GNSS TEC, GUVI, Swarm, digisonde drift data) to find the
possible cause of the detected features. The results of this study
are summarized below: The 2015 storm was followed by a second SSC by an HSSWS
happened on 21 March at night. During that day, a negative phase
was detected at all stations in both TEC and foF2 but not any
disturbance
was
observed
in
GUVI
data. One
day
later
(Dstmin = −56 nT; Kpmax = 6.33; AEmax = 1078 nT) a slight
positive phase was present both in TEC and in foF2 at all
stations. During the remaining days in the recovery phase,
foF2 data do not show significant departure from the QD values,
while in TEC a slightly positive effect was recorded. The possible
reason for the late recovery phase behavior may be linked to the fact
that the examined European stations were then inside the
plasmasphere
during
the
daytime. Thus
the
effect
of
the
composition change could have been suppressed by other factors. Another possible reason could be that the HSSWS caused electron
density increase during the first 2 days. Nava et al. (2016) found that
the HSSWS event is added some energy into the magnetosphere,
extending the lifetime of the recovery phase. [1] On 13 November 2012: the thermospheric composition
changes and PPEFs contributed to the evolution of the
daytime positive phase at all stations. Drift measurements
from Pruhonice indicate the presence of the ionospheric
trough (MIT) between 20:30–10:00 UT, reflected in the
westward motion of the F-layer plasma with a velocity of
350 m/s, which was the main cause of the depletion in
foF2 during the postmidnight sector at JR, PQ and SO. [2] On 17 March 2015, a daytime positive ionospheric storm
developed at all stations except at JR. We can conclude that the
negative phase at JR is a consequence of the equatorward
motion of the MIT during the daytime. By GUVI data we
assume that the composition change possibly had no effect. 5 Conclusion We
assume that the possible cause of the positive phase was mainly
connected to the Joule-heating of the auroral thermosphere,
which lifted up the plasma through enhanced equatorward
winds to altitudes where chemical loss rate of ions are slow. Besides, after previous studies, one of the cause was most
possibly the PPEFs. The observed virtual height variation
and the vertical drift data along with previous studies also
confirms the uplifting scenario. [1] On 13 November 2012: the thermospheric composition
changes and PPEFs contributed to the evolution of the
daytime positive phase at all stations. Drift measurements
from Pruhonice indicate the presence of the ionospheric
trough (MIT) between 20:30–10:00 UT, reflected in the
westward motion of the F-layer plasma with a velocity of
350 m/s, which was the main cause of the depletion in
foF2 during the postmidnight sector at JR, PQ and SO. [2] On 17 March 2015, a daytime positive ionospheric storm
developed at all stations except at JR. We can conclude that the
negative phase at JR is a consequence of the equatorward
motion of the MIT during the daytime. By GUVI data we
assume that the composition change possibly had no effect. We
assume that the possible cause of the positive phase was mainly
connected to the Joule-heating of the auroral thermosphere,
which lifted up the plasma through enhanced equatorward
winds to altitudes where chemical loss rate of ions are slow. Besides, after previous studies, one of the cause was most
possibly the PPEFs. The observed virtual height variation
and the vertical drift data along with previous studies also
confirms the uplifting scenario. We detail the observed diversities between foF2 and TEC during
the daytime and nighttime of the two storms in the Supplementary
section. The differences between the behavior of the foF2 parameter
and TEC can be linked to the fact that TEC includes the plasma not
only from the topside ionosphere (from above the height of
foF2 parameter - hmF2) but also from plasmasphere. It is
generally accepted that about 2/3 of the integrated TEC comes
from the region above hmF2 (Mendillo, 2006). Due to this fact TEC 19 frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 and AE-index are from OMNIWEB (http://omniweb.gsfc.nasa. gov/form/dx1.html) with a resolution of 1 h and 1 min, as well as
from
WDC
Kyoto
(https://wdc.kugi.kyoto-u.ac.jp/dst_final/
index.html). International Q-days (QD) list (https://wdc.kugi. Acknowledgments We thank the data centers (OMNIWEB and WDC for
Geomagnetism, Kyoto) and the Széchenyi István Geophysical
Observatory at Nagycenk, Hungary, Athens (AT138), Rome
(RO041), Pruhonice (PQ052) and Juliusruh (JR055) station for
supplying high quality ionosonde and digisonde data for the
research. The GUVI instrument was designed and built by The
Aerospace Corporation and The Johns Hopkins University. The
Principal Investigator is Dr. Andrew B. Christensen and the Chief
Scientist and co-PI is Dr. Larry J. Paxton. GNSS RINEX files for
the GNSS-TEC processing are provided from many organizations
listed by the webpage (http://stdb2.isee.nagoya-u.ac.jp/GPS/
GPS-TEC/gnss_provider_list.html). Also we are grateful for
the reviewers for their valuable advices, which improved our
article. The GUVI data used here are provided through support
from the NASA MO&DA program. [11] In future studies, we found important to compare these
results with some existing models/simulations in order to analyze
and test the aforementioned processes. Funding This article is supported by the NKM55/2018 MTA mobility
tender. Global GNSS-TEC data processing has been supported by
JSPS KAKENHI Grant Number 16H06286. The contribution of
VB was supported by OTKA, Hungarian Scientific Research Fund
(grant no. PD 141967) of the National Research, Development
and Innovation Office and by Bolyai Fellowship (GD, no. BO/
00461/21). Furthermore, her work was supported by the GINOP-
2.3.2-15-2016-00003 project. [8] By the 2012 storm” recovery phase, the variation from the
daytime positive to negative phase in foF2 and TEC is probably
due to the change in the neutral composition. [9] The foF2 and TEC data are similar in their main patterns
during the two investigated geomagnetic storms. [10] The combined analysis of Swarm, rTEC, drift and
foF2 measurements give another method beside the existing
ones of previous researchers to monitor the evolution and
movement
of
the
ionospheric
features
like
MIT
(the
ionospheric footprint of the plasmapause), SED and SAPS. This can deepen our understanding of the processes during
different geomagnetic storms, and could be use in the future
in space weather prediction models. Frontiers in Astronomy and Space Sciences Author contributions KB was the main organizer and writer of this article, and
responsible for the manual correction of all ionosonde data used
for the analysis. VB contributed to the structure of the research, the
basic concept, checked the workflow and improved the manuscript. BH performed the analysis of Swarm satellite data, added figures. JU
selected and plotted the GNSS TEC data. DK manually corrected the
digisonde drift measurements and wrote the description for it. ÁK
supported the first author with ideas and fundings. All members
contributed substantially to science discussion and manuscript
development. [6]
Swarm
data
together
with
the
digisonde
drift
measurements support our hypothesis that the extreme
decrease in foF2 and TEC at night of 17 March is related
to the equatorward motion of the MIT along with the intense
SAPS westward ion drift, as MIT went down to 44.6° (or even
below 42°) geomagnetic latitude at 0.72 h MLT. This condition
resulted in an even pronounced electron density drop in the
ionosphere above SO and PQ. The decreased O/N2 ratio also
contributed to the depletion effect. [7] After the observation of previous studies, the SED plume
over America (at subauroral latitudes) between 20 and 24 UT
on 17 March can be linked to the positive phase (SED) from
18–22 UT in foF2 and rTEC over Southern Europe (RO and
AT): the westward winds could bring the plasma from Europe
to North America leading to the detected SED plume over
there. 5 Conclusion kyoto-u.ac.jp/qddays/),
geomagnetic
coordinate
calculator
(https://wdc.kugi.kyoto-u.ac.jp/igrf/gggm/index.html),
GIRO
scaled
digisonde
parameters
(https://giro.uml.edu/didbase/
scaled.php) and for drift data (https://giro.uml.edu/driftbase/),
GUVI data available at http://guvitimed.jhuapl.edu/data_fetch_
l3_on2_gif, Swarm satellite constellation, Langmuir Probe data
are
available
at:
(https://earth.esa.int/eogateway/missions/
swarm/product-data-handbook/preliminary-level-1b-plasma-
dataset). [3] On 14 November 2012: in foF2 data the negative phase is
extremely significant at all stations from 6 UT till the afternoon
hours. Based on the GUVI data, this effect is related to a very
strong decrease in O/N2 ratio during the day. [4] During the night of the 14 November, 2012 we found that
the presence of the midlatitude ionospheric trough (MIT) was
the primary reason for the formation of the negative phase at
JR,
PQ,
SO
stations. The
rTEC
maps
and
the
drift
measurements at PQ also confirms the presence of a
nighttime trough. In addition, based on the GUVI data the
daytime compositional disturbance zone seems to extend
equatorward during the night, contributing to a more
pronounced electron density drop. [5] The location of MIT minima (coincides with the ionospheric
footprint of the PP) is likely reached the latitude of Sopron at
night of 14 November according to the data. On the contrary AT
and RO remained inside the plasmasphere, appearing as increase
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March 2023): The IMF Bz and SW data and also Dst-, Kp-, 20 frontiersin.org Berényi et al. 10.3389/fspas.2023.1092850 Conflict of interest 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. The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest. Publisher’s note The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fspas.2023.1092850/
full#supplementary-material All claims expressed in this article are solely those of the authors
and
do
not
necessarily
represent
those
of
their
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Identification of ATP binding residues of a protein from its primary sequence
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BioMed Central BioMed Central Identification of ATP binding residues of a protein from its primary
sequence Address: Institute of Microbial Technology, Chandigarh, India
Email: Jagat S Chauhan - jagat@imtech.res.in; Nitish K Mishra - nitish@imtech.res.in; Gajendra PS Raghava* - raghava@imtech.res.in
* Corresponding author Email: Jagat S Chauhan - jagat@imtech.res.in; Nitish K Mishra - nitish@imtech.res.in; Gajendra PS Raghava* - raghava@imtech.res.in
* Corresponding author Email: Jagat S Chauhan - jagat@imtech.res.in; Nitish K Mishra - nitish@imtech.res.in; Gajendra PS Raghava* - raghava@imtech.res.in
* Corresponding author Received: 6 August 2009
Accepted: 19 December 2009 This article is available from: http://www.biomedcentral.com/1471-2105/10/434 This article is available from: http://www.biomedcentral.com/1471-2105/10/434 © 2009 Chauhan 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. © 2009 Chauhan et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: One of the major challenges in post-genomic era is to provide functional
annotations for large number of proteins arising from genome sequencing projects. The function
of many proteins depends on their interaction with small molecules or ligands. ATP is one such
important ligand that plays critical role as a coenzyme in the functionality of many proteins. There
is a need to develop method for identifying ATP interacting residues in a ATP binding proteins
(ABPs), in order to understand mechanism of protein-ligands interaction. Results: We have compared the amino acid composition of ATP interacting and non-interacting
regions of proteins and observed that certain residues are preferred for interaction with ATP. This
study describes few models that have been developed for identifying ATP interacting residues in a
protein. All these models were trained and tested on 168 non-redundant ABPs chains. First we
have developed a Support Vector Machine (SVM) based model using primary sequence of proteins
and obtained maximum MCC 0.33 with accuracy of 66.25%. Secondly, another SVM based model
was developed using position specific scoring matrix (PSSM) generated by PSI-BLAST. The
performance of this model was improved significantly (MCC 0.5) from the previous one, where
only the primary sequence of the proteins were used. Conclusion: This study demonstrates that it is possible to predict 'ATP interacting residues' in a
protein with moderate accuracy using its sequence. The evolutionary information is important for
the identification of 'ATP interacting residues', as it provides more information compared to the
primary sequence. This method will be useful for researchers studying ATP-binding proteins. Based
on this study, a web server has been developed for predicting 'ATP interacting residues' in a protein
http://www.imtech.res.in/raghava/atpint/. Page 1 of 9
(page number not for citation purposes) Open Ac
Research article
Identification of ATP binding residues of a protein from its primary
sequence
Jagat S Chauhan, Nitish K Mishra and Gajendra PS Raghava* Open Access Background binding sites is a micro-environment where ATP is cap-
tured and hydrolyzed to ADP, releasing energy which is
utilized by the protein to "do work" by changing the
shape of the protein and/or making the enzyme catalyti-
cally active. These proteins are powered by the hydrolysis
of ATP and convert this chemical energy for mechanical
work [1]. Many ATP Binding proteins are transmembrane Adenosine-5'-triphosphate (ATP) is an important mole-
cule in cell biology as an energy molecule and coenzyme. This molecule interacts with large number of proteins dur-
ing cellular activities and plays a crucial role in various
biological reactions. ATP binding proteins (ABPs) have a
binding site that allows ATP molecule to interact. This Page 1 of 9
(page number not for citation purposes) Page 1 of 9
(page number not for citation purposes) http://www.biomedcentral.com/1471-2105/10/434 http://www.biomedcentral.com/1471-2105/10/434 BMC Bioinformatics 2009, 10:434 proteins and responsible for transport of a wide variety of
substrates (e.g. lipids, sterols) across extra and intracellu-
lar membranes [2]. In summary, ATP binding proteins
have important roles in membrane transport, muscle con-
traction, cellular motility and regulation of various meta-
bolic processes. out cross-validation (LOOCV) [4,20,21]. In this tech-
nique one sequence is used for testing and remaining
sequences for training, this process is repeated in such a
way that each sequence is used once for testing. Though
this is the best technique for evaluation, it is time consum-
ing and computer intensive. Thus, we have used 5-fold
cross-validation in this study where sequences were ran-
domly divided into five sets. One set was used for testing
and the remaining four sets were used for training. This
process was repeated five times in such a way that each set
was used once for testing [9,22]. The final performance
was obtained by averaging the performance of all five sets. Thus it is important to identify ATP binding proteins and
'ATP interacting residues' in these proteins. The experi-
mental identification of residues that interacts with ATP in
a protein is costly and time consuming. Thus there is need
to use alternate techniques such as computational tech-
nique, which have been used successfully for predicting
function of proteins [3-12]. In past, methods have been
developed for the prediction of polynucleotide (DNA/
RNA) interacting residues [8,13,14]. Support Vector Machine (SVM) In most of our studies including this one, we have imple-
mented SVM using SVM light [23], which is freely down-
loadable package from http://svmlight.joachims.org/. SVM is a machine learning approach based on structural
risk minimization principle of statistics learning theory
[24]. The main reason of using this package frequently by
us is that it allows implementing various kernels and
parameters. Position Specific Scoring Matrix (PSSM) We extract 360 ATP binding protein chains from SuperSite
encyclopedia [18]. After removing the redundant
sequences using the program CD-HIT, a total of 267 non-
redundant PDB chains were obtained where no two
sequences have more than 40% identity. In the next step,
we examined these proteins using software Ligand Protein
Contact (LPC) [19] and remove those proteins, which are
not ATP binding proteins according to LPC. Our final
dataset have 168 non-redundant ATP binding protein
chains, available at http://www.imtech.res.in/raghava/
atpint/atpdataset p
f
g
(
)
In this work, PSSM profiles were generated using PSI-
BLAST [25] where a protein sequence was searched against
SWISS-PROT dataset using E-value cut-off of 0.001. This
profile contains the probability of occurrence of each type
of amino acid at each position along with insertion/dele-
tion. Hence, PSSM is considered as a measure of residue
conservation in a given location. This means that evolu-
tionary information for each amino acid is encapsulated
in a vector of 21 dimensions where the size of PSSM
matrix of a protein with M residues is 21 × M, where M is
the length of the target sequence, and each element repre-
sents the frequency of occurrence of each of the 20 amino
acids and one dummy amino acid "X" at one position in
the alignment [16]. Pattern or window size We have generated overlapping patterns (segments) of
different window sizes from 7 to 25 for every ATP binding
protein sequences. If the central residue of the pattern was
a 'ATP interacting residue', then we assigned the pattern as
positive pattern (ATP interacting) otherwise it was
assigned as negative pattern (non-ATP interacting). To
generate the pattern corresponding to the terminal resi-
dues in a protein sequence, we have added (L-1)/2
dummy residue "X" at both terminals of protein (where L
is the length of pattern) [9]. As an example, for window
size 17, we have added 8 "X" before N-terminal and 8 "X"
after C-terminal, in order to create M patterns from
sequence of length M [16,17]. Finally we have obtained a
total of 3056 unique windows/patterns of length 17 out
of 3082 ATP interacting residues. In this study, a systematic attempt has been made to
develop a highly accurate and reliable method for predict-
ing 'ATP interacting residues' in a protein. Initially, Sup-
port Vector Machine (SVM) based models have been
developed using proteins sequence. In the past, it has
been shown that the evolutionary information provided
more information [16,17] than protein sequence, thus we
have also used evolutionary information in the form of
PSSM profile for developing a prediction method. All the
models developed in this study were evaluated using five-
fold cross validation technique. Background Saito et al [15] devel-
oped a general method for predicting nucleotide-binding
sites in a protein, which successfully predicts 31% ATP
binding sites (not ATP interacting residues). To the best of
our knowledge, no prediction method has been devel-
oped for detecting specifically the residues interacting
with ATP from a protein sequence. Thus, there is a need to
develop method for predicting 'ATP interacting residues'
in a protein in order to understand protein-ATP interac-
tion. Page 2 of 9
(page number not for citation purposes) Average accessible surface area The accessible surface area is the surface area of a protein
that is accessible to another protein or ligand. The average
accessible surface area scale values of each amino acid
were obtained from Janin et al [32]. Accuracy y
Percentage of correctly predicted ATP interacting and non-
interacting residues Five-fold cross-validation Evaluation of a newly developed method is a major chal-
lenge for researchers. One of the commonly used tech-
niques for evaluating a model is jack-knife or leave-one- Page 2 of 9
(page number not for citation purposes) Page 2 of 9
(page number not for citation purposes) http://www.biomedcentral.com/1471-2105/10/434 BMC Bioinformatics 2009, 10:434 http://www.biomedcentral.com/1471-2105/10/434 Hydrophobicity The hydrophobicity effect is often a major contributor of
binding affinity between a protein and its ligand. All
Hydrophobicity
calculations
were
obtained
from
Fauchère and Pliska scale [26]. Sensitivity This parameter allows computation of the percentage of
correctly predicted ATP interacting residues. Sensitivity
TP
TP FN
=
+ Residue interface propensities The residue interface propensity is an important feature of
protein binding sites that shows the propensity of each
amino acid residues in the interface area. Residue interface
propensities for each of the 20 amino acids were com-
puted from Jones and Thornton [30]. MCC
TP
TN
FP
FN
TP FP
TP FN
TN FP
TN FN
=
−
+
+
+
+
(
)(
) (
)(
)
(
)(
)(
)(
) Where TP is the number of correctly predicted ATP inter-
acting residues, TN is the number of correctly predicted
non-interacting residues, FP is the number of non-inter-
acting residues predicted as interacting residues and FN is
the number of interacting residues wrongly predicted as
non-interacting. Specificity p
f
y
This parameter allows computation of the percentage of
correctly predicted non-ATP interacting residues. Specificity
TN
TN FP
=
+ Polarity Specificity
TN
TN FP
=
+ Polarity is a separation of electric charge leading to a mol-
ecule having an electric dipole. It results from the uneven
partial charge distribution between various amino acids in
a protein. We have used Grantham R polarity scale values
[28]. Solvation potential Accuracy
TP TN
TP FP TN FN
=
+
+
+
+ The solvation potential is an important parameter of pro-
teins that gives an idea about the preference of amino acid
residues to be exposed to solvent or buried in the inter-
face. For calculation of solvent potential for each amino
acid, we have used Jones et al scale [29]. Matthews's Correlation Coefficient (MCC) Net charge The surface of a protein has a net charge that depends on
the number and identities of the charged amino acids, and
on pH. At a specific pH the positive and negative charges
will balance and the net charge will be zero. Net charge of
amino acid obtained from Klein et al [31]. All the parameters described above are threshold depend-
ent parameters, thus performance of a model depend on
threshold. In order to provide the comprehensive view of
performance of a model, we have calculated these param-
eters on different threshold (range from +1 to -1). Beta-Sheet Sensitivity
TP
TP FN
=
+ Many nucleotide-binding proteins having a P-loop or
phosphate-binding loop, is an ATP binding site motif. It
is a glycine-rich loop preceded by a beta sheet. Thus the
Beta-Sheet may be important feature in the ATP binding
protein. It is obtained from Chou and Fasman scale [27]. Matthews's Correlation Coefficient (MCC) It is the statistical parameter to assess the quality of predic-
tion and to take care of the unbalancing in data. An MCC
equal to 1 is regarded as a perfect prediction, whereas for
a completely random prediction this value becomes 0. Evaluation Parameter For evaluation of the performance of methods, we have
used standard parameter that is routinely used in this type
field. Following is a brief description of the threshold
dependent parameters which was used for evaluation. In this study we have used following seven important
structural feature as SVM input feature - Area under the ROC Curve (AUC) All the measures described above have a common draw-
back that their performance depends on threshold
selected. A known threshold independent parameter is
Receiver Operating Curve (ROC). It is a plot between true
positive proportion (TP/TP+FN) and false positive pro- All the above features, parameter, scale values were taken
from http://www.genome.jp/aaindex[33] and used as
input features in SVM. Page 3 of 9
(page number not for citation purposes) Page 3 of 9
(page number not for citation purposes) http://www.biomedcentral.com/1471-2105/10/434 http://www.biomedcentral.com/1471-2105/10/434 BMC Bioinformatics 2009, 10:434 portion (FP/FP+TN). We have used SPSS package to plot
ROC and calculate AUC. against database of ATP-binding proteins. In order to eval-
uate the performance of BLAST on dataset used in this
study, we have searched each ATP binding protein chain
against remaining ATP binding protein chains. It was
observed that only 71 ATP interacting protein chains
showed similarity (BLAST hit) with other ATP binding
protein chains. Thus, BLAST cannot be used to predict any
ATP interacting residues in 97 ATP binding protein chains
out of 168 chains in our dataset. In order to evaluate per-
formance of BLAST on those protein chains which,
showed similarity, we randomly picked 10 proteins,
which have similarity with other ATP-binding protein
chains. Even on these proteins, the performance of BLAST
was very poor, where the sensitivity was 44% and the
probability of correct prediction was 43.37%. This result
suggests that BLAST is not suitable for predicting ATP
interacting residues in a protein. SVM Modules using single sequence It has been shown in previous studies on nucleotide inter-
acting proteins that they perform best for 17-window size
(pattern length) [16,9]. Thus we have used pattern length
17 for developing our prediction model. All possible over-
lapping peptides of 17 amino acids were generated from
ATP binding proteins/chains, a peptide/pattern is assigned
ATP interacting or positive if the residue at its center is ATP
interacting otherwise it was assigned as negative. After clas-
sifying them as positive and negative patterns, they were
converted into binary patterns. The peptide of length N was
represented by a vector of dimensions N × 21, where each
residue is represented by a vector of dimension 21 (e.g. Ala by 1,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0; Cys by Composition analysis We have analyzed the composition of interacting and
non-interacting residues by computing the amino acid
composition of 17 amino acid pattern where the central
residue was interacting or non-interacting and observed
that the occurrence of Gly and positively charged amino
acids Arg, Lys and His were significantly different in ATP
interacting residue and non-interacting residues (Figure
1) with respective p-value 0.00186, 0.00862, 0.00254 and
0.07941. It can be inferred that the Gly and positively
charged amino acids are important for the interaction
with ATP. Beside above residues, significant composi-
tional difference was observed for following residues Leu,
Pro, Ala and Val with p-value 0.00511, 0.00011, 0.00049
and 0.02253 respectively (Table S1; see in Additional file
1). It shows that non-polar and hydrophobic amino acid
residues such as Val, Leu are important in protein-ATP
binding. We have used two-tailed unpaired T-test to check
the significant in difference of amino acid compositions
in binding and non-binding residues. Page 4 of 9
(page number not for citation purposes) SVM Module based on physico-chemical parameters SVM Module based on physico-chemical parameters
In this study we have also developed SVM module using
various physico-chemical features, which, are important
for protein structure and function. Seven physico-chemi-
cal parameters have been used for this (see methods sec-
tion). We have normalized these parameters [36] before
using them for developing SVM classifier. Performance of
SVM Module based on physico-chemical parameters is
shown in Table 4. As shown in Table 4, performance
(maximum MCC 0.26) was lower than SVM module
based on sequence. Prediction using BLAST One of the methods which is routinely used for predicting
function of a new protein sequence is BLAST. It is a simi-
larity based method and identifies segments/regions in
the query sequences which are similar to the target
sequence. This method can be used to predict ATP inter-
acting residues in a protein by searching a query protein Percentage composition of ATP interacting and non-interacting residues
Figure 1
Percentage composition of ATP interacting and non-interacting residues. Percentage composition of ATP interacting and non-interacting residues
Figure 1
Percentage composition of ATP interacting and non-interacting residues. Percentage composition of ATP interacting and non-interacting residues
Figure 1
Percentage composition of ATP interacting and non-interacting residues. Page 4 of 9
(page number not for citation purposes) Page 4 of 9
(page number not for citation purposes) http://www.biomedcentral.com/1471-2105/10/434 BMC Bioinformatics 2009, 10:434 http://www.biomedcentral.com/1471-2105/10/434 We have achieved 66.25% accuracy with minimum differ-
ence between sensitivity and specificity and MCC 0.33 by
17 window patterns (Table 1) at threshold 0.0. Normally
we select a threshold where sensitivity and specificity are
nearly equal, in order to make the balance between sensi-
tivity and specificity. The performance of SVM model for
window size 17 using single sequence is shown in Figure 2. We have achieved AUC 0.725 which was significantly better
than random (AUC 0.5). 0,1,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0);
contains
20
amino acids and one dummy amino acid "X". Our SVM
module predict a score for each residue in protein (in range
of -1.0 to 1.0), we define a threshold to discriminate ATP
interacting and non-interacting residues. The performance
of SVM module developed using a single sequence for win-
dow size 17 is shown in Table 1. We have also tried various
window sizes from 7 to 25 residues and observed that 17
window size patterns gave better performance (Table 2). Table 1: The performance of SVM model (learning parameter: g:
0.1 c: 2 j: 3) using amino acid sequence (The SVM parameter g
(in RBF kernel), c: parameter for trade-off between training
error & margin, j: cost-factor) Table 1: The performance of SVM model (learning parameter: g:
0.1 c: 2 j: 3) using amino acid sequence (The SVM parameter g
(in RBF kernel), c: parameter for trade-off between training
error & margin, j: cost-factor) SVM Modules using evolutionary information Table 1: The performance of SVM model (learning parameter: g:
0.1 c: 2 j: 3) using amino acid sequence (The SVM parameter g
(in RBF kernel), c: parameter for trade-off between training
error & margin, j: cost-factor)
Thres
Sen
Spec
Accuracy
MCC
-1
100
1.73
50.87
0.09
-0.9
99.87
2.88
51.37
0.11
-0.8
99.67
4.39
52.03
0.13
-0.7
99.25
6.51
52.88
0.15
-0.6
98.36
10.31
54.34
0.18
-0.5
96.89
15.78
56.33
0.22
-0.4
93.75
23.54
58.64
0.24
-0.3
88.94
32.9
60.92
0.26
-0.2
83.4
43.31
63.36
0.29
0
65.53
66.97
66.25
0.33
0.1
54.6
76.99
65.79
0.32
0.2
43.11
84.98
64.04
0.31
0.3
33.94
91.16
62.55
0.31
0.4
25.7
94.7
60.2
0.28
0.5
18.07
97.15
57.61
0.25
0.6
12.64
98.53
55.58
0.22
0.7
8.71
99.18
53.94
0.19
0.8
6.22
99.54
52.88
0.16
0.9
3.93
99.77
51.85
0.13
1
1.87
99.84
50.85
0.08
(Bold values indicate the point where sensitivity and specificity is
roughly equal with maximum MCC.) In the past, it has been shown in several studies that evo-
lutionary information gives more information about a
protein than single sequence [34,35]. In this study the
evolutionary information in the form of PSSM profile has
been used for predicting ATP interacting protein residues. The PSSM profile for each sequence was generated using
PSI-BLAST where sequence was search against of SWISS-
PROT. Each element of PSSM matrix was normalized
before using it as an input feature of SVM module. The
performance of SVM module that was developed using
PSSM, at different threshold, is shown in Table 3. We have
achieved maximum MCC 0.51, with accuracy 75.25% at
threshold -0.1. These results indicated that the evolution-
ary information was very important for predicting ATP
interacting residues as performance increase significantly
from MCC 0.33 to 0.51. The performance of SVM model
based on evolutionary information is shown by ROC plot
in Figure 2 which indicates an improvement from AUC
0.725 to 0.823 and thereby clearly suggesting that SVM
model based on PSSM profile perform better than module
based on single sequence. In the past, it has been shown in several studies that evo-
lutionary information gives more information about a
protein than single sequence [34,35]. In this study the
evolutionary information in the form of PSSM profile has
been used for predicting ATP interacting protein residues. (Bold values indicate the point where sensitivity and specificity is
roughly equal with maximum MCC.) Discussion The ATP interacting proteins play a significant role in sig-
naling pathways, in which ATP is used as a substrate by
kinases that phosphorylate proteins. The identification of
ATP interacting residues is difficult using experimental
techniques. There is a need for developing computational
techniques for identifying ATP interacting residues in a
protein from its protein sequence. Saito et al [15] devel-
oped a general method for predicting binding site using
empirical scores system. Though this method allows Page 5 of 9
(page number not for citation purposes) http://www.biomedcentral.com/1471-2105/10/434 BMC Bioinformatics 2009, 10:434 Table 2: The performance of SVM model using binary pattern of different window size patterns. Window size
Threshold
Sensitivity
Specificity
Accuracy
MCC
parameters
7
0
60.99
64.47
62.73
0.25
g:0.1 c:1 j:1
9
0
61.25
67.41
64.33
0.29
g:0.1 c:1 j:1
11
0
63.2
64.01
63.61
0.27
g:0.1 c:3 j:3
13
0
63.4
64.81
64.11
0.28
g:0.1 c:2 j:1
15
0
61.62
67.11
64.37
0.29
g:0.1 c:1 j:1
17
0
65.53
66.97
66.25
0.33
g:0.1 c:2 j:3
19
0
61.98
69.44
65.71
0.32
g:0.1 c:1 j:1
21
0
60.86
69.96
65.41
0.31
g:0.1 c:1 j:1
23
0
63.65
68.52
66.08
0.32
g:0.1 c:2 j:2
25
0
63.32
70.26
66.79
0.34
g:0.1 c:2 j:1
(Bold values indicate the values where accuracy highest and sensitivity and specificity are roughly equal) Table 2: The performance of SVM model using binary pattern of different window size patterns. (Bold values indicate the values where accuracy highest and sensitivity and specificity are roughly equal) section we obtained poor performance both in terms of
sensitivity and probability of correct prediction. Thus the
routinely used similarity search technique like BLAST is
not suitable for this problem. In the next step, we examine
motif-based techniques for predicting ATP interacting res-
idues. We search motifs using FingerPRINTScan [38] in
168 ATP binding protein chains used in this study and got
motifs only in 54 proteins. No motif was found in the
remaining 114 proteins. These motifs only cover around
11% ATP interacting residues (Table S2; see in Additional
file) and no common motif was found in ATP binding
protein (Table S3; see in Additional file). These results
shows that motifs based method cannot be used for iden-
tifying of ATP interacting residues. detection of ATP binding sites on a protein with low accu-
racy but provides no information about ATP interacting
residues. Page 6 of 9
(page number not for citation purposes) Discussion There are methods, which allow identifying ATP
interacting residues in a protein if its structure is known
[19,37]. These methods are basically assignment method,
which assign ATP interacting residues in a PDB file. In this
study an attempt has been made to predict ATP interacting
residue in a protein with high accuracy. One of the obvi-
ous question arise can we used existing techniques for pre-
dicting ATP interacting residues. First we used BLAST for
predicting ATP interacting residues. As shown in result ROC plot shows performance of SVM modules developed
using amino acid sequence and PSSM profile
Figure 2
ROC plot shows performance of SVM modules devel-
oped using amino acid sequence and PSSM profile. This study is a systematic attempt to understand and pre-
dict ATP interacting residues in a protein First we analyzed
ATP interacting residues and its neighbors, and found that
there is a significant difference in interacting and non-
interacting residues. This means ATP interacting residues
can be predicted using any machine leaning techniques. It
has been shown in previous studies that SVM perform bet-
ter than other artificial intelligence technique particularly
on small dataset. Thus SVM based model has been devel-
oped for predicting ATP interacting residues in a protein
from its primary structure and achieved reasonable accu-
racy. As PSSM based evolutionary information provide
better information [9], hence we also made an attempt to
develop method using evolutionary information for pre-
dicting ATP interacting residues. The performance of SVM
module increases significantly when evolutionary infor- ROC plot shows performance of SVM modules developed
using amino acid sequence and PSSM profile
Figure 2
ROC plot shows performance of SVM modules devel-
oped using amino acid sequence and PSSM profile. ROC plot shows performance of SVM modules developed
using amino acid sequence and PSSM profile
Figure 2
ROC plot shows performance of SVM modules devel-
oped using amino acid sequence and PSSM profile. Page 6 of 9
(page number not for citation purposes) Page 6 of 9
(page number not for citation purposes) http://www.biomedcentral.com/1471-2105/10/434 BMC Bioinformatics 2009, 10:434 mation is in place of single sequence. This demonstrates
that evolutionary information is important for predicting
ATP interacting residues. In this study we used window
size 17; the question arises why we have used 17. Discussion Though
window size 17 is frequently used in prediction of second-
ary structure of interacting residues, it does not mean that
window size 17 is applicable to each problem. One
should try different window size in order to find out opti-
mize window size for a given problem. We try various
window sizes from 7 to 25 residues for predicting ATP
for window size 17. Although accuracy of binary pattern
of 25 window size is better than 17 but difference in sen-
sitivity and specificity is much higher. This means that
window size 17 is most suitable for predicting ATP inter-
acting residues. This is first study of this kind so it is diffi-
cult to compare its performance with existing methods. We hope this study will be useful for researchers working
in this area. There is a high probability that other
researcher will work on this problem and will develop bet-
ter method. Conclusion
In this study we have develop method, for the first time,
Table 3: The Performance of SVM model (Learning Parameter:
g: 0.01 c: 4 j: 1) Using PSI-BLAST Profile
Threshold
Sensitivity
Specificity
Accuracy
MCC
-1
98.52
15.47
57
0.25
-0.9
97.93
20.43
59.18
0.29
-0.8
96.55
25.2
60.87
0.31
-0.7
95.07
30.68
62.88
0.34
-0.6
93.27
36.96
65.11
0.37
-0.5
90.87
43.59
67.23
0.39
-0.4
88.44
50.1
69.27
0.42
-0.3
85.48
56.34
70.91
0.44
-0.2
82
63.34
72.67
0.46
0
74.44
75.79
75.11
0.50
0.1
70.01
80.39
75.2
0.51
0.2
65.41
84.4
74.9
0.51
0.3
60.32
87.78
74.05
0.5
0.4
55.85
90.6
73.23
0.5
0.5
51.22
92.97
72.09
0.49
0.6
46.39
94.58
70.48
0.47
0.7
40.21
96.12
68.17
0.44
0.8
34.63
97.44
66.03
0.41
0.9
28.65
98.03
63.34
0.37
1
21.78
98.92
60.35
0.33
(Italic-bold values indicate the point where sensitivity and specificity is
roughly equal and Bold values indicate point where maximum
Accuracy and MCC.)
Table 4: The Performance of SVM model (Learning Parameter:
g: 0.001 c: 4 j: 1) Using seven physiochemical properties. Discussion (Italic-bold values indicate the point where sensitivity and specificity is
roughly equal and Bold values indicate point where maximum
Accuracy and MCC.) mation is in place of single sequence. This demonstrates
that evolutionary information is important for predicting
ATP interacting residues. In this study we used window
size 17; the question arises why we have used 17. Though
window size 17 is frequently used in prediction of second-
ary structure of interacting residues, it does not mean that
window size 17 is applicable to each problem. One
should try different window size in order to find out opti-
mize window size for a given problem. We try various
window sizes from 7 to 25 residues for predicting ATP
interacting residues and achieved maximum performance mation is in place of single sequence. This demonstrates
that evolutionary information is important for predicting
ATP interacting residues. In this study we used window
size 17; the question arises why we have used 17. Though
window size 17 is frequently used in prediction of second-
ary structure of interacting residues, it does not mean that
window size 17 is applicable to each problem. One
should try different window size in order to find out opti-
mize window size for a given problem. We try various
window sizes from 7 to 25 residues for predicting ATP
interacting residues and achieved maximum performance Discussion Threshold
Sensitivity
Specificity
Accuracy
MCC
-0.9
93.68
18.71
56.19
0.19
-0.8
92.35
22.05
57.2
0.2
-0.7
90
26.85
58.43
0.22
-0.6
87.35
31.23
59.29
0.22
-0.5
84.3
36.72
60.51
0.24
-0.4
80.43
41.79
61.11
0.24
-0.3
76.32
47.35
61.84
0.25
-0.2
72.45
52.68
62.57
0.26
-0.1
68.25
57.78
63.01
0.26
0
63.18
62.95
63.06
0.26
0.1
57.95
68.08
63.01
0.26
0.2
52.55
72.75
62.65
0.26
0.3
47.15
77.05
62.1
0.25
0.4
41.56
80.86
61.21
0.24
0.5
36.56
84.77
60.66
0.24
0.6
31.79
87.42
59.6
0.23
0.7
26.99
89.57
58.28
0.21
0.8
23.15
92.55
57.85
0.22
0.9
19.44
94.34
56.89
0.21
(Bold values indicate point where maximum Accuracy and MCC.) Table 4: The Performance of SVM model (Learning Parameter:
g: 0.001 c: 4 j: 1) Using seven physiochemical properties. Discussion Threshold
Sensitivity
Specificity
Accuracy
MCC
-0.9
93.68
18.71
56.19
0.19
-0.8
92.35
22.05
57.2
0.2
-0.7
90
26.85
58.43
0.22
-0.6
87.35
31.23
59.29
0.22
-0.5
84.3
36.72
60.51
0.24
-0.4
80.43
41.79
61.11
0.24
-0.3
76.32
47.35
61.84
0.25
-0.2
72.45
52.68
62.57
0.26
-0.1
68.25
57.78
63.01
0.26
0
63.18
62.95
63.06
0.26
0.1
57.95
68.08
63.01
0.26
0.2
52.55
72.75
62.65
0.26
0.3
47.15
77.05
62.1
0.25
0.4
41.56
80.86
61.21
0.24
0.5
36.56
84.77
60.66
0.24
0.6
31.79
87.42
59.6
0.23
0.7
26.99
89.57
58.28
0.21
0.8
23.15
92.55
57.85
0.22
0.9
19.44
94.34
56.89
0.21
(Bold values indicate point where maximum Accuracy and MCC.) Table 3: The Performance of SVM model (Learning Parameter:
g: 0.01 c: 4 j: 1) Using PSI-BLAST Profile
Threshold
Sensitivity
Specificity
Accuracy
MCC
-1
98.52
15.47
57
0.25
-0.9
97.93
20.43
59.18
0.29
-0.8
96.55
25.2
60.87
0.31
-0.7
95.07
30.68
62.88
0.34
-0.6
93.27
36.96
65.11
0.37
-0.5
90.87
43.59
67.23
0.39
-0.4
88.44
50.1
69.27
0.42
-0.3
85.48
56.34
70.91
0.44
-0.2
82
63.34
72.67
0.46
0
74.44
75.79
75.11
0.50
0.1
70.01
80.39
75.2
0.51
0.2
65.41
84.4
74.9
0.51
0.3
60.32
87.78
74.05
0.5
0.4
55.85
90.6
73.23
0.5
0.5
51.22
92.97
72.09
0.49
0.6
46.39
94.58
70.48
0.47
0.7
40.21
96.12
68.17
0.44
0.8
34.63
97.44
66.03
0.41
0.9
28.65
98.03
63.34
0.37
1
21.78
98.92
60.35
0.33
(Italic-bold values indicate the point where sensitivity and specificity is
roughly equal and Bold values indicate point where maximum
Accuracy and MCC.) Table 4: The Performance of SVM model (Learning Parameter:
g: 0.001 c: 4 j: 1) Using seven physiochemical properties. Table 3: The Performance of SVM model (Learning Parameter:
g: 0.01 c: 4 j: 1) Using PSI-BLAST Profile (Bold values indicate point where maximum Accuracy and MCC.) for window size 17. Although accuracy of binary pattern
of 25 window size is better than 17 but difference in sen-
sitivity and specificity is much higher. This means that
window size 17 is most suitable for predicting ATP inter-
acting residues. This is first study of this kind so it is diffi-
cult to compare its performance with existing methods. We hope this study will be useful for researchers working
in this area. There is a high probability that other
researcher will work on this problem and will develop bet-
ter method. Page 7 of 9
(page number not for citation purposes) Additional file 1 Additional file 1
Table S1. P-value for compositional difference in ATP interacting residue
and non-interacting residue. Table S2, S3. Motifs based analysis. Fprint-
Scan AccNumber list and its frequency. Probability of percentage coverage
of FprintScan in ATP interacting residue. Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2105-10-434-S1.DOC] 17. Kaur H, Raghava GPS: A neural-network based method for pre-
diction of gamma-turns in proteins from multiple sequence
alignment. Protein Sci 2003, 12:923-929. Table S1. P-value for compositional difference in ATP interacting residue
and non-interacting residue. Table S2, S3. Motifs based analysis. Fprint-
Scan AccNumber list and its frequency. Probability of percentage coverage
of FprintScan in ATP interacting residue. Click here for file g
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important for predicting the structural component of a
protein, first time we have demonstrated that the evolu-
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the best model trained on our data set. This server will
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https://link.springer.com/content/pdf/10.1007%2Fs00384-018-3108-5.pdf
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English
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Meta-analysis of the prognostic value of CpG island methylator phenotype in rectal cancer
|
International journal of colorectal disease
| 2,018
|
cc-by
| 4,497
|
International Journal of Colorectal Disease (2018) 33:995–1000
https://doi.org/10.1007/s00384-018-3108-5 International Journal of Colorectal Disease (2018) 33:995–1000
https://doi.org/10.1007/s00384-018-3108-5 REVIEW REVIEW REVIEW 1
ABMU Singleton Hospital, Sketty Lane, Swansea, Wales SA2
8QA, UK Abstract bst act
Purpose The pathological and prognostic importance of CpG island methylator phenotype (CIMP) in rectal cancer, as a sub-
population of colorectal cancer, is unknown. A meta-analysis was preformed to estimate the prognostic significance of CIMP in
rectal cancer. Methods A systematic search was performed of PubMed, Embase, MEDLINE, PubMed Central, and Cochrane electronic
databases for articles pertaining to CIMP and rectal cancer. Articles were analysed and data extracted according to PRISMA
standards. Results Six studies including 1529 patients were included in the analysis. Following dichotomisation, the prevalence of CIMP-
positive tumours was 10 to 57%, with a median of 12.5%. Meta-analysis demonstrated the pooled odds ratio for all-cause death
for CIMP-positive tumours vs CIMP-negative tumours was 1.24 (95% CI 0.88–1.74). Z test for overall effect was 1.21 (p = 0.23). Heterogeneity between the studies was low (X2 5.96, df 5, p = 0.31, I2 = 16%). A total of 15 different loci were used for assessing
CIMP across the studies, with a median of 6.5 loci (range 5–8). Conclusions No significant association between CIMP and poor outcomes in rectal cancer was demonstrated. There was a high
degree of heterogeneity in CIMP assessment methodologies and in study populations. Rectal cancer datasets were frequently not
extractable from larger colorectal cohorts, limiting analysis. Keywords Methylation . CIMP . Rectal cancer . Biomarkers Keywords Methylation . CIMP . Rectal cancer . Biomarkers Meta-analysis of the prognostic value of CpG island methylator
phenotype in rectal cancer R. F. Kokelaar1
& H. Jones1 & J. Beynon1 & M. E. Evans1 & D. A. Harris1 Accepted: 14 June 2018 /Published online: 20 June 2018
# The Author(s) 2018 Introduction techniques and the judicious use of neoadjuvant chemoradia-
tion (nCRT) [3]. The potential for organ preservation is also
increasingly accepted due to the introduction of minimally
invasive surgery and ‘watch-and-wait’ strategies of non-
operative management [4–7]. Despite advances, there is how-
ever an inability to determine which patients may benefit from
one treatment modality or another based upon anything other
than traditional radiological and histopathological staging [8,
9]. As the era of personalised medicine advances, there is an
increasing need for molecular biomarkers that will aid deci-
sion making in the preoperative phase. Colorectal cancer (CRC) is a significant health problem due to
high prevalence and mortality, representing the third most
common cause of cancer death in the USA [1]. Rectal cancer
(RC) as a sub-population of approximately 30% of CRC poses
additional significant mortality, morbidity, and management
challenges, due to the anatomical confines of the bony pelvis
and proximity of significant neurovascular structures and oth-
er organ systems [2]. The management of rectal cancers is
currently undergoing a paradigm shift; previously inoperable
locally advanced and locally recurrent rectal cancers are in-
creasingly rendered operable due to improved operative The molecular and genetic aberrations that underlie CRC
carcinogenesis are complex and not fully understood, al-
though there is a consensus that there are divergent processes
responsible for tumour development at different sites through-
out the colon and rectum [10]. DNA methylation is one epi-
genetic process implicated in CRC, as well as other cancers,
and CpG island methylator phenotype (CIMP) has drawn in-
terest as a potential mechanism underlying both carcinogene-
sis and as a potential biomarker [11, 12]. CIMP, however, has * R. F. Kokelaar
rory.kokelaar@gmail.com Int J Colorectal Dis (2018) 33:995–1000 996 primarily been associated with carcinogenesis in the right co-
lon that is characterised by hypermethylation and microsatel-
lite instability (MSI) (the serrated pathway), rather than the
traditional chromosomal instability pattern typical of other
sites in the colon and rectum [13–15]. Despite the preponder-
ance in the right colon, CIMP tumours are known to occur in
the rectum, although the clinical significance of this molecular
tumour type occurring at this site is poorly understood, al-
though some authors have suggested they represent a poor-
prognostic subgroup [16, 17]. Statistical analysis Analysis was conducted using RevMan statistical software (v
5.3. Pub: The Cochrane Collaboration). Heterogeneity was
calculated as the chi-squared value (X2, df −1) and the I2
statistic [24]. Overall effect was calculated by Z test with sig-
nificance set as p < 0.05, based on meta-analysis employing
Mantel-Haenszel odds ratio (OR) for CIMP status and 5-year
overall survival rates with 95% confidence intervals (CI). Definition of CIMP No consensus definition of CIMP exists across the published
literature. Studies variable report a binary CIMP +ve or -ve, or
groupings of CIMP-high(-H), CIMP-intermediate(-I), CIMP-
low(-L), or CIMP-negative(-N). For the purposes of this re-
view, dichotomisation of different CIMP classifications was
performed so to classify results into CIMP +ve (including
CIMP +ve, CIMP-high, and CIMP-intermediate groups) and
CIMP -ve (including CIMP-low and CIMP -ve/-N). This pro-
cess is keeping with that of other authors [21]. Results All types of study were included in the analysis, although
reviews, meta-analysis, and book chapters were excluded. Exclusion criteria were single-locus or gene methylation stud-
ies, studies where DFS and OS outcomes were not reported,
and studies where the rectum was not defined as in identifiable
cohort within colorectal cancers. Studies where surgery was
not performed with curative intent were also excluded. Electronic search yielded 203 original articles, once duplica-
tions had been removed. No additional studies were found in
the grey literature. Following review of abstracts, 160 articles
were excluded on the basis of not relating to colorectal cancer
(2), CIMP not basis of analysis or not assessed (89), single
locus/gene methylation only (3), survival outcomes not
assessed (59), and being review articles (7). The remaining
43 articles then underwent full manuscript screening for eligi-
bility, where a further 37 were excluded on the basis of the
colon and rectum being assessed as a single cohort (13), and
the rectum not being defined as an individual cohort separable
from the left or distal colon (24) (Fig. 1). The remaining six
studies were included, and baseline and CIMP/outcomes data
extracted (Table 1) [16, 17, 25–28]. Introduction Many of the studies that have
examined the role of methylation in RC or indeed CRC have
focused on a single gene locus or have relied upon small
cohorts, making outcomes interpretation challenging [18,
19]. The aim of this paper is to review and meta-analyse the
prognostic value of CIMP in adenocarcinoma in the rectum. institution, country, total number of patients, sex, TNM stag-
ing, method of determining CIMP, and methodology. Numbers and/or percentages of patients expressing CIMP
was extracted, as were DFS and OS. Search protocol Two reviewers independently assessed the quality of included
studies using the Newcastle-Ottawa (N-O) scale [22]. A score
of 6 out of 9 criteria fulfilled on assessment of selection, com-
parability, and outcome was used as a cutoff for inclusion in
the analysis, which was itself conducted according to
PRISMA guidelines [23]. An online search was performed to retrieve original research
articles where CIMP was assessed in rectal adenocarcinoma
specimens, and where outcomes data were assessed (disease-
free and overall survival (DFS and OS)). PubMed, Embase,
MEDLINE, PubMed Central, and Cochrane databases were
searched using the Boolean terms (CpG island methylator
phenotype OR CIMP) AND (cancer OR carcinoma OR ade-
nocarcinoma OR tumor OR tumour) AND (colorectal OR
rectal) AND (prognosis OR outcome). A cutoff for inclusion
was January 2018, results were compiled in a reference man-
ager, and duplicates were removed. The grey literature was
examined for additional contributions. Study design and
search strategy was registered pre-emptively at PROSPERO
(registration number CRD42018099569) [20]. Data extraction Two reviewers independently applied the exclusion criteria to
retrieved abstracts, and discrepancies were agreed by consen-
sus. Data was extracted by one author from full-text manu-
scripts, and each dataset was verified by an independent re-
viewer. Baseline data for each study included author, date, Int J Colorectal Dis (2018) 33:995–1000 997 Retrieved Arcles: 203
Screened for eligibility:
43
Analysis: 6
Excluded based on full text assessment: 37
Large bowel assessed as single cohort: 13
Rectum not separated from le / distal colon: 24
Excluded based on tle/abstract: 160
Not colorectal cancer - 2
Non-CpG methylaon focus of analysis – 89
Single gene methylaon only – 3
Not related to prognosis – 59
Fig. 1 Consort diagram showing selection criteria for inclusion in the
analysis Jo (II–IV). The approximate mean age of the patients was 62–
65 years, and 68% were male. Three studies specify that all
patients were nCRT naïve, two make no statement regarding
nCRT prior to tissue sampling, and one specifies all patients
received nCRT (Table 1). Excluded based on tle/abstract: 160
Not colorectal cancer - 2
Non-CpG methylaon focus of analysis – 89
Single gene methylaon only – 3
Not related to prognosis – 59 CpG island methylation status was assessed in a median of
6.5 loci (range 5–8), with a total of 15 different genes
employed across all studies. CIMP characterisation is de-
scribed in all papers, two employing a +ve/ve strategy and
the others employing variable strategies of CIMP-H/-I/-L/-N
(Table 2). Following dichotomisation, the prevalence of CIMP
+ve tumours ranged from 10 to 57%, with a median of 12.5%. Two studies reported a positive association between CIMP
and overall poor survival, and four reported no association. Meta-analysis demonstrated that the pooled OR for all-
cause death for CIMP +ve tumours versus CIMP -ve tumours
was 1.24 (95% CI 0.88–1.74). Z test for overall effect was
1.21 (p = 0.23) (Fig. 2). Heterogeneity between the studies
was low (X2 5.96, df 5, p = 0.31, I2 = 16%). Single and mul-
tivariate analysis of individual genes used in assessing CIMP
did not demonstrate any association with outcomes. Pooled
analysis of clinicopathological factors assessed in relationship
to CIMP and survival was not possible due to inconsistent
reporting and non-extractable data. Meta-analysis demonstrated that the pooled OR for all-
cause death for CIMP +ve tumours versus CIMP -ve tumours
was 1.24 (95% CI 0.88–1.74). Data extraction Z test for overall effect was
1.21 (p = 0.23) (Fig. 2). Heterogeneity between the studies
was low (X2 5.96, df 5, p = 0.31, I2 = 16%). Single and mul-
tivariate analysis of individual genes used in assessing CIMP
did not demonstrate any association with outcomes. Pooled
analysis of clinicopathological factors assessed in relationship
to CIMP and survival was not possible due to inconsistent
reporting and non-extractable data. Fig. 1 Consort diagram showing selection criteria for inclusion in the
analysis The median N-O score for the included studies was 7
(range 6–7). No studies were excluded on the basis of quality
assessed by this metric. Five studies were based on retrospec-
tive cohorts and one was based on a cohort extracted from an
ongoing phase III clinical trial [17]. CIMP analysis was per-
formed on resected specimens in all but one study (Jo [17];
pre-treatment biopsies) and all employed poly-locus method-
ologies. Two studies assessed methylation in CRC but had
extractable primary outcome data for a RC sub-cohort, al-
though extractable clinicopathological data was often not
available for this sub-cohort [16, 27]. The remaining four
studies only assessed RC. Discussion This study demonstrates the significant heterogeneity in
methods used to assess CIMP in RC, but that across a range
of gene panels, CIMP has not been shown to be associated
with worse overall survival in rectal cancer. Confidence inter-
vals for individual studies were broad and each range crossed
OR 1.0. Of the two studies that did report an increased risk of
death, the 5-year survival rates were reported as 63.2 and 25%
respectively, although numbers in the second group was ex-
tremely small (n = 5). DFS was not reported consistently or
extractably across studies and was therefore not meta-
analysed, although again, there was conflicting data presented
between studies. Jo reports that although there is no difference The six studies included 1529 patients with a mean sample
size of 254 (range 78–864). Only one paper reported a cohort
of less than 100 (Kim; 87). Each of the studies included pa-
tients with AJCC stage I–IV tumours, except Williamson and Table 1 Characteristics
of included studies
Reference
No. of
patients
Study
interval
Age range
(mean)
Men
(%)
AJCC
nCRT
N-O
score
Samowitz et al. 2009 [24]
864
1997–2001
30–79 (nr)
nr
I–IV
nr
6
Jo et al. 2011 [17]
150
2004–2006
nr (61)
71
II–IV
No
6
Bae et al. 2013 [16]
168
2004–2006
36–87 (62)
67
I–IV
No
7
Williamson et al. 2017 [25]
160
2002–2011
nr (65)
71
II–IV
Yes
7
Kim et al. 2017 [26]
87
2006–2007
31–88 (65)
59
I–IV
nr
7
Kokelaar et al. [27]
100
2010–2013
24–89 (71)
70
I–IV
No
7
nr not recorded/extractable Int J Colorectal Dis (2018) 33:995–1000 998 Table 2
CIMP methodologies
Reference
CIMP markers
CIMP classification
CIMP association
with outcome
Samowitz et al. 2009 [24]
hMLH, MINT1, MINT2,
MINT31, CDKN2A
CIMP-positive vs CIMP-negative
CIMP-high poorer survival
(p < 0.040)
Jo et al. 2011 [17]
SOCS1, RUN3, NEUROG1,
IGF2, CACNA1G
CIMP-positive vs CIMP-negative
No statistical significance
(p > 0.050)
Bae et al. 2013 [16]
hMLH1, CDKN2A, SOCS1,
RUNX3, NEUROG1, IGF2,
CACNA1G, CRABP1
CIMP-high vs CIMP-low vs
CIMP-negative
CIMP-high poorer survival
(p = 0.019)
Williamson et al. 2017 [25]
hMLH1, MINT1, SOCS1,
NEUROG1, THBD, HAND1,
ADAMTS1, IGFBP3
CIMP-high vs CIMP-intermediate
vs CIMP-low
No statistical significance
(p > 0.050)
Kim et al. 2017 [26]
SOCS1, RUN3, NEUROG1,
IGF2, CACNA1G
CIMP-high vs CIMP-low vs
CIMP-negative
No statistical significance
(p > 0.050)
Kokelaar et al. Discussion [27]
hMLH1, MINT1, SOCS1,
NEUROG1, THBD, HAND1,
ADAMTS1, IGFBP3
CIMP-high vs CIMP-intermediate
vs CIMP-low
No statistical significance
(p > 0.050) The factors accounting for the differences in these obser-
vations are likely to be multifaceted, although differences in
patient cohorts and lack of statistical power make interpreta-
tion difficult. In this analysis, one study relied upon a popula-
tion set drawn from a randomised trial of nCRT, and thus may
not be representative of the wider population [17, 29]. There
was also a wide geographical variation in datasets,
representing populations with likely significant differences
in clinical factors such as body mass index and smoking sta-
tus. Clinicopathological variables may account for some if the
variation in results was not able to be sub-analysed due to data
being non-extractable, frequently because it was not consis-
tently presented across the studies or was not presented sepa-
rately for rectal cancers within a larger colorectal cohort, thus
making multivariable analysis impossible. These factors are
keeping with the wider experience in CRC methylation re-
search [18]. Despite none of the studies being excluded based
on their respective N-O scores, the overall quality of the in-
cluded studies was only good–fair, with a median score of 7
out of a possible 9. in OS, there was a statistically significant relationship between
poorer DFS and CIMP-positive tumours (p = < 0.010), in
agreement with Bae and Kim, who also report a worse DFS
with CIMP-H (p = 0.042 and p = 0.018, respectively). This
finding is however directly contradicted by Kokelaar, where
pooled CIMP-H and CIMP-I were not associated with DFS
(p = 0.10). in OS, there was a statistically significant relationship between
poorer DFS and CIMP-positive tumours (p = < 0.010), in
agreement with Bae and Kim, who also report a worse DFS
with CIMP-H (p = 0.042 and p = 0.018, respectively). This
finding is however directly contradicted by Kokelaar, where
pooled CIMP-H and CIMP-I were not associated with DFS
(p = 0.10). Within the broader context of CRC, CIMP has been
demonstrated to be significantly related to both DFS and
OS. A systematic review and meta-analysis by Juo analy-
ses 33 studies and extracted data representing 10,635 pa-
tients, finding that the OR for DFS and OS was 1.45 (95%
CI 1.07–1.97) and 1.43 (95% CI 1.18–1.73), respectively,
for CIMP-positive tumours on the basis of pooled
dichotomised analysis [18]. Fig. 2 Pooled analysis between
CIMP and OS References 1. Jemal A et al (2017) Annual report to the nation on the status of
cancer, 1975–2014, featuring survival. J Natl Cancer Inst 109(9). https://doi.org/10.1093/jnci/djx030 2. Kokelaar RF et al (2016) Locally advanced rectal cancer: manage-
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(2017) Management of the complete clinical response. Clin Colon
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Proscurshim I, Gama-Rodrigues J (2013) Transanal endoscopic mi-
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vant chemoradiation therapy: another word of caution. Dis Colon
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Bwatch and wait^ for rectal cancer after neoadjuvant therapy: a
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management of rectal cancer. Cancer 122(1):34–41 8. Dayde D, Tanaka I, Jain R, Tai M, Taguchi A (2017) Predictive and
prognostic molecular biomarkers for response to neoadjuvant che-
moradiation in rectal cancer. Int J Mol Sci 18(3):573 9. Karagkounis G, Kalady MF (2017) Molecular biology: are we get-
ting any closer to providing clinically useful information? Clin
Colon Rectal Surg 30(5):415–422 10. Dienstmann R, Vermeulen L, Guinney J, Kopetz S, Tejpar S,
Tabernero J (2017) Consensus molecular subtypes and the evolu-
tion of precision medicine in colorectal cancer. Nat Rev Cancer
17(2):79–92 Compliance with ethical standards thirteen); the most commonly employed panels being the
‘classic’ (MINT1, MINT2, MINT31, CDKN2A, and
hMLH1), or the Weisenberger panel (CACNA1G, IGF2,
NEUROG1, RUNX3, and SOC1) [30]. Jia and colleagues
also reported that up to 15 different methylation markers
were employed in studies of CRC in their systematic re-
view of methodologies, and that the prevalence of CIMP
ranged between 6.4 and 48.5% [31]. Studies investigating
the relationship between a single methylated locus and sur-
vival have remain inconclusive, although hypermethyla-
tion of the promotor regions of CDNK2A and IGFBP3
has shown the greatest association with poor outcomes in
some small studies, although the data is conflicting and
often not replicated in studies with larger cohorts
[32–34]. The relationship between CIMP and MSI is also
the subject of ongoing investigation, with some evidence
suggesting that methylation silencing of hMLH1 is the
common factor in sporadic colorectal cancers [35]. The
CIMP+/MSI+ phenotype has been associated with poor
outcomes in gastric cancer based on pooled analysis of
panels that included hMLH1 [21], although similar analy-
sis in CRC has in some populations indicated the converse
[36]. The complexity reflected in these studies is represen-
tative of the overlying complexity of the genetic and epi-
genetic profiles of CRC genetics. The Cancer Genome
Atlas Network describes a pool of 125 colorectal tumours,
including 62 rectal tumours that were subjected to whole-
genome methylation and mutational analysis [37]. Their
findings describe a complex pattern of four sub-groups
characterised by overlapping but variable patterns of
hypermutation and MSI [38]. However, no difference in
the tumour site was noted and no outcomes were assessed
relating to methylation and tumour site, contradicting other
analyses [10]. Conflict of interest
The authors declare that they have no conflicts of
interest. Conflict of interest
The authors declare that they have no conflicts of
interest. Open Access This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give appro-
priate credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made. Discussion The reported prevalence of
CIMP in the included studies was 4.6 to 46.5%, with a
median of 18.2%. Despite the pooled analysis demonstrat-
ing a worse prognosis for DFS and OS, 8 out of 11 and 13
out of 19 studies in this meta-analysis failed to identify any
significant relationship between DFS and OS, respectively. Thirty-seven studies were excluded from the analysis due
to the RC sub-cohort not being presented separately from
either from a whole CRC or left-sided cancer cohort, sig-
nificantly limiting the power of this analysis. Fifteen genes were used to assess CIMP across the 6
studies included in this analysis (median six), which is
keeping with the findings of other authors. Juo reported a
median of five genes in their analysis (range of three to Fig. 2 Pooled analysis between
CIMP and OS Int J Colorectal Dis (2018) 33:995–1000 999 Conclusions Jo P, Jung K, Grade M, Conradi LC, Wolff HA, Kitz J, Becker H,
Rüschoff J, Hartmann A, Beissbarth T, Müller-Dornieden A,
Ghadimi M, Schneider-Stock R, Gaedcke J (2012) CpG island
methylator phenotype infers a poor disease-free survival in locally
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